Université Victor Segalen Bordeaux THÈSE DOCTORAT DE L UNIVERSITE BORDEAUX SEGALEN

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1 Université Victor Segalen Bordeaux Année 2013 Thèse n 2073 THÈSE Pour le DOCTORAT DE L UNIVERSITE BORDEAUX SEGALEN Le 22 novembre 2013 Par Sara ELRAMAH Née le 17 Juin 1981 au Soudan Mention : Science Biologique et Médicales Option : Neurosciences Présentée et soutenue publiquement Towards a Better Understanding of mirna Function in Neuronal Plasticity: Implications in Synaptic Homeostasis and Maladaptive Plasticity in Bone Cancer Pain Condition MicroRNAs et Plasticité Neuronale: Rôle dans l Homéostasie Synaptique et la Plasticité Dysfonctionnelle en Condition de Douleur Cancéreuse Membre du Jury : M. Martin Teichmann Professeur - INSERM U869, Université Bordeaux Segalen, Président Mme. Marzia Malcangio Professeur - King s College of London, Rapporteur M. Alain Trembleau - Professeur - UMR CNRS 7102 Université Pierre et Marie Curie, Rapporteur Mme. Valérie Fénelon - Professeur - INSERM U862 - Université Bordeaux 1, Examinateur Mme. Florence Rage - Docteur UMR CNRS Montpellier, Examinateur M. Alexandre Favereaux Docteur UMR CNRS Université Bordeaux Segalen

2 ACKNOWLEDGEMENTS Three years of my life was not just about scientific degree, it has been a life experience that I learned a lot from.. It was challenging for me to start new domain in my PhD, and I was always thinking that this must be even more challenging for the thesis supervisor. I am grateful for everything Alexandre Favereaux taught me, and for the patience in doing it. Thank you.. I would like to thank Marc Landry for all the help and opportunities he gave me, but mostly, I would like to thank him for believing in me. This was so important for me to know from Marc as Marc and as head of the team. Many thanks for Frédéric Nagy and André Calas for their helpful comments and fruitful discussions. Christel Baudet; I am grateful for all the help you gave me with experiments. Thank you so much for your sincere concern, and the support you gave me during the 3 years. Thank you for listening. Thank you so much Rabia Benazzouz for the help, especially with organizing animal experiments. I am truly grateful for all the help Olivier Lapirot gave me. Even when he was super busy with projects and students, he always managed to cut a time explaining and helping me with experiments. Thank you I would like to thank Yves Le Feuvre for the electronic help and for his enlightening discussions that participated significantly to this work. It will always be linked in my mind Bordeaux and Cherine Abd Alsalam I will always treasure the time we spent together. Thanks for everything Cheri.. The project was that you help me practicing the language, and you did you part perfectly. I am sorry that I am leaving 3 years later with the same vocabulary, yet, am so grateful for the nice evenings out. Thank you Fanny Farrugia for everything, you truly helped me. I can t imagine a scenario of me doing PhD in Bordeaux without Petra Horakova. Thank you so much for been there. I don t know what I would have done without your support. Amira Zaki and Ahmad Bassiouny; it has been a real pleasure meeting you. I really enjoyed my time in your company. Thank you so much Paul Robillard for the help with cell culture, you made my life easier. Marie Moftah; I don t think I would have accomplished this work without you. Thank you for the opportunity. I would like to thank Matthieu Bastide for the efforts, and for his significant participation in this thesis projects. I would like to thank all master students who spent their internships in the lab during the 3 years. Especially I would like to thank Charline Kambrun and Alienor Ragot for their help.

3 I would like to thank members of Nagy Team for the help they gave me to settle down, when I first arrived the city. I would like to thank members of Group Le Masson; especially, Virginie Roques, Vanessa Charrier, Claire Leger, Ludivine Allard, Alexia Roux. Importantly I would like to thank Jean-Marie Cabelguen for the nice discussions and the help he is always willing to give. My Family, I wanted to start mentioning my gratitude for each thing you did for me, I didn t know whether I should start with thanking for listening, being by my side, support you gave me, looong phone calls, coping with the drama this is going to be long list. I will just thank you for the unconditional love. If this work is considered an achievement, it s yours as it is mine. If I asked to whom I would like to dedicate this work, it will be again and always to the beautiful souls of Tita and Hano.

4 ABSTRACT MicroRNAs (mirnas) are a type of small RNA molecules (21-25nt), with a central role in RNA silencing and interference. MiRNAs function as negative regulators of gene expression at the posttranscriptional level, by binding to specific sites on their targeted mrnas. A process results in mrna degradation or repression of productive translation. Because partial binding to target mrna is enough to induce silencing, each mirna has up to hundreds of targets. mirnas have been shown to be involved in most, if not all, fundamental biological processes. Some of the most interesting examples of mirna activity regulation are coming from neurons. Almost 50% of all identified mirnas are expressed in the mammalian brain. Furthermore, mirnas appear to be differentially distributed in distinct brain regions and neuron types. Importantly, mirnas are reported to be differentially distributed at the sub-cellular level. Recently, mirnas have been suggested to be involved in the local translation of neuronal compartments. This has been derived from the observations reporting the presence of mirnas and the protein complexes involved in mirna biogenesis and function in neuronal soma, dendrites, and axons. Deregulation of mirnas has been shown to be implicated in pathological conditions. The present thesis aimed at deciphering the role of mirna regulation in neuronal plasticity. Here we investigated the involvement of mirna in synaptic plasticity, specifically in homeostatic synaptic plasticity mode. In addition, we investigated the involvement of mirnas in the maladaptive nervous system state, specifically, in bone cancer pain condition. We hypothesized that local regulation of AMPA receptor translation in dendrites upon homeostatic synaptic scaling may involve mirnas. Using bioinformatics, qrt-pcr and luciferase reporter assays, we identified several brain-specific mirnas including mir-92a, targeting the 3 UTR of GluA1 mrna. Immunostaining of AMPA receptors and recordings of miniature AMPA currents in primary neurons showed that mir-92a selectively regulates the synaptic incorporation of new GluA1- containing AMPA receptors during activity blockade. Pain is a very common symptom associated with cancer and is still a challenge for clinicians due to the lack of specific and effective treatments. This reflects the crucial lack of knowledge regarding the molecular mechanisms responsible for cancer-related pain. Combining mirna and mrna screenings we were able to identify a regulatory pathway involving the nervous system-enriched mirna, mir Thus, mir-124 downregulation was associated with an upregulation of its predicted targets, Calpain 1, Synaptopodin and Tropomyosin 4 in a cancer-pain model in mice. All these targets have been previously identified as key proteins for the synapse function and plasticity. Clinical pertinence of this finding was assessed by the screening of cerebrospinal fluid from cancer patient suffering from pain who presented also a downregulation of mir-124, strongly suggesting mir-124 as a therapeutic target. In vitro experiments confirmed that mir-124 exerts a multi-target inhibition on Calpain 1, Synaptopodin and Tropomyosin 4. In addition, intrathecal injection of mir-124 was able to normalize the Synaptopodin expression and to alleviate the initial phase of cancer pain in mice.

5 INDEX 1. MiRNA INTRODUCTION MiRNAs Genome MiRNAs Biogenesis MiRNA:Target mrna Recognition MiRNA:Target mrna Binding Sites MiRNA:Target Interaction ( G) MiRNA:Target Interaction (G:U Wobble base pairs) Additional Features Influencing Site Efficacy MiRNA Targeting Coding Regions and 5 UTR MiRNAs Target Multiplicity Multiple Target Sites Synergistic effect MiRNA Non-conserved Target Sites Approaches for Target Recognition MiRNAs Modes of Regulation Translational Repression Inhibition at Pre-Initiation Stage Repression at Post-Translation Initiation Stage Co-translational Protein Degradation Inhibition of Translation Elongation Premature Termination & Ribosome Drop-off Target Degradation Repression or Degradation Mode of Regulation Cleavage/Slicing Explanations for Conflicted MiRNA Regulation Data P-bodies or GW-bodies Stress Granules MiRNA-mediated Target Upregulation Functional Aspects of MiRNAs Models of mirna Function

6 Switch Targets Tuning Targets Neutral Targets Antitargets Degree of Repression Micromanagers of Protein Output MiRNAs Physiological Function Intermingled Relation with Transcription Factors Methods of Identifying mirna Functions Implications in different Physiological Functions Local Protein Translation in Neurons Expression of mirnas in the Nervous System MiRNA & Synaptic Plasticity The Factors behind Expression Alteration MiRNAs involvement in the Nervous System Maladaptive States Neurodegenerative diseases and Psychiatric Disorders Central Nervous System Cancer Pain Manuscript: MicroRNAs are Regulators of Neuronal Plasticity with Implications in Pain Mechanisms MIRNAS REGULATION IN HOMEOSTATIC SYNAPTIC PLASTICITY Homeostatic Plasticity Introduction Homeostatic Synaptic Scaling in Excitatory Neurons (In Vitro) Homeostatic Synaptic Scaling in Inhibitory Neurons (In Vitro) Signaling Pathways Underlying Synaptic Scaling Evidences for Local Translation and UTR Involvement Homeostatic Synaptic Scaling (In Vivo) MiRNAs Regulation in Homeostatic Plasticity Homeostatic Synaptic Plasticity & MiRNAs (In Vitro)..103

7 Homeostatic Plasticity & MiRNAs (In Vivo) Manuscripts: MiRNA mir-92a regulates translation and synaptic incorporation of GluA1 containing AMPA receptors during Homeostatic scaling MiRNA Regulation in Homeostatic Synaptic Scaling Discussion The Presence of mrna in Dendrites Experiments indicating Local Translation Experiments confirming Local Translation GluA1 mrna Regulation upon Activity Blockade Messenger GluA1 MiRNA(s) Targeting Prediction and Candidate MiRNAs Regulatory Role of mir-92a on GluA1 upon Activity Blockade MIRNAS REGULATION IN BONE CANCER PAIN Pain Introduction Nociception Classification of Primary of Afferents The Neurochemistry of Nociceptors Central Projections of the Nociceptor Neuronal Transmission and Receptors The Ascending Pain Pathways Descending Inputs Plasticity of Nociception Peripheral Mechanisms of Plasticity Peripheral Nociceptive Plasticity following Inflammation Peripheral Nociceptor Plasticity following Nerve Injury Central Mechanisms of Plasticity Glial-Neuronal Interactions. 153

8 3.2 Bone Cancer Pain Introduction Models of Bone Cancer Pain Pain Development and Maintenance Inflammatory Point of View Neuropathic Point of View Neurochemical Changes in the Spinal Cord Bone Cancer Pain Transmission to Higher Centers of the Nervous System Neurochemical Changes in the Brain Descending Influence in Cancer-induced Bone Pain Comparison of Neurochemical Signatures between Inflammatory, Neuropathic, and Bone Cancer Pain Therapeutic Targets Available Analgesics MiRNA and Pain Introduction Manuscript: MicroRNA mir-124 regulates Calpain 1, Synaptopodin and Tropomyosin 4 in bone Cancer Pain MIR-92a AND -124 IN SYNAPTIC PLASTICITY DISCUSSION MiRNAs Function in Neuronal Synaptic Plasticity Findings from the Present Work Local Protein Translation and MiRNA Presence at Synaptoneurosome MiRNA as Synaptic Tag and its Localization to Synapses Targeted mrna Point of View MiR-124 Potential Role in Local Translation The Reason behind MiRNA Alteration upon Activity REFRENCES

9 1. MiRNA INTRODUCTION Non-protein coding RNAs account for ~98% of the transcriptional output in humans [1]. They have important cellular functions [2], participating in a diverse collection of regulatory events [3]. Their regulatory impact can occur at some of the most important levels of genome function, including chromatin structure, chromosome segregation, transcription, RNA processing, RNA stability, and translation [4]. In the last decade, RNA molecular biology area was transformed thoroughly by the discovery of small (~20 30 nucleotide [nt]) noncoding RNAs [4]. Diverse classes of small RNAs have been found in animals, plants, and fungi [1]. They have been classified into different groups based on their origin, structure, the components to which they are coupled, or their biological roles [4, 5]. These small RNA groups include micrornas (mirnas), small-interfering RNAs (sirnas), trans-acting sirnas (tasirnas), small-scan RNAs (scnrnas), repeat-associated sirnas (rasirna), and Piwi-interacting RNAs (pirnas) [2, 5, 6]. Regardless of their type and size, small non-coding RNAs share one unifying function in the cellular physiology: the epigenetic regulation of gene expression. They can behave as sequence-specific triggers for mrna degradation, translation repression, heterochromatin formation, and transposon control [5]. Many of these small RNAs function through several pathways on gene expression, with corresponding regulatory mechanisms collectively subsumed under the heading of RNA silencing [4-6]. They have been implicated in RNAi and RNA silencing by serving as specificity factors that direct bound effector proteins to target nucleic acid molecules via base-pairing interactions [1, 4]. Two types of small RNA molecules, micrornas (mirnas) and small interfering RNAs (sirnas) have a central role in RNA silencing and interference [1]. The downregulation of gene expression by mirnas and sirnas is a complex process involving both translational repression and accelerated mrna decay, each of which appears to occur by multiple mechanisms [7]. SiRNAs and mirnas are the most broadly distributed in both phylogenetic and physiological terms and are characterized by the doublestranded nature of their precursors [4], however, they mainly differ in their biogenesis [6]. MiRNAs have emerged as critical player in the field of life sciences [1]. They are between 21 and 25nt long, and comprise a large family of endogenous molecules that powerfully regulate eukaryotic gene expression (Fig. 1) [8]. They have been identified in the unicellular algae Chlamydomonas reinhardtii [9, 10], indicating that mirnas are probably evolutionarily older than originally thought [11]. 1

10 Figure1. Examples of Metazoan mirnas Shown are predicted stem loops involving the mature mirnas (red) and flanking sequence. The mirnas* (blue) are also shown in cases where they have been experimentally identified [12]. (A) Predicted stem loops of the founding mirnas, lin-4 and let-7 RNAs [13, 14]. (B) Examples of mirnas from other metazoan genes, mir-1, mir-34, and mir-124. Shown are the C. elegans stem loops, but close homologs of these mirnas have been found in flies and mammals [15-18]. (C) Examples of mirnas from plant genes, MIR165a, MIR172a2, and JAW. Adapted from Bartel [8]. The founding member of the mirna family is lin-4, which was first identified in C. elegans through a genetic screen for defects in the temporal control of post-embryonic development [19, 20]. Series of molecular and biochemical studies demonstrated that this is involving direct, but imprecise, base pairing between lin-4 to its target lin-14 3 UTR. In addition, it has been reported that this imperfect binding was essential for the ability of lin-4 to control LIN-14 expression through the regulation of protein synthesis [8, 21-23]. Almost 7 years later, a second mirna was discovered, let-7 [24], as lin-4, let-7 was reported with a role in the developmental control of C. elegans. It acts to promote the transition from late-larval to adult cell fates in the same way that the lin-4 RNA acts earlier in development to promote the progression from the first larval stage to the second [14, 25]. Similar to lin-4, let-7 was found to perform its function by binding to the 3 UTR of lin-41 and hbl-1 (lin-57), inhibiting their translation [14, 25-28]. Because of their common roles in controlling the timing of developmental transitions, the lin-4 and let-7 RNAs were dubbed small temporal RNAs (strnas) [29]. Less than one year later, three labs cloned small RNAs from flies, worms, and human cells, thus, reporting a total of over one hundred additional genes for tiny noncoding RNAs. However, unlike lin-4 and let-7 RNAs, many of 2

11 the newly identified ~22 nt RNAs were not expressed in distinct stages of development, thus the term microrna (mirna) was used instead of the strnas [15, 17, 18]. MiRNAs have been found to be conserved in closely related animals, such as human and mouse, or C. elegans and C. briggsae [12, 30, 31]. This statement remains true even when ignoring evolutionary conservation as a criterion for classifying clones as mirnas. Many are also found to be conserved among more broadly animal lineages [12, 30, 32, 33]. For instance, more than a third of the C. elegans mirnas have their homologs among the human mirnas [12]. When comparing distant lineages, considerable expansion or contraction of gene families is apparent, the most striking example being the let-7 family, which has four identified members in C. elegans and at least 15 in human, but only one in Drosophila [12, 29, 33, 34]. This high conservation characteristic across a wide range of species [12, 29, 32], has been suggested to indicate a role of mirnas participation in essential biological processes [35]. MiRNAs are known to mainly function as negative regulator of gene expression at the posttranscriptional level, by binding to sites on their target mrnas [11, 24, 36, 37]. They use antisense mechanism to guide RNA-induced silencing complexes (RISCs) to specific messages with fully or partly complementary sequences [4, 6, 38, 39]. A process that results, in inducing mrna degradation or repression of productive translation [8, 11, 36, 40-43]. Since the discovery of let-7, more than 1000 distinct mirnas have been described and annotated in various organisms ranging from algae to humans ( [44, 45]. An important aspect of mirna expression is the sheer abundance of certain mirnas in the cells. For example, mir-2, mir-52, and mir-58 are each present on average at more than 50,000 molecules per adult worm cell a greater abundance than the U6 snrna of the spliceosome [12]. Whether this high expression is attributable to very robust transcription or to slow decay is not yet known. Some mirnas are expressed at much lower levels. For instance, mir-124 is present in the adult worm on average at 800molecules per cell [12]. This low average level (though still higher than that of the typical mrna) could be due to low expression in many cells or high expression in just a few cells [8]. The rapid increase in mirnas numbers in the recent years, in addition to their important functional roles in biological processes, led to the necessity of standardization of a universally accepted nomenclature system. MiRNAs are designated with unique numerical identifiers indicative of the series of their discovery. The initials of the species are borne as a prefix to mir, while the numerical identifier is written as the suffix [46]. Furthermore, mature mirnas are denoted by mir while mir is allocated to precursors or genes. The genes encoding the mirna are named using the same three letter prefixes, with capitalization, hyphenation and italicization (e.g. mir-1) in D. melanogaster). 3

12 However, there are few exceptions in the mirna nomenclature system including lin-4 and let-7 which do not follow the conventional naming process for historic reasons [47]. MiRBase ( plays an important role in mirna nomenclature. The mirbase database is a searchable database of published mirna sequences and annotation. The numbers of individual mirnas expressed in different organisms are reported to be comparable to those of transcription factors or RNA-binding proteins (RBPs). In addition, many mirnas are expressed in a tissue-specific or developmental stage-specific manner, thereby greatly contributing to cell-type-specific profiles of protein expression [39]. In addition to the examples from the founding members of mirna family (lin-4 and let-7 RNAs) [12, 16, 17, 29, 48], other interesting examples include mir-1, which is primarily found in mammalian heart [16, 18]; mir-124 is nearly exclusively expressed in the brain [16]; mir-122, which is primarily in the liver [16]; mir-223, which is primarily in the granulocytes and macrophages of mouse bone marrow [49]; mirnas of the mir-35 mir- 41cluster, which are preferentially in the C. elegans embryo [17]; and those of the mir-290 mir-295 cluster, which are expressed in mouse embryonic stem cells but not in differentiated cells [50]. With all the different genes and expression patterns, it has been proposed that every metazoan cell type at each developmental stage might have a distinct mirna expression profile; providing ample opportunity for micromanaging the output of the transcriptome [8]. Because partially bind to target mrna is enough to induce silencing, each mirna has several, possibly up to hundreds of targets [51-53]. Thus, individual mirnas can coordinate or fine-tune the expression of proteins in a cell [39]. Despite the fact that mirnas constitute only 1%-3% of each organism genome [35, 54], they are estimated to regulate more than 60% of genes in many and perhaps all metazoans [8, 11, 13, 55-57]. In addition to their first discovered functions in the regulation of developmentally timed events [58-65], they have been shown to be involved in most, if not all, fundamental biological processes. These include timing of developmental transitions [14, 22], induction of organ asymmetry [66], tumor suppression [67], oncogenic activity [68], invasion and metastasis [69], modulation of embryonic stem cell differentiation [70-72], and neurodegeneration [73]. Moreover, extensive profiling of different tissues has shown that, in various combinations, subsets of the currently known mirnas are dysregulated in disease versus normal states [74, 75]. More recently, mirnas are being investigated both as therapeutics and targets by the pharmaceuticalindustry which is hoping to harness the power of RNA-guided gene regulation to combat disease and infection [76, 77]. 4

13 1.1 MiRNAs Genome: micrornas have been mapped on all chromosomes, with the exception of the human Y chromosome [35]. Their presence within the genome, suggests that their transcription might be tightly coordinated with the transcription of other genes including the protein coding genes that serve either as a source of mirnas or as their targets [78]. mirna genes have been reported to be transcribed in a similar mechanism as for protein-coding genes [39]. The promoter regions of autonomously expressed mirna genes are found to be, as well, highly similar to those of protein-coding genes [79, 80]. Another common feature with protein-coding genes is that the promoters of mirna genes are controlled by transcription factors (TFs), enhancers, silencing elements and chromatin modifications (Fig. 2). This has been concluded because of the presence of CpG islands, TATA box sequences, initiation elements and certain histone modifications, in the mirna genome [39]. The evidence demonstrating their tissue-specific or development-specific expression, indicates that such expression is under major level of control [39]. mirna genes have been categorized based on their genomic location into; intergenic, intronic and exonic [81]. Figure2. Gene regulation by transcription factors and micrornas. Adapted from [82]. Around 50% of mammalian mirna-coding genes are localized within the intergenic space [78]. It has been hypothesized that these genes derive from independent transcription units, because they are found to come from regions of the genome quite distant from previously annotated genes [15, 17, 18]. Most of the intergenic mirnas have been reported to be expressed autonomously, possess their own regulatory elements [13, 15, 17, 18, 80]. The majority of mirna genes within this category are transcribed by RNA Polymerase II (RNA PolII) [83, 84]. According to experimental observed and predicted data, most of the intergenic mirnas should have a primary transcript of 3-4 kb in length, with a delineated transcription start site and a poly(a) signal [80, 85]. 5

14 Approximately 40% of mirna genes are localized within gene introns [81, 86]. In contrast to intergenic mirnas, they are found in the introns of annotated genes, both protein-coding and noncoding [15, 17, 87]. Observations revealing the preferential orientation of these genes as predicted mrnas, led to the suggestion that most of these mirnas are not transcribed from their own promoters, but are instead processed from the introns, as the case for many snornas [12, 30, 33, 34]. The initiation of RNA PolII- or, in some cases, RNA PolIII-dependent transcription [79, 88] within an intron, might lead to preventing the transcription and splicing of the protein coding genes. On the other hand, different approaches show the ability of numerous intronic mirnas to be coexpressed with their host genes [15, 17, 81, 89-91]. Furthermore, intronic mirnas can be transcribed from the same promoter as their host genes and processed from the introns of the host gene transcripts [30]. This arrangement provides a convenient mechanism for the coordinated expression of a mirna and a protein [8]. For instance, the intronic mir-208a, which is coexpressed with its heart-specific host gene Myh6, controls the suppression of negative regulators of muscle growth and hypertrophy in mice [92]. Similarly, transcriptional activation of apoptosis-associated tyrosine kinase (AATK), which is essential for neuronal differentiation, leads to expression of its intronic mirna mir-338 that suppresses mrnas whose protein products are negative regulators of neuronal differentiation [93]. In addition to the possible role of mirnas as capacitators of their host gene function [94], intronic mirnas can also function as direct negative regulators of their host gene expression. This has been exemplified in mir-128, which can regulate the expression levels of its host gene Arpp21 in the brain [95, 96]. Regulatory scenarios are easy to imagine in which such coordinate expression could be useful, thus, explaining the conserved relationships between mirnas and host mrnas [8]. Interestingly, the location of some intronic mirnas is well conserved among diverse species, indicative of their significance in coordinating various physiological processes [35]. An example of this conservation involves mir-7, found in the intron of hnrnp K in both insects and mammals [33]. Although the previous examples, it should be noted that the simultaneous presence of the mirna and its host mrna in a given tissue does not automatically imply co-transcription of the mirna and its host gene(s). In these cases, the potential impact of transcription on host gene splicing might be avoid, where the transcription does not occur simultaneously but rather in a seesaw -like fashion [97]. Furthermore, some mirnas (mir-1839) are known to be generated from small nucleolar RNAs (snornas; [98]). SnoRNAs are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs [35]. Exonic mirnas are loocated within exons, representing approximately 10% of known mirna genes [35, 78]. These mirnas are also thought to be transcribed by their host gene promoter and their maturation often excludes host gene function [81]. 6

15 A single mirna transcript might code for more than one product. Many mirna genes are organized into operons: clusters of open reading frames that are transcribed together in a single polycistronic transcript. Polycistronic transcripts are more abundant in bacteria than in eukaryotes [99]. Half of all mirnas are identified in clusters that can be transcribed as polycistronic primary transcripts [8]. It has been predicted that approximately one-third of mirnas are closer than 10 kb to another mirna in animal genomes [45, 99]. Although the majority of worm and human mirna genes are isolated and not clustered [12, 31], over half of the known Drosophila mirnas are clustered [33]. Furthermore, mirna genes that are clustered within kb from each other display common expression patterns, while those that are spaced more than 50 kb apart tend to be expressed in a noncoordinated fashion [89]. Common expression patterns of clustered mirnas might reflect their generation from a single PolII-dependent polycistronic transcript [84]. Orthologs of C. elegans lin-4 and let-7 are clustered in the fly and human genomes and are coexpressed, sometimes from the same primary transcript, leading to the idea that the genomic separation of lin-4 from let-7 in nematodes might be unique to the worm lineage [33, 48, 100]. The coordinated mirna genes expression may have a functional significance, individual mirna derived from the same cluster might contribute jointly to regulate common physiological process [101]. On the other hand, other evidences revealed that in some clusters, individual functions of each mirna in the cluster can be different [102]. This led to the conclusion that mirnas within a genomic cluster are often, though not always, related to each other; and related mirnas are sometimes but not always clustered [15, 17]. Although, mirnas genes that share the same promoters as the host transcript usually have similar expression profiles as those of the host gene [89], examples of mirna genes and clusters that reside within the introns but that are under the control of their own promoters are also known [103]. Interestingly, in both flies and mammals, some mirnas are convergently transcribed from both DNA strands of a single locus, giving rise to two mirnas with distinct seed sequences. In Drosophila melanogaster, sense and antisense transcripts of mir-iab-4 are expressed in non-overlapping embryonic segments, and processed mirnas regulate development by targeting homeotic Hox genes expressed in specific embryonic domains [104, 105]. 1.2 MiRNA Biogenesis: Although, the mechanisms of mirna action remained elusive, their biogenesis rapidly became clear (Fig. 3) [106]. Indeed, understanding mirna biogenesis and function has been greatly facilitated by analogy and contrast to the sirna system, and vice versa [8]. RNA polymerases were shown to 7

16 produce different coding or noncoding RNAs. MiRNAs are first transcribed into pri-mirna by two candidate RNA polymerases; pol II and pol III. In general, Pol II produces the mrnas and some noncoding RNAs, including the small nucleolar RNAs (snornas) and some of the small nuclear RNAs (snrnas) of the spliceosome. Pol III produces some of the shorter noncoding RNAs, including trnas, 5S ribosomal RNA, and the U6 snrna. Intronic mirnas are processed by pol II. Although most of the metazoan mirna genes do not have the classical signals for polyadenylylation [107], observations provided indirect evidence that many mirnas primary transcripts are capped pol II transcripts [16, ]. These observations include that; (1) the pri-mirnas can be longer than one 1 kb, which is longer than typical pol III transcripts. (2) The frequent presence of internal runs of uridine residues in pri-mirnas is expected to prematurely terminate pol III transcription. (3) Many mirnas are differentially expressed during development, as it is often observed for pol II but not pol III products. (4) The robust reporter protein expression that has been cloned downstream to the 5 portion of mirna genes. Nevertheless previous evidences indicate that others mirna primary transcripts might still be pol III transcripts. Thus, it has been shown that exogenous expression of mirnas from a pol III promoter produces efficiently and precisely processed mirnas that function in vivo [49], indicating that there is no obligate link between the identity of the polymerase and downstream mirna processing or function [8]. Mature mirnas are generated from long primary transcripts by a multi-step biogenesis pathway, with many variants [99]. In the canonical pathway, mirna transcript is processed in two steps, catalyzed by two members of the RNase III enzyme family, Drosha and Dicer, operating in complexes with dsrna-binding proteins (dsrbps) [39, 87]. The first maturation step of the mammalian mirnas is the nuclear cleavage of the nascent folded hairpin mirna transcript (pri-mirna), liberating a nt stem loop intermediate, known as the mirna precursor (pre-mirna) [87, 111]. This processing is performed by the Drosha DGCR8 complex, which cleaves both strands of the stem at sites near the base of the primary stem loop. Drosha is predominantly localized in the nucleus and contains two tandem RNase-III domains, a dsrna binding domain and an amino-terminal segment of unknown function [112]. Drosha cleaves the RNA-duplex with a staggered cut typical of RNase III endonucleases, and thus the base of the premirna stem loop has a 5 phosphate and ~2nt 3 overhang [112, 113]. The mechanism by which Drosha recognize pri-mirna are largely unknown but include the stem loop secondary structure, the stem structure and the flanking sequence of the Drosha cleavage site [49, 111, 112]. 8

17 Figure3. MicroRNA biogenesis. Adapted from [39]. 9

18 Another pathway of mirna maturation is the noncanonical pathway of mirtron [78]. MiRtrons are the regulatory RNAs which get processed to form pre-mirs using the splicing machinery (hence, bypassing the Drosha DGCR8 step) whereas the pri-mirs are processed using the Drosha-DGCR8 complex. However, both of which result in the formation of pre-mirs of 70nt length [39, ]. This pathway has been initially discovered in Drosophila [118, 119], and recently identified in diverse mammals, drosophila and nematodes [8, 24, 39]. It was subsequently shown to be conserved in vertebrates [ ], however, only few mirtrons have been described in mammals so far and these include mir-877, mir-702, and mir-1124 mir-1141 [120, 121]. Pre-miRNAs are then actively exported from the nucleus to the cytoplasm by Exportin 5 (Exp5), a Ran-GTP dependent nucleo/cytoplasmic cargo transporter [39, ]. Once inside the cytoplasm, these hairpin precursors are further cleaved into a small, imperfect dsrna duplex [58-60]. The nuclear cut by Drosha defines one end of the mature mirna, while in the second event of mirna maturation, the other end is processed in the cytoplasm by the enzyme Dicer [112]. Dicer, was first characterized for its role in generating the small interfering RNAs (sirnas) that mediate RNA interference (RNAi) [123] and was later shown to play a role in mirna maturation [58, 60]. Dicer contains a putative helicase domain, a DUF283 domain, a PAZ (Piwi Argonaute Zwille) domain, two tandem RNase-III domains and a dsrna-binding domain (dsrbd) [123]. Although Dicer alone is sufficient to cleave pre-mirnas in vitro, it associates with various proteins in the cell, including TRBP2 (transactivation response element RNA-binding protein 2), PACT (PRKRA; interferon-inducible dsrna-dependent protein kinase activator A) and AGO proteins (also known as EIF2C proteins) to perform pre-mirna cleavage, mature strand selection and loading into an AGO protein to form an active RISC [124]. Dicer first recognizes the double-stranded portion of the premirna, perhaps with particular affinity for a 5 phosphate and 3 overhang at the base of the stem loop. Then, at about two helical turns away from the base of the stem loop, it cuts both strands of the duplex. This cleavage by Dicer lops off the terminal base pairs and loop of the pre-mirna, leaving a 5 phosphate and ~2nt 3 overhang characteristic of an RNase III. Thus, it produces an imperfect duplex (mirna:mirna*), that comprises the mature mirna and a similar-sized fragment derived from the opposing arm of the pre-mirna [8, 58-60]. Two noncanonical pathways that relay on AGO2 protein for the generation of mirna have been reported recently. This pathway relies on Ago2 unique endonuclease activity required for the premirna cleavage event [ ]. In mammals, AGO2, which has robust RNaseH-like endonuclease activity, has been shown to support Dicer processing by cleaving the 3 arm of some pre-mirnas. This result in forming an additional processing intermediate called AGO2-cleaved precursor mirna (ac-pre-mirna), which serves as Dicer substrate (Fig. 3) [128]. However, the other noncanonical pathway for mirna generation is Drosha-dependent but Dicer-independent. In this case, mirna that 10

19 is processed by Drosha, its maturation does not require Dicer. Instead, the pre-mirna becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3' end, which is then further exonucleolyticly trimmed. To date, only a sole mirna, mir-451, has been shown to employ this mechanism [125, 127]. Thus, the full contribution of the Ago2-dependent pathway remains to be explored [78]. Because of the evidence indicating that the specificity of the initial cleavage mediated by Drosha determines the correct register of cleavage within the mirna precursor, Drosha is suggested to be responsible for defining both mature ends of the mirna [112]. This idea that Drosha, not Dicer, imparts the specificity is appealing because studies have shown that generic double-stranded RNA is refractory to Drosha cleavage and that Dicer progressively chops up an RNA double strand, irrespective of its sequence [123, ]. Furthermore, mirna variants can be produced from the same arm of the precursor, and called isomirs [132], where they are frequently detected in high-throughput sequencing of small RNAs [99]. Deep sequencing data suggests that the 3 end of the mature mirna is subjected to more variation than the 5 end [133, 134]. Consequently, it has been suggested that different isomirs have, in principle, different targeting properties [99]. Although plant mirnas seem to be producted from long, primary transcripts, the biogenesis of the mirna duplex appears to differ from mirna maturation in animals. Neither Drosha homologue nor pre-mirnas have been detected in plants [8, 24]. However, the Dicer family showed a unique level of complexity [24]. In A. thaliana, four Dicer homologues were found (DCL1, DCL2, DCL3 and DCL4), with two of which (DCL1 and DCL4) that contain nuclear localization signals [135]. The lack of premirna, together with the apparent nuclear localization of the DCL1 protein [135], suggests that DCL1 provides the Drosha functionality in plants, making the first cut that sets the register for mirna maturation. DCL1 (or another enzyme yet to be identified) then makes the second cut, which corresponds to metazoan Dicer cleavage, before the mirna leaves the nucleus. It has been suggested that that HASTY, the plant ortholog of Exportin-5, is responsible for exporting the mirna:mirna* duplex from the nucleus [114, 115, 136]. Following cleavage and nucleo-cytoplasmic export, the mirna pathway of plants and animals appears to be biochemically indistinguishable from the central steps of RNA silencing pathways known as post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNAi in animals [8]. The generated mature mirnas range from 21 to 25 nucleotides, such differences in size possibly result from the presence of bulges and mismatches on the pre-mirna stem [24]. The mirna strand of the mirna:mirna* duplex is then loaded, in a selective way, into the mirna ribonucleoprotein 11

20 complex (mirnp) [8, 137, 138]. The effector complex for mirnas shares core components with that of sirnas, so much that both are collectively referred to as the RNA-induced silencing complex (RISC) [24]. The mirisc consists of an mirna strand, the Argonaute (AGO) protein family, the GW182 protein family (glycine-tryptophan [GW] repeat-containing protein of 182 kda) [35], that are key factors in the assembly and function of miriscs [39], in addition to other accessory proteins [35, 139]. The proteins of the Argonaute family contain three evolutionarily conserved domains, namely PAZ, MID and PIWI, which are known to interact with the 3 - and 5 -ends of the mirna, respectively [140, 141]. In mammals, four AGO proteins, namely AGO1 to AGO4, function in mirna-mediated protein translational repression [35]. When the mirna strand is loaded into the RISC, the opposing arm, mirna* sequence [17], appears to be peeled away and degraded. The degradation fate of mirna* has been concluded from libraries of cloned mirnas, where typically mirnas* are found to accumulate in lower frequency than are the mirnas [12, 16, 33]. With approximately 100-fold difference in mirna:mirna* duplex cloning frequency [12], indicating that the mirna* is generally short-lived compared to the mirna strand [8]. Although mature mirnas can reside on either strand of the hairpin stem [24], the mechanism for choosing which of the two strands enters the RISC, lies in the relative stability of the two ends of the duplex [8]. The strand that enters the RISC is nearly always the one whose 5 end is less tightly paired the mirna* strand [137, 138]. These findings indicate that the relative instability at the 5 end of the mature mirna might facilitate its preferential incorporation into the RISC [137, 138]. Therefore, the thermodynamic properties of the mirna precursor determine the asymmetrical RISC assembly, and therefore, the target specificity for post-transcriptional inhibition [24]. Interestingly, several lines of evidence suggest that both mature products from a hairpin have the potential to function as mirnas. Several studies reported that both mirna and mirna* strands of the mirna duplex accumulate at equal frequencies proposing that both enter the RISC, raising the prospect that either or both might be functional [16, 24, 137, 142]. Studies in Drosophila revealed that a substantial fraction of mirna genes are highly conserved along both mirna and mirna* sequences and that seed matches of both mirna and mirna* species exhibit preferential conservation in 3 UTRs [143]. Recently, reports revealed that mirna* molecules have targeting activity in vivo and in vitro [ ]. Some studies identified functional targets of specific mammalian mirna* species, such as mir-9a* [146, 147], mir-199* [ ], mir-30a* [151], and mir-18a* [152]. Moreover, a recent study demonstrated that a substantial fraction of mirna* species are stringently conserved in their seed regions over vertebrate evolution, their impact has been further revealed using functional tests [145]. Generated data suggest that, as a general rule, a single mirna precursor encodes two mature mirnas, with distinct targeting properties [99]. These data indicate that the regulatory impact of mirna* species on mammalian genomes is much more substantial than 12

21 is currently envisaged. Although the new nomenclature system for mirna, many studies are still using the old mirna/mirna* terminology, since this nomenclature provides important information regarding strand preference of mirna biogenesis, which is almost always asymmetric. It has been stated that, it is abundantly clear that mirna* species cannot be ignored as regulatory molecules and support the notion of a broader usage of 5p-3p nomenclature that acknowledges fluidity rather than sole use of mirna* nomenclature [145]. 1.3 MiRNA:Target mrna Recognition: The importance of complementarity to the mirnas 5 portion has been suspected since the observation that the lin-14 UTR has core elements of complementarity to the 5 region of the lin-4 mirna [13, 14, 23]. Later on, this idea supported by several observations reporting the residues nt complementarity to 3 UTR elements mediating posttranscriptional repression [153], and the conservation of this mrna residues complementary sites [12, 154, 155]. The importance of this mirna:target mrna complementary has been further supported by in silico analysis, which use evolutionary conservation of putative target sites to filter signal (functional mirna target sites) from noise (non-target sites that have mirna sequence complementarity by chance) [38]. Endogenous mirna has been shown to have their targeted sites on UTRs and ORF of mrnas. Nevertheless, experimental data revealed an enrichment of mirna binding sites in 3 UTR of messages of mirnadownregulated proteins (Fig. 4) [52, 55, 156, 157]. Figure 4. MiRNA target recognition in plants and animals [158]. The importance of the 5 end of the small RNA in gene regulation has been attributed to its specific accessibility to the RISC, presenting this core region for base-pairing to the mrnas. Presentation of 13

22 an A-form helix segment of ~7 nt length would be a reasonable compromise between the topological difficulties associated with a longer fragment and the drop in specificity that would result from a shorter core [6, 8]. Many biochemical studies indicate that affinity of the RISC to the seed is stronger than that to other regions of the RNA [159, 160], in addition the presence of protein contacts to the guide-strand backbone have been suggested to preorganize the seed region prior to target binding [161, 162]. Nevertheless, additional pairing outside the core, might participate in adding mirna:target specificity [8, 106]. The degree of gene regulation has been shown to vary substantially, depending on the specific mirna/mrna target combination [163]. The target-site recognition has been shown to be primarily determined by the quality and stability of core match (mirna:mrna binding site) [ ], in addition to other factors that may participate in determining base pairing specificity. This includes proteins or mrna structure that could restrict mirnp accessibility to the UTRs [106, 164, 165]. Furthermore, mrnas tend to contain several binding sites for the mirna, emphasizing the potential importance of synergistic binding of the mirna to the target. This synergism has been directly demonstrated, as addition of multiple binding sites into a 3 UTR resulted in more efficient inhibition of translation than that expected from the sum of the effect of each binding site individually [166]. Previous studies showed that an sirna can translationally repress a target mrna with imperfectly complementary binding sites in its 3 UTR, concluding that sirna functions as mirna [ ]. Hence, sirna has been used to investigate mirna:mrna pairing [106]. Several investigations were systematically performed in vivo and in vitro, to determine which region of the mirna:mrna interaction was of primary importance, using designed 3 UTR constructs cloned downstream to reporter gene. These experiments led to identifying two broad categories of functional mirna target sites; 5 dominant sites and 3 compensatory sites (Fig. 5) [37]. Many observations confirmed and supported the suggestions that extensive base-pairing to the 5 end of the mirna is crucial for target site function [37, 106, , 169]. Thus, applying mismatch mutations in the 5 region of the mirna inactivated the reporter repression, in compare to other sites mutations [106]. 14

23 Figure 5. Types of mirna Target Sites (A C) Canonical, 7 8nt seed-matched sites. Vertical dashes indicate contiguous Watson Crick pairing. (D E) Marginal, 6nt sites matching the seed region. These 6mer sites typically have reduced efficacy (Figure 4A) and are conserved by chance more frequently than the larger sites. Therefore, when prioritizing site efficacy and prediction specificity, prediction algorithms with stringent seed-pairing criteria disregard 6mer sites. (F G) Sites with productive 3 pairing. For 3 -supplementary sites (F), Watson Crick pairing usually centering mirna nucleotides (orange) supplements a 6 8 nt site (A E). At least 3 4 well-positioned contiguous pairs are typically required for increased efficacy, which explains why 3 -supplementary sites are atypical. For 3 - compensatory sites (G), Watson Crick pairing usually centering on mirna nucleotides (orange) can compensate for a seed mismatch and thereby create a functional site. (H) Number of preferentially conserved mammalian sites matching a typical highly conserved mirna (Friedman et al., 2008). For each site matching the seed region, orange-hatched subsectors indicate the fraction of conserved sites with preferentially conserved 3 -supplementary pairing. Analysis was performed with the 87 mirna families highly conserved in vertebrates. A 7mer site is counted only if it is not part of an 8mer site, and a 6mer site is counted only if it is not part of a larger site. Values plotted are the number of preferentially conserved sites confidently detected above background, calculated as the average number of conserved sites minus the upper 95% confidence limit on the sites estimated to be conserved by chance. Thus for each site type of panels (A) (E), there is 95% confidence that the actual average of preferentially conserved sites is higher than that plotted [6]. 15

24 1.3.1 MiRNA:Target mrna Binding Sites: Testing the minimal length of 5 seed matches, revealed a single 8mer seed (mirna positions 1 8) was sufficient to confer strong regulation by the mirna, while a single 7mer seed (positions 2 8) was also functional, although less effective [37, 170]. In contrast, 6mer seeds showed no regulation [37, 170]. Another study investigated which type of heptanucleotide match is most associated with decreased protein output. Results showed that the motif that is the most associated with messages destabilization is a 6-nucleotide match to the mirna seed supplemented by a match to mrna nucleotide 8, and was named the 7mer-8 seed matched site [52]. The translation repression of mrna with single sites revealed that a 7mer-A1 match was more effective than a Watson-Crick 1-7nt match. Besides, it was demonstrated that nucleotide at the position 1 of the mirna is critical to induce regulation. Thus, an Adenosine at position 1 favors mirna-mediated protein downregulation, even when it cannot participate in a Watson-Crick interaction [55]. It has been shown that proteins from messages with single 7- or -8-mer sites had a significant propensity to be downregulated when compared to those from messages without 3 UTR sites (Fig. 6) [52]. These sites that are consistently associated with both performed conservation and mrna destabilization after mirna introduction are named the 6mer, 7mer-A1 and 8mer seed-matched sites [55, 171, 172]. Figure 6. Relative Mean Efficacy of mirna Sites [6]. It has been proposed that target specificity may therefore be determined during mirna biogenesis by the exact cleavage point of the pre-mirna to form the mature mirna. Consequently, different isomirs (heterogeneous mirna variants [173]) have, in principle, different targeting properties. This phenomenon is known as seed shifting [174]. It has been suggested that isomirs are likely to be 16

25 functionally relevant [132, 175]. As high-throughput technologies and analysis methods become more robust, the contribution of isomirs to differential gene regulation will be better understood [99]. Brennecke et al [37] investigated the minimal 5 sequence complementarity necessary to confer target regulation when strong base-pairing to the 3 end of the mirna is allowed. Surprisingly, under these conditions, as few as four base-pairs in positions 2-5 were able to confer efficient target regulation [37]. The study suggested that a functional seed requires a continuous helix of at least 4 or 5 nucleotides and that there is some position dependence to the pairing, since sites that produce comparable pairing energies differ in their ability to function [37]. Unlike the 5 region of the mirna, interactions in the second category 3 end of mirna were shown of minimal importance [37, 106]. Thus, extensive 3 pairing up to 17 nucleotides in the absence of the minimal 5 element was not sufficient to confer regulation [37]. However, the 3 region can modulate target interaction, with regard to the pairing at the 5 region [106]. Hence, in the context of a strong 5 end pairing, sites with 3 pairing below the random noise level are functional in contrast, insufficient 5 pairing, requires strong 3 pairing for function [37]. Hence, a classification of mirna and their target regions has been defined (Fig 5). Targets in the first category, 5 dominant sites, base-pair well to the 5 end of the mirna [37]. This group has been further subgrouped into: (i) those with good pairing to both 5 and 3 ends of the mirna (canonical sites) and (ii) those with good 5 pairing but with little or no 3 pairing (seed sites). Canonical sites have strong seed matches supported by strong base-pairing to the 3 end of the mirna. Canonical sites can thus be seen as an extension of the seed type (with enhanced 3 pairing in addition to a sufficient 5 seed) [37]. Systematically, 3 pairing of mirna has been categorized into 3 -supplementary sites and 3 -compensatory sites [6]. 3 -supplementary sites : are 3 pairing optimally centered on mirna nucleotides and it is shown to enhances seed pairing. MiRNA 3 pairing appears to be poorly predicted with thermostability criteria and more reliable to pairing geometry, preferring at least 3 4 contiguous Watson Crick pairs uninterrupted by bulges, mismatches or wobbles. However, such sites are atypical, and tend to be only slightly more effective than those without the supplementary pairing [171], suggesting that supplementary 3 pairing plays a modest role in target recognition [6]. 3 -compensatory sites : are those pairing to the 3 portion of the mirna which; compensate for a single nucleotide bulge or mismatch in the seed region. In 3 -compensatory sites, the pairing centered on mirna nucleotides extends to at least nine contiguous Watson Crick pairs, substantially 17

26 more than the number needed to observe effective supplementary pairing [6]. However, 3 - compensatory pairing are only rarely under selective pressure to be conserved [37, 55, 56]. It has been proposed that such sites with extensive pairing to the 3 portion of the mirna possess much more informational complexity than do the 7 8mer perfect matches and therefore emerge much less frequently and are harder to maintain in evolution [6]. Members of mirnas families are mirnas sharing a common 5 dominant canonical and seed site, but with different 3 compensatory sites. Hence, the mirna 3 ends were shown to be key determinants of target specificity within mirna families, and used to discriminate among individual of families members in vivo [37]. Another type of mirna:target mrna binding includes highly complementary target sites. Highly complementary target sites have extensive central pairing which are rarely documented in animals which results in target regulation through slicing. This kind of regulation is found more in plants, with very few examples of mirna-dependent target cleavage reported in mammals [ ]. Slicing is thought to provide an efficient means of RNA degradation or clearance [38]. Argonaute protein Ago2 is the main actor, catalyzing cleavage of the target strand in the context of extensive base pairing [130, 176, 179]. Recently, Shen et al [180] have reported a new class of mirna target sites. Interestingly this site lacks both perfect seed pairing and 3 -compensatory pairing, instead have contiguous Watson- Crick pairs to the center of the mirna at either nt 4 14 or This study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. It has been proposed that this expanded repertoire of cleavage targets and the identification of the centered site type might help explaining why central regions of many mirnas are evolutionarily conserved. Although centered sites were able to cleave mrna in vitro (in elevated Mg²+), in cells they repress protein output without AGO2-catalyzed cleavage [180]. The study further revealed that central region of vertebrate mirnas, although less conserved than other mirna regions, is more conserved than opposite arm of the premirna double stranded hairpin. Because both arms participate equally in the pairing required to form the pre-mirna hairpin, preferential conservation of the mirna observed in this region suggested that these central nucleotides play a role beyond that of mirna biogenesis [180]. The reason why centered sites had not been described previously can be their relatively low abundance, which resembles that of 3 compensatory sites and is far lower than that of seed-matched sites [180]. Taken together these data, illustrate both the broad scope of seed-type regulation and the widespread influence of this targeting mode on mrna evolution [180]. 18

27 1.3.2 MiRNA:Target mrna Interaction (ΔG): Studies using the free energy as a means of quantifying the potential mirna:mrna interaction, showed controversial results. Doench et al [106], showed that calculated free energy ΔG varies with different mutated construct on the 5 end of the mirna, demonstrating a strong correlation with reporter inhibition. Interactions with a low free energy were not active in reporter repression, whereas greater free energy was optimally active [106]. However, the study by Brennecke et al [37] did not find a clear correlation between 5 pairing energy and function. Further support might come from previous studies, where the pairing and energy-based criteria designed to identify and rank 3 - supplementary pairing [155, 181, 182], were found to lack predictive value [52, 171] MiRNA:Target mrna Interaction (G:U wobble base pairs): The role of G:U wobble base pairs, which are thermodynamically favorable and are common in RNA secondary structure, was investigated in the context of mirna:mrna interactions [106]. MiRNA target genes can contain predicted binding sites with seeds that are interrupted by G:U base-pairs or simple nucleotide bulges [14, 22, 26, 27, 154, 183]. A single G:U wobble was found to be highly detrimental to translational repression, and more than one G:U wobble pairing eliminated activity entirely [37, 106]. Interestingly, the study by Brennecke et al [37] showed that the degree of repression upon applying bulged nucleotide, differs depending on the bulged nucleotide [37]. In the study by Doench et al [106], supporting the importance of free energy in determining mirna/target interaction, G:U wobble pairing was found to be highly detrimental to mirna function despite its favorable contribution to RNA:RNA duplexes represented by favorable ΔG [106] Additional Features Influencing Site Efficacy: In addition to the sequence specificity provided by mirna guides, other factors have been shown to influence target site selection (Fig 6). This includes the nucleotide composition in the immediate vicinity of the site, where AU richness was found the most influential component that enhances accessibility to the mirna complex [6, 171, 184]. Examining the effect of local AU composition, it was found to be significantly correlated with protein translation [52]. When the site has a match to position 8, an A or U across from position 9 is particularly favorable, suggesting a non-watson Crick recognition of this nucleotide as the one in position 1 [55, 172]. The remaining benefit of local AU composition might be, as suggested for site position, to place the site within a more accessible UTR context [6]. Another factor is the location of the target site; sites within the 3 UTR and out of the path of the ribosome tend to be most effective if they do not fall in the middle of long UTRs [171]. It has been suggested that sites in the middle of long UTRs might be less accessible to the silencing complex 19

28 because they would have opportunities to form occlusive interactions with segments from either side, whereas sites near the UTR ends would not [6]. RNA-binding proteins (RBPs) were also reported to influence mirna:target accessibility (example; the FMRP protein) [164, 165]. They can associate with target mrnas thus interfering with mirisc binding or function [184]. Another factor can be the mirisc regulation, indeed, several members of the TRIM NHL family of proteins directly associate with Argonaute and regulate the ability of mirisc to repress target gene expression [184]. Finally, an important factor is the target site accessibility, which can be influenced by stable secondary structures [160, 185]. Generally unstructured areas seem to enhance accessibility to the mirna complex [6] MiRNA Targeting Coding Regions and 5 UTR: In plants, most of the reported mirna interactions were with the amino acid coding region (CDS) of the target genes [8, 186], which has been thought for years to be the distinction between plants and animals mirnas systems [187]. Although most investigations into metazoan mirna function has been for sites in 3 UTRs, evidence using artificial sites show that targeting can occur in 5 UTRs and open reading frames (ORFs) [188, 189]. Furthermore, computational and experimental genome-wide analyses indicate that a significant amount of mrnas, could be targeted in ORFs [52, 55, 156, 157, 171, 190, 191]. Overall, endogenous ORF targeting appears to be less frequent and less effective than 3 UTR targeting but still much more frequent than 5 UTR targeting [6]. One reason why 5 UTRs and ORFs may be less hospitable for targeting is that the physical presence of the microribonucleoprotein (mirnp) complex bound to these regions would be displaced by the translation machinery with ribosome scanning during initiation and/or with reading of the message [8, 189]. Support for this notion comes from the observation that the transition to more effective and more selectively conserved sites occurs ~15nt into the 3 UTR [171]. It has been suggested that targeting of sites perfectly complementary to artificial sirnas is not hampered by ribosome interference, presumably because the ribosome has more difficulties to disrupt extensive pairing or because sites cleavage doesn t need silencing complex to remain associated for long [6]. In zebrafish, exogenous reporter mrnas containing target sites for let-7 mirna in either the coding region or the 5 UTR were both silenced after injection of exogenous let-7 [188]. In a study by Lytle et al [189], authors introduce mirna target sites into the 5 UTR of luciferase reporter mrnas containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a mirnp complex would not interfere with the initiation of translation. Results showed; that HeLa cells expressing endogenous let-7a mirna efficiently repressed the translation of IRES-containing 5 UTR reporters. These results suggest that association with any position on a target mrna is 20

29 mechanistically sufficient for a mirnp to exert repression of translation at some step downstream of initiation [189]. Although mirna target sites in ORFs were previously noticed, they did not get the attention deserved because they were thought to be rare, weak or of uncertain importance in vivo [157, 171]. The fact that validated 3 UTR target sites currently outnumber those in coding regions has been proposed to reflect a potentially flawed bias of most bioinformatics tools towards 3 UTRs [38]. Nucleotides within the ORF have been proposed to be good candidates to form persistent mirna:mrna heteroduplexes. This is supported by the fact that CDS nucleotides were constrained through their participation in codons that endows them stability against mutations [187]. Recent findings by different groups reported supporting evidences for mirna targeting of CDS [71, 72, ], which has been confirmed using reporter assays [167, 188, 190]. Thus, CDS targets can be as physiologically relevant as their 3 UTR counterparts. Just as in 3 UTRs, one mirna can have multiple targets in the CDS of the same mrna, and multiple mirnas can target the same CDS in a combinatorial and cooperative manner [187]. Possible interactions between target sites in coding sequences and 3 UTRs have been further studied by Fang et al [197]. Results showed that mirna target sites in coding regions enhance regulation mediated by target sites in 3 UTRs. These effects were stronger for conserved target sites of 7 8nt length in coding regions compared to non-conserved sites. Nevertheless, this synergistic effect of seeds in coding sequences is significant but weaker than the effect generated by additional 3 UTR seeds [197] MiRNAs Target Multiplcity: A genome-wide statistical analysis showed evidence for a surprisingly large number of mirna target sites genome wide [37]. Combinatorial regulation, in which two factors simultaneously regulate a single gene, is a common feature of euokaryotic cells [106]. Several hundred mirnas have been characterized, and each mirna is thought to have several hundred mrna targets (Fig. 7) [43]. In addition to potential regulatory effect each mirna on hundreds of target transcripts, owing to their relaxed base-pairing requirements [38], more than one mirna can simultaneously translationally repress a single mrna [106]. Estimation of the total number of targets approaches the number of protein-coding genes, suggesting that regulation of gene expression by mirnas plays a greater role in biology than previously anticipated [37]. 21

30 Figure 7. MiRNAs target multiple genes and genes are targeted by multiple mirnas. (a) MiRNAs have multiple targets. (b) Many genes have seed matches for multiple mirnas in their 3 UTRs. (c) A complex network of mutual interactions between mirnas and mrnas. Adapted from Pater [198] Multiple Target Sites Synergistic effect: Messenger RNA multiple sites have been shown to be more responsive to mirna regulation [52]. Target site multiplicity is thought to enhance the degree of repression by animal mirnas [38]. Sites that are close together (within 40nt, but no closer than 8nt) tend to act cooperatively, leading to marked enhancement in repression over that expected from the independent contributions of each site [171, 199]. In the study by Doench et al [106], two internal sites in 3 UTR, spaced by 4 or 0nt showed similar repression. Interestingly, a construct with the binding site for the 3 region overlapping with the binding site for the first four 5 nucleotides of another site, did not show further decrease in repression. However, if this overlap between the two sites was increased to 9nt, the construct gave the same amount of repression as only one internal site. Because a binding site can prevent access to sufficiently close binding site, these results suggest that a factor stably associates with the mrna [106]. The cooperative action of multiple RISCs appears to provide the most efficient translational inhibition [166]. By analogy to transcription factors, cooperative mirna function provides a mechanism by which repression can become more sensitive to small changes in mirna expression levels, and it greatly enhances the regulatory effect and utility of combinatorial mirna expression [6]. This explains the presence of multiple mirna complementary sites in most genetically identified targets of metazoan mirnas [13, 14, 23, 26, 27]. Experiments investigating the spacing requirements on the mrna for mirna interaction, revealed that the magnitude of regulation for 8mer and 7mer seeds - but not 6mer seeds - was strongly increased when two copies of the site were introduced in the UTR 22

31 [37]. Although not the norm, hundreds if not thousands of sites fall within a cooperative configuration with a site for the same or a co-expressed mirna [6] MiRNA Non-conserved Target Sites: Poorly conserved 7nt sites have been shown to outnumber preferentially conserved ones by about ten to one [6], and a large fraction of these sites can be functional [156]. However, these 3 UTRs are most often found in genes primarily expressed in tissues where the cognate mirna is absent [156]. An evolutionary explanation for this strategy has been provided, known as selective avoidance [6]. In this case, messages selectively avoid targeting by mirna, and are called the antitargets of that mirna [200]. The phenomenon of selective avoidance has been suggested to have a widespread impact on UTR evolution, especially when considering the thousands of messages avoiding targeting to particular mirnas together with those avoiding targeting to all mirnas. The estimated number of antitargets has been shown to be comparable to the number of conserved targets [156]. Nevertheless, recent work revealed that mirnas can target extensively the amino acid coding region of animal mrnas at locations that are not necessarily conserved across organisms [187]. There is now considerable evidence that many such non-seed target sites of high biological relevance exist [38] Approaches for Target Recognition: The abundant expression of mirnas in diverse multicellular species raised many questions about their functional importance within expressing cells. The answer for these questions are suggested to be in finding their regulatory targets [6]. Large-scale approaches such as computational analysis of conserved complementary sites, have revealed important insights into target recognition and function [37, 55, 155, 156, 171, 172, 182, 201]. Nevertheless, this kind of approach has many drawbacks, in reflecting functional mirnas targets. This might be due to the fact that many important features of mirna:target mrna are not taken in consideration, while other controversial features are considered. For instances, computational approaches for target identifications based primarily on the extent of base-pairing or the total free energy of duplex formation will include many nonfunctional target sites [ ], and ranking target sites accordingly to overall complementarity or free energy of duplex formation might not reflect their biological activity [154, 155, ]. On the other hand, scoring local AU content has been considered more successful in predicting responsive targets [52, 171]. Perhaps because of RNAbinding proteins, RNA tertiary structure, and multiple competing RNA pairing conformations, the details of intracellular UTR structures might differ substantially from predicted structures, such that 23

32 scoring local AU content is more reliable for predicting site accessibility [6]. Computational prediction would be eased by taking into consideration expression profiling of both mirna and mrna levels [106]. Thus, it has been demonstrated that a binding site that is not repressed by endogenous levels of mirna becomes repressed upon addition of exogenous mirna. Exchanging the mrna promoter with a stronger one led to dramatic decrease in reporter repression [106]. This is leading to serious concern regarding those target site tests that rely exclusively on transfected mirnas and reporter constructs [38]. Validation of predicted mirna:mrna interactions by ectopic expression of either mrna target at artificially low levels, or the mirna at artificially high levels, may confirm an interaction that does not exist in vivo. The potential target must be evaluated in its cellular context [106]. Further factors that should be taken in consideration are; target accessibility, base-pairing topology, and nucleotide composition of adjacent regions [171, 185, 205]. Additional factors that are ignored by computational filters with a possible significance are chaperone-like molecules, tissuespecific expression, temporal dependence, etc. [187] There is still no sufficiently refined understanding of mrna targeting by mirnas in vivo to explain why some experimentally discovered sites are recognized by mirnas, whereas other regions that show similar or higher complementarity are not. This is exemplified by the surprising finding that a target site that is engineered to perfectly complement to the lsy-6 mirna in its cog 1 target is completely non-functional [66]. Developments in the computational approaches accuracy will fill the gap in understating the functional aspects of endogenously expressed mirnas. 1.4 MiRNAs Modes of Regulation: The translation of mrna has been divided into three steps: initiation, elongation and termination. The translation initiation starts with the recognition of the mrna 5 terminal cap structure m7gpppn (in which N is any nucleotide) by the eif4e subunit of the eukaryotic translation initiation factor (eif) eif4f, which also contains eif4g, an important scaffold for the assembly of the ribosome initiation complex. The eif4g interacts with eif3, facilitating the recruitment of the 40S ribosomal subunit [206, 207]. In addition, eif4g is able to interact simultaneously with eif4e and the polyadenylatebinding protein 1 (PABP1), resulting in bringing the two ends of the mrna in close proximity in a process called circularization [208, 209]. Circularization can stimulate translation initiation by increasing the affinity of eif4efor m7gpppn, and might facilitate ribosome recycling [209]. However, translation can be initiated independently of the m 7 G cap and eif4e, if ribosomes are recruited to the mrna through an interaction with an internal ribosome entry site (IRES) [210]. Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation [11]. 24

33 MiRNAs bind to the target mrnas and interfere with their translation. Although much is known about mirna biogenesis and biological functions, the mechanisms of mirna regulation are still under debate [36, 189]. The difficulties in the understanding the molecular mechanisms by which mirnas alter gene expression, is likely to be due to the diversity of these mechanisms and the sometimes the inconsistency in experimental findings [7]. MiRNAs perform their regulatory functions, by assembling together with Argonaute family proteins into mirna Induced Silencing Complexes (miriscs) [36]. After maturation mirna [58, 112], load into the mirisc [179], pairs with target mrna to drive its regulation [6]. MiRNAs can induce the RISC to regulate gene expression by several mechanisms [8], depending on the degree of complementary (fully or partially), between loaded mirna and its target [38, 166, 168, 179, 211, 212]. Once incorporated into a cytoplasmic RISC, the mirna will specify cleavage if the mrna has sufficient complementarity to the mirna, or it will repress productive translation if the mrna is imperfectly complementarity to mirna (Fig. 8) [166, 179, 211]. Figure 8. Mechanisms of mirna-mediated repression. Adapted from Nilsen T.W. [43]. 25

34 1.4.1 Translational Repression: Early reports revealed that lin-4 RNA expression coincides with a drop in LIN-14 protein without a change in lin-14 mrna in C. elegans. An observation suggesting that lin-4 RNA regulates lin-14 mrna, through translational repression mechanism [23]. Later experiments showed that the polysome profile of lin-14 mrna is indistinguishable at different larval stage, even when LIN-14 protein levels have dropped [21]. The base pairing between lin-4 and lin-14 has proved to be crucial for their interaction in vivo, as mutations that affect their complementarity compromise or abolish this negative regulation [13]. Further studies gave evidence that metazoan mirnas mediate translational repression. MiRNAs were reported to repress the expression of heterologous reporter transcripts without decreasing mrna levels. This mode of action was conserved if these messages contain either the natural mirna complementary sites from the mirna target [183] or multiple artificial complementary sites that have bulges or mismatches at their center when paired to the mirna [166, 168, 211]. Later on, several studies reported that translational repression appears to be the predominant mechanism by which mirnas negatively regulate their targets throughout the animal kingdom [14, 111, 183]. Two possibilities were put forward to explain translation repression [21]. The first includes repression of the translation at a step after the initiation of the translation, in a manner that does not perceivably alter the density of the ribosomes on the message. This can be by slowing or stalling of the ribosomes on the message. The second possibility includes continuity of the translation, however, accompanied by degradation of the newly synthesized polypeptide. Both of these mechanistic possibilities are lumped together as translational repression, even though in the second possibility polypeptide synthesis per se is not repressed [8] Inhibition at Pre-Initiation Stage: The notion that mirnas inhibit the initiation of translation is supported by a number of observations [7], reporting that mirnas-regulated mrnas were found to be unengaged with polysomal fraction in sucrose gradients but rather with the free mrnp pool [213, 214]. The observation of a shift of targeted mrnas to translationally active polysomes of lower mass (i.e., fewer ribosomes per message), was suggested as a consequence of impaired initiation [213]. Furthermore, it has been suggested that mirnas could induce a blockade of the translation initiation at the cap-recognition step [36]. Recent successes in reconstituting mirna-dependent translational repression in cell-free systems have likewise led to evidences indicating that mirnas cause an m7g cap-dependent impediment to the recruitment of 80S ribosomes to mrna [ ]. The introduction of an internal ribosome entry site (IRES) into an mrna bypasses mirna-mediated repression, hence, the translation of these constructed mrnas was found to be refractory/insensitive to repression by mirnas [ , 217]. 26

35 Furthermore, Argonaute proteins exhibit sequence similarities to the cytoplasmic cap-binding protein eif4e, which is essential for cap-dependent translation initiation. Translational repression has been shown to be correlated with the cap-binding affinity of the mirna-binding protein Ago, suggesting a concrete mechanism by which mirnas may inhibit translation initiation. The mechanism underlying the blockade of translation initiation is supposed to occur through the displacement of eif4e from the cap structure by AGO2. This has been further confirmed by the results from amino acid residues substitution, which led to abrogating the silencing activity [218]. An alternative mechanism has been suggested by Eulalio et al [219], showing that Ago proteins do not function in cap-binding but is rather important for the interaction of AGO with GW182 protein. The interaction of AGO with GW182 protein is required for repression but occurs downstream of AGO proteins. A latter study by Chendrimada et al [220] reported an association of AGO2 with both eif6 and the large ribosomal subunit. By binding to the large ribosomal subunit, eif6 prevents this subunit from prematurely joining with the small ribosomal subunit. Thus the recruitment of eif6 by AGO2, might prevent the association of the large and small ribosomal subunits [220] Repression at Post-Translation Initiation Stage: On the other hand, early evidence indicated that mirnas may be able to inhibit translation at a step after initiation [7]. Thus, studies in the worm Caenorhabditis elegans showed that, the lin-4 mirna was able to inhibit the synthesis of the LIN-14 protein but failed to affect the synthesis, polyadenylation state or abundance of lin-14 mrna [21]. The initiation of lin-14 translation seems to occur normally in the presence of lin-4, because lin-14 mrnas are efficiently incorporated into polyribosomes regardless of lin-4 expression [21].Therefore, it is reasonable to speculate that the translational repression by lin-4 occurs after translational initiation, probably during translational elongation and/or the subsequent release of the LIN-14 protein [21]. Further studies supported the possible role of mirna in blocking translation at postinitiation steps [189]. In addition to the examples form the founding members of the mirna family in C. elegans, subsequent studies showed that the majority of mirnas in Drosophila melanogaster cells and in C. elegans sediment with ribosomes [221]. Studies in mammalian cell cultures present persuasive evidence that mirnas repress protein synthesis after translation is initiated [163, ]. Although these studies differ in the details [36], their conclusions of post-initiation repression mechanism has been stemmed from common observation showing the association of repressed mrnas with polysomes [21, 36, 163, 221, 222, ]. In contrast to the results showing a decrease in the polysome mass, in this case, no change was detected in the polysome profile of messages targeted by mirnas [21, 163, ]. Furthermore, they found that the sedimentation of mirnas was sensitive to a variety of conditions that affected translation suppression, indicating that the mirnas were associated with actively translating mrnas [163]. For example, they dissociate into monosomes or 27

36 ribosomal subunits following brief incubation with translation inhibitors, such as hippuristanol, puromycin, or pactamycin [163, 223, 224]. Conflicting reports have argued for and against the hypothesis that translation initiation is the step affected by the presence of mirna-binding sites in an mrna [189]. Interestingly, evidence exists revealing that repression still occurs when translation is initiated at internal ribosome entry sites (IRES) [189, 224]. Although, in contrast to translation of mrnas containing 5 -terminal m7gpppn cap, initiation at IRESs does not require a whole complement of initiation factors [229, 230]. RNA transfection data indicate that mirna-binding sites in the 5 or 3 UTR of an IRES-containing mrna lead to repression, whether the 5 end carries a G-cap or A-cap [231]. Because IRESs initiate translation of mrnas independently of the mrna cap structure, these results indicate that mirnas repress translation at a step downstream of cap recognition [224]. These observations have led to proposals that mirnas might cause retarded elongation by translating ribosomes, possibly coupled to premature termination, or induce cotranslational degradation of nascent polypeptides [21, 163, 213, ]. Systematically, post-initiation mechanisms include: (1) cotranslational protein degradation; (2) inhibition of translation elongation; and (3) premature termination of translation (ribosome drop-off) [36] Cotranslational Protein Degradation: The paradoxical observation that the targets of mirnas appear to be actively translated while the corresponding protein product remains undetectable prompted the proposal that the nascent polypeptide chain might be degraded cotranslationally. This mechanism has been proposed to occur through recruiting proteases by mirnas, hence promoting degradation of nascent polypeptides [223]. This possibility has been supported by the failure to immunoprecipitate the repressed mrna with antibodies against the growing polypeptide [230]. However, other investigators indicated that this proposal is mainly based on negative rather than direct positive evidence. For instance, the identity of the putative protease remains unknown; and the proteasome was excluded as a possibility because proteasome inhibitors do not restore protein expression from silenced reporters and other proteases have not been identified [11, 213, 223, 224]. In addition, results demonstrating that repression by mirnas is not prevented when reporter proteins are cotranslationally targeted into the endoplasmic reticulum (ER) argue against the idea that nascent proteins are degraded in the cytosol [213]. Likewise, Selbach et al. [53] found that the genes belonging to ontology categories intrinsic to membrane and endoplasmic reticulum are significantly over-represented among identified mirna targets undergoing predominantly translational repression. 28

37 Inhibition of Translation Elongation: MessengerRNA repression has been proposed to occur by mechanisms that include stalling or slowing down of elongating ribosomes. A mechanism that has been reported to involve the mrna UTR elements [232, 233]. Nevertheless, until now and to our knowledge, there is no performed study that clearly demonstrates the inhibitory control of mirna on the translation elongation of target gene. On the other hand, some of the studies that investigated the mirnas mode of regulation rejected the theory [170, 234] Premature Termination & Ribosome Drop-off: Another study has reported a role for mirna inhibition of protein synthesis after initiation, where the defect in protein synthesis has been ascribed to premature termination of translation, or ribosome drop-off [224]. Petersen et al [224], used mirna target reporter containing several partially complementary sites, in its 3 UTR. When this reporter was transiently expressed, it was associated with polyribosomes, although its expression was repressed by the synthetic mirna [36, 189]. The inhibition appears to be postinitiation because translation driven by the cap-independent processes of targets IRES is repressed by short RNAs. Silencing by short RNAs causes a decrease in translational readthrough at a stop codon, and ribosomes on repressed mrnas dissociate more rapidly after a block of initiation of translation than those on control mrnas. These results suggest that repression by short RNAs, and thus probably mirnas, is primarily due to ribosome drop off during elongation of translation [224]. However, it has been argued that the repression was not accompanied by a shift of target mrna toward smaller polysomes, something to be expected if indeed fewer ribosomes were associated with mrna [230]. Although evidences support translational repression by mirnas through post-initiation mechanisms, they do not demonstrate unequivocally that the initiation and post-initiation mechanisms are mutually exclusive. It has been suggested that initiation might always be inhibited, but when the elongation step is also repressed, ribosomes would queue on the mrna, thereby masking the effect of an initiation block [11] Target Degradation: The mechanistic picture is further complicated by the fact that mirnas clearly exert other effects on mrna metabolism [163]. MiRNA with partially complementarity to target mrna has been shown to direct target degradation [36]. Several studies indicated that mirna-mediated mrna destabilization is an important mechanism of translation inhibition [35, 52, 53, 170]. The recent development of ribosome profiling has provided a sensitive method for examining the role of RNA degradation versus 29

38 translational inhibition [235]. Ribosome profiling of mammalian cells in culture gave evidences supporting that regulation by mirnas occurs largely through mrna destabilization, as opposed to translational repression [236]. The mechanism of mirna-mediated target degradation has been reported in a wide range of animal kingdom, from worms to human [220, ]. This effect was further demonstrated in cells grown in culture [157, 237, 239, 240, ], and in cell extracts [217]. MiRNA-silencing machinery directs its targets to the cellular decay pathway by inducing deadenylation (removal of the 3 poly(a) tail ) and subsequent 5 decapping of the target mrnas [ , 248]. The poly(a) tail and the poly(a)-binding protein (PABP) are known to enhance capdependent translation of mrna by interacting with the eif4g of the eif4f complex [249]. The mechanism of mirna-mediated target degradation includes the exposure of the message to exonucleolytic digestion from the 5 end [237, 240]. MiRNAs-mediated mrna degradation requires Argonaute proteins, the P-body component GW182, the CAF1-CCR4-NOT deadenylase complex, the decapping enzyme DCP2, and several decapping activators including DCP1, Ge-1, EDC3, and RCK/p54 [237, ]. Direct interactions of GW182 with the Argonaute proteins, lead to its recruitment to mirna targets. GW182 is thought to mark the transcript as a target for decay via deadenylation and decapping [250, 254]. Furthermore, PABP interacts with mirna-silencing complex via GW182 and this interaction is important in mirna-mediated silencing of the target mrna [255, 256]. The deadenylation of the target mrna was shown to be carried out by the deadenylase complex (CCR4-CAF1-NOT1; [ ]). It has been proposed that mirnas primarily cause target message to be deadenylated, unable them to bind PABPC, hence preventing their circularization [217]. In mammalian cells, accelerated deadenylation has been shown to result in a reduced abundance of mirna-repressed mrnas [239]. However there are controversial results regarding deadenylation involvement in target degradation, in cultured cells and cell extracts. In contrast to cultured cells, in which deadenylated mrnas are committed to decapping and 5 -to-3 exonucleolytic degradation [172, 188, 206, 213, 257], in cell extracts deadenylated mrnas are shown not to be further degraded and remain in a deadenylated, translationally repressed state [158, 163]. These data indicate that deadenylation either maintains the target mrna in the repressed state or triggers the decapping and subsequent decay [35]. In addition, deadenylation does not account for all the mirna-mediated translational repression [237, 239]. Messages that were not able to be deadenylated because they are engaged in a histone mrna stem loop rather than a poly(a) tail are nonetheless susceptible to the same degree of translational repression [239]. Moreover, reduction in cellular deadenylase activity that diminishes the influence of mirnas on message stability does not impair their ability to repress translation [237]. It is still unknown, whether the deadenylation and the consequent mrna decay is primary or secondary to the 30

39 translational repression [234]. It has been proposed that translational repression and mrna deadenylation may be two independent effects of mirnps [230]. Several experiments [ ], indicated that mirna-dependent mrna deadenylation and decay is not dependent on active translation [11], since it still occurs when translation is inhibited by cycloheximide [217, 240]. However, in some cases, mirna-induced mrna decay requires ongoing translation [240]. Following deadenylation, target mrnas are decapped by the DCP1/DCP2 decapping enzyme complex [253]. DCP2 requires additional co-factors for full activity or stability. In metazoan, these include DCP1, edc4 (also known as Ge1), PAT and the DeAD-box protein RCK (also known as Me31B) [158]. However, investigating the role of decapping is more difficult to demonstrate, because decapping factors are redundant [240], and depleting decapping factors does not lead to restore of protein levels. The blocking of decapping causes deadenylated mirna targets to accumulate - since deadenylation precedes decapping - these deadenylated mrnas are not translated efficiently, so protein levels are not fully restored [240]. However, transcriptome analysis showed that in cells depleted of decapping factors, mrna levels of predicted and validated mirna targets increase [240] [158]. In vivo, decapped mrnas are ultimately degraded by the major cytoplasmic 5 -to-3 exonuclease, which has been confirmed by observations showing that the abundance of mirna targets increases when these factors are depleted or when dominant-negative forms are overexpressed [237, 240, 248, 253, 258, 259]. In cells depleted of components of the CAF1 CCR4 NOT complex, most mirna targets (both predicted and validated) are upregulated [237, 248]. All these data support the idea that deadenylation is a widespread consequence of mirna regulation [248] Repression or Degradation Mode of Regulation: Repression could be entirely due to either mrna degradation or translational repression, but it can also be a combination of both processes [237, 240]. The question raised is whether degradation is an independent mechanism by which silencing is accomplished, or is it a consequence of a primary effect on translation? [36]. Studies of mrna reporters containing 5 terminal hairpins that block translation initiation showed that exclusion of mrnas from the translated pool per se is not enough to cause mrna deadenylation and degradation, implying that the effect of mirnas on mrna stability is not a simple consequence of translational repression [238, 239, 248]. In human cells, insertion of a large/strong stem loop into the 5 UTR, led to translation repression, nevertheless the mrna was found to be deadenylated in an mirna-dependent manner [239]. Likewise, in zebrafish embryos and human cell extracts, mirna targets were deadenylated despite having a defective cap structure (Appp-cap) that impairs translation [217, 242]. Furthermore, in D. melanogaster cells and human cell extracts, mirna-mediated mrna decay could occur in the absence of active translation [217, 240]. 31

40 On the other hand, it has been suggested that if translational repression occurs before degradation, mrna destabilization must immediately follow. Therefore, regardless of whether destabilization occurs before or immediately after a translational block, it nevertheless provides the main contribution to the reduction in protein output [158]. Moreover, accessory proteins bound to the 3 UTR might be involved, or structural subtleties of imperfect mirna mrna duplexes, particularly of their central regions, could be important [246, 260]. It will be important also to establish what makes some mirna targets very sensitive to degradation while others are mainly repressed translationally, with no or only minimal effect on mrna decay [230] Cleavage/Slicing: MiRNAs that are fully complementary to their mrna targets have been shown to induce endonucleolytic target cleavage (Fig. 9) [129, 176, 261]. Plants targets were shown to undergo slicing [262], however, only the very few highly matched animal targets (in contrast to all of the centrally mismatched ones) are sliced [176, 177]. Nevertheless, a perplexing observation revealed that the plant mir172 regulate APETALA2 via translational repression despite the near-perfect complementarity between the mirna and its single complementary site in the APETALA2 ORF [263, 264]. Evidence supporting mirna-mediated mrna cleavage showed that cleavage activity from sirna-loaded human RISCs is strongly reduced by single central mismatches [265], while let-7 is made cleavagecompetent simply by engineering its target to be centrally matched [179]. In mammals, slicing activity is catalyzed by Argonaute2 (AGO2), which leaves a 3 hydroxyl on the 5 cleavage fragment and a 5 monophosphate group on the other fragment [63, 266, 267]. The cleavage performed by mirna guides is found at precisely the same site as the one from sirna-guided, i.e., between the nucleotides pairing to residues 10 and 11 of the mirna [179, 261, 262, 268]. Furthermore, the cleavage site is not affected when the mirna is not perfectly paired to the target at its 5 terminus [262, 269]. Therefore, the cut site appears to be determined relative to mirna residues, not mirna:target base pairs [8]. This event leads to rapid decay of the entire message by generating a pair of RNA fragments, each bearing an unprotected end that is susceptible to 5 - or 3 -exonuclease attack [270]. After cleavage of the mrna, the mirna remains intact and can guide the recognition and destruction of additional messages [179, 271]. 32

41 Figure 8. A speculative model explaining the roles of each mirna region (A) MicroRNA (red) bound by Argonaute (AGO) such that nucleotides 2 8 are preorganized to favor efficient pairing. Nucleotide 1 is twisted away from the helix and permanently unavailable for pairing, and nucleotides 9 11 are facing away from an incoming mrna and unavailable for nucleation; the remainder of the mirna is bound in a configuration that has not been preorganized for efficient pairing. (B) Recognition of an 8mer site by the preformed binding pocket. An A at position 1 of the site presumably is recognized directly by AGO or another protein of the silencing complex. (C) Massive conformational accommodation of extensively paired sites. At very rare sites of endogenous mirnas (and the intended sites of exogenous sirnas) pairing is extensive. In this model, pairing anchored and nucleated at the seed extends to the central region of the mirna causing the protein to loosen its grip on the 3 region of the mirna and thereby allowing the mirna and mrna to wrap around each other. (D) Accommodated pairing suitable for mrna cleavage. The Argonaute protein locks down on the extensively paired duplex, which places the active site (black arrowhead) in position to cleave the mrna. (E) 3 -supplementary pairing. The message can pair to nucleotides 13 16, incorporating them into a short helical segment without major perturbation of the Argonaute protein or the remainder of the mirna. Importantly, in this mode of target recognition, the mirna and mrna are not wrapped around each other. Adapted from Bartel D.P. [6]. MiRNA-mediated target mrna decay has been proposed to have two important consequences. First, the decreased efficiency of message translation through diminishing targeted transcripts concentration; which results in a greater overall reduction in protein synthesis. Second, inducing message degradation renders mirnas, irreversible to their inhibitory influence on gene expression, an outcome not achievable by translational downregulation alone [7]. The relative contribution of mrna degradation to the overall effect of mirnas differs depending on the message [237]. Some messages appear to principally apply mechanism of downregulation, whereas in others the effect can be quite modest compared to that of translational repression. The coexistence of the two mechanisms has been suggested to relate, at least in part, to the innate longevity of the targeted mrnas. All else being equal, one would expect messages that are intrinsically long lived to be destabilized more than others that are inherently labile if mirnas engaged in equivalent interactions with distinct 3 UTRs cause similar rates of deadenylation and decay [7]. From the experimental point of view, mirna decay is a process that participates to mirna:mrna targets validations, whether by inhibition of mirna [238, 246, 247], or mirna ectopic expression [157], followed by analyzing the abundance of targeted transcripts [238]. 33

42 It has been proposed that sequence parameters alone might not be sufficient to determine the regulatory output of mirna mrna interactions [38]. The interplay of mirnas and their associated factors with the complement of proteins bound to specific 3 UTRs, have been suggested to play a role in determining the mechanism and extent of regulation [163]. This might include, in addition to the AGO core elements of RISC, the protein composition of the target mrna protein particle (or messenger ribonucleoprotein particle (mrnp)) [38]. Consistent with this, it has demonstrated that mirna-mediated regulation can be modulated by protein binding to the 3 UTR [272], and that mrna-specific fates (enhanced turnover, translational inhibition or a combination of both) might occur as a consequence of mirna-mediated regulation [237] Explataions for Conflicted MiRNA Regulation Data: It is hard to reconcile the different reported modes of mirna regulation of gene expression [7, 36]. Studies comparing natural full-length 3 UTRs suggest that the final outcome of mirna regulation depends on the features and sequence of the target s 3 UTR [171, 237, 240]. In these studies, the contribution of translational repression or mrna degradation to gene silencing differed for each mirna-target pair [36]. Furthermore, neither a cap-dependent repression mechanism nor a cap-independent mechanism is easily dismissed, the evidence for each appear convincing. These two modes of regulation have been suggested not to be mutually exclusive, and some experiments designed to detect one may have obscured the other. Thus, it is possible that mirnas employ multiple mechanisms to repress the translation of targeted messages, including one that inhibits the earliest events of cap-dependent initiation and another that impedes a later, cap-independent event in translation [7]. Another suggestion is that mirnas silence gene expression through a common and unique mechanism, and that the multiple modes of action reflect secondary consequences of this primary event rather than independent mechanisms. These secondary effects might vary in a cell- or targetdependent manner. For instance, translational repression may represent a primary event that in a target- or cell-specific manner may or may not lead to mrna decay. Another explanation is that multiple mechanisms exist, and particular mechanisms take pre-eminence according to conditions that we do not yet understand [4, 36]. Cell-specific effects might also affect the mode of regulation: for example, zebrafish nanos1 mrna is deadenylated and degraded by mir-430 in somatic cells but is refractory to mir-430 regulation in the germline [36, 242]. 34

43 In addition to the possibility that mirnas silence gene expression via multiple mechanisms, the discrepant results of mechanistic studies, might reflect the different interpretations and experimental approaches [36, 230]. Thus the differences in mirna mechanisms between animal species, cell types, or growth conditions maybe real but could also result from the disparate systems and methodologies used [7]. Most of the experiments described were carried out in transfected cells grown in culture, using artificial mrna reporters bearing mirna sites in the 3 UTR. However, it should be noted that currently available in vitro systems are not very robust and the observed repression being at most only threefold to fourfold (on the other hand, stronger effects are perhaps not to be expected as mirnas generally induce only a moderate repression) [230]. Another potential source of discrepancy is the nature of the reporter. Some studies used artificial reporters containing multiple identical mirnabinding sites, systematically inserted in a heterologous 3 UTR, which might not be the case for natural 3 UTRs. Nevertheless, when such reporters were used, different mechanisms could be observed; so these reporters alone do not explain all of the discrepancies [36, 214, 217, 224]. Another possible explanation of some contradictory data is referred to the type of promoter used for driving expression of the reporter gene [230, 273]. Most evidence that translation initiation is inhibited comes from studies of mrnas synthesized in vitro and incubated in cell-free extracts or transfected into cultured cells. In vivo, mirna targets are not naked mrnas but exist as mrnps. The RNA-binding proteins are known to be deposited on mrnas cotranscriptionally or during processing. Therefore, the full complement of proteins associating with mrnas transcribed in vivo is likely to be different from that bound to the same mrna in an in vitro system or following mrna transfection [36]. It will be crucial to understand the regulation of mirna function through modulation of the activity of RISC components and associated factors, possibly by phosphorylation and other protein modifications. Thus, the involvement of different signaling pathways in the control of mirna function should be studied. It will also be important to determine the precise contributions of different cellular structures, such as P-Bodies and stress granules, to mirna-mediated repression of translation [11]. In addition, repression in some of the extracts required either overexpression of mirnp components [217], or preannealing of the synthetic mirna to the target mrna [274], making it difficult to know how far these systems recapitulate the physiological mirna response [230]. Furthermore, there are different human Argonaute paralogs; all are presumed to repress translation. However, it has not been investigated whether different Argonaute proteins silence partially complementary targets through similar or different molecular mechanisms. The corollary is whether 35

44 discrepancies between different studies reflect the action of different Argonaute proteins or Argonaute complexes with distinct protein compositions. Biochemical purification of Argonaute-containing mrna-protein complexes revealed partially non-overlapping sets of mrnas in association with individual Argonaute proteins, suggesting some degree of specificity in target selection [36, 275]. A rcent study by Iwasaki et al. [276] suggests that the two Drosophila AGO proteins may regulate protein synthesis via different mechanisms, with AGO2 repressing translation initiation by interfering with eif4e eif4g interaction and AGO1 functioning via the recruitment of GW182 leading to mrna deadenylation and translational block downstream of cap recognition [230]. It has been proposed that a complete and accurate understanding of the mechanism of mirna function depends on the elucidation of three-dimensional structures of AGO proteins, their complexes with the mirna and the cap structure, and ultimately the structure of mirnp bound to mrna. Structural information would help validate or refute the current models for mirna function [11]. It has been reported in a study by Lytle et al [189], that transfection using either cationic lipids or electroporation, gave discrepant results on target reporter. This might be due to the fact that transfected RNAs would likely remain cytoplasmic, whereas an RNA transcribed from a DNA plasmid would assemble into a nuclear RNA protein complex that is later exported to the cytoplasm. Also, cationic lipid transfections can be more efficient than electroporation [277], so the possibility exists that a large amount of DNA taken up by the cell might saturate the ability of the mirna system to repress translation. It has been concluded that the method of transfection affects the ability of an mrna to be repressed by endogenous mirnas. Thus, a single transfection method may not be reliable for drawing conclusions concerning the efficacy of translational repression by mirnas [189]. Previously published data showed that repression might follow different mechanisms even in a single test system. It has been argued that divergent results reported in the literature could be explained by differences in rate-limited steps in translation under different experimental protocols [278], which could mask the true effect of mirnas on translation [230]. Moreover, some models have been based on studies in which only one or a few targets were studied, which introduces the possibility of generalizing the behavior of a single mirna:mrna interaction that may not represent the dominant biological mechanism [170] P-bodies or GW-bodies: The appropriate subcellular localization of a protein or an RNP is essential to their function and regulation. Compartmentalization can control access to binding partners, concentrate factors that act 36

45 together or temporarily segregate pathway components away from the rest of the cellular environment [39]. A significant fraction of translationally silent mrnas, including those repressed by mirnas, were found to accumulate in discrete cytoplasmic foci known as mrna processing bodies (P-body) [36, 78, 279, 280], or glycine-tryptophan bodies (GW-bodies) [ ]. These ribonucleoprotein aggregates contain mrna-degrading enzymes and are implicated in the catabolism and/or storage of nontranslated mrnas [176, 213, 251, 252, 280, 282, ]. The accumulation of repressed mrnas targeted by mirnas in P-bodies [213, 237, 252, 280, 284, 288, 289], suggested their critical role [189]. This has been confirmed by applying a variety of approaches, revealing a central role for P- bodies in mirna-mediated mrna translational suppression and/or degradation processes [43]. The core P-body protein components are conserved from yeast to mammals [290]. MiRNAs and mirna targets, in addition to other protein components, are found to be colocalized in P-bodies. The translational machinery has been reported to be excluded from P-bodies [36, 280, 289]. Furthermore, components of the mirna repression complex RISC, including the Argonaute proteins (Ago1 and Ago2) have been found to be highly enriched in P-bodies, indicating a role in mirna/sirnamediated gene silencing [7, 230, 237, 280, 284, 285]. P-bodies also contain proteins that are known to be involved in mrna decay machinery; decapping enzymes Dcp1p/Dcp2p, the activators of decapping, Edc3p, RAP55/Scd6p, and the Lsm1p-7p complex, the enhancers of decapping, deadenylating enzymes [282, 290], the 5 to 3 exoribonuclease, Xrn1/Pcm, and general translational repressors, Me31B/RCK/Dhh1p and Pat1p [283]. Importantly, P-bodies are also enriched for members of the GW182 protein family [36, 279, 281, 283], a family that is involved in mirna-mediating repression [237, 253] [219, 251, 252, ]. Although there were previous attempts to identify proteins localized to P-bodies [259, 288, 294, 295], the protein composition of these particles has not been determined yet, likely because P-bodies are refractory to biochemical purification [43]. P-bodies are highly dynamic structures, with proteins and mrnas continuously moving in and out [ ], and the number and size of P-bodies fluctuate during cell cycle and depending on the translational activity of the cell [11, 39, 296]. Another role of P-bodies in translational repression is to provide a storage space for sequestered mrna. Under stress conditions, mirna-repressed mrna has been shown to be released from P-bodies [289], suggesting that the mirna-repressed mrna is still capable of resuming translation [5]. The inhibition of mirna biogenesis or depletion of GW182 proteins led to the disappearance of P-bodies [250, 259, 296, 297]. A positive correlation exists between mirna-mediated repression and the accumulation of target mrnas in P-bodies [213, 280, 289, 298]. The cationic amino acid transporter-1 (CAT 1) mrna, a target of mir-122, localizes to P-bodies when it is translationally repressed, however upon stress, its repression is relieved and it exits P-bodies [289]. Other studies link P-body status to mirna function. 37

46 In mature mouse oocytes and early embryos, mirna function is globally suppressed even though mirnas are abundant [299, 300], which has been found to coincide with the loss of P-bodies [301, 302]. The P-bodies disappear in developing oocytes and reappear around the blastocyst stage [301]. This is paralleled by a dispersal of AGO and the relocalization of GW182 to the cell cortex, a mechanism that possibly uncouples miriscs from translational repression. A rationalized model has been proposed, for the mechanism by which mirna function [237, 281, 303]. In this model; mirna binds to an Argonaute protein and recognizes its mrna targets by base pairing. In turn, the Argonaute protein interacts with GW182, and the complex containing mrna/mirna is delivered to P-bodies. In the P-body, the targeted mrna is de-adenylated by resident de-adenylases, then either decapped and degraded or stored. Importantly, that the translational machinery will be removed, because P bodies are devoid of any ribosome [43, 281, 303]. Although many evidences clearly link P-bodies to mirna silencing, other findings exist, indicating that microscopically visible P-bodies are not essential for mirna function, and that the formation of P-bodies is rather a consequence than the cause of mirna-mediated repression [39, 250, 259, 297]. Several observations indicated that P-body-dependent processes, does not account for all mirnamediated regulation [7, 43]. A quantitative microscopic analysis of Argonaute protein localization revealed that only a small proportion (<2%) of Argonaute proteins are found in P bodies [286]. Furthermore, several studies reported that mirnas are associated with polysomes [163, 223, 227, 228], and a recent report demonstrated that most mirnas are associated with mrnas undergoing translation [163]. Additionally, recent report indicates that mirna function does not require P-body structural integrity [163, 259], and mirna pathway remind unaffected in cells lacking detectable microscopic P-bodies [250, 259]. In addition, approximately 20% of RNA was found to be localized to visible P-bodies, indicating that the repression either involves submicroscopic P-bodies or occurs outside them [11]. Finally, knockdown of certain P-body component was found to cause the disappearance of visible foci but did not compromise mirna function [259]. The depletion of cellular Lsm1 or Lsm3 has been found to disaggregate P-bodies, either completely or at least to a submicroscopic size, without impairing the ability of mirnas to downregulate gene expression [7]. In contrast to knockdowns of LSM1 and LSM3, depletion of other P-body components such as DCP1 or DCP2, GW182, and various decapping activators, either individually (for example, RCK/p54) or in combinations, prevents efficient inhibition of target mrnas in cultured cells [237, 240, , 259, 288, 304]. This is implying that silencing is initiated in the soluble cytoplasmic fraction, and that the localization of the silencing machinery in P bodies is a consequence rather than a cause of silencing [213, 214, 250, 259]. P-bodies were suggested to play a less pivotal role either as graveyards where mirna-associated messages that already have been translationally inactivated (and possibly also 38

47 deadenylated), are sent to decompose or as depots where messages transiently repressed by mirnas can be stored until needed [250, 259, 289]. Some investigators gave explanations for the above mentioned debate; whether the localization to P- bodies is a cause or a consequence of silencing [36]. The findings that only a minority of Argonaute proteins and mirnas localize to P-bodies [163, 286], could suggest that regulated mrnas exit translation and are delivered to P-bodies by a process that involves the removal of the mirna/argonaute complex. In this case, no marked accumulation of mirnas or Argonaute proteins would be expected, and the apparent localization of these factors would be the result of transitory interactions. The demonstration that P-body structural integrity was not required for mirna-mediated repression [259] is also not definitive, because of the possible presence of P-bodies in a submicroscopic foci [43]. An explanation regarding the presence of repressed mrnas associated with polysomes, has been proposed. The absolute level of repression of most mirna-regulated mrnas seems to be relatively mild (e.g. about twofold [156, 157]) [200]. If regulation is mediated exclusively by accelerated mrna degradation, then a decrease in steady-state mrna levels would result. However, if regulation occurs without mrna destabilization, then it is predicted by the P-body hypothesis that regulated mrnas would partition between nontranslating (sequestered) and translating states proportional to the degree of regulation. Thus, at steady state, assuming twofold repression, half of a specific regulated mrna would be expected to be associated with P bodies, and the other half would be expected to be in the translating pool. At present, there are two examples in which this prediction is fulfilled [213, 289] and several in which it is not [21, 163, ]. It will be of considerable interest to determine the subcellular distribution (whether ribosome associated) of additional mrnas that are regulated in the absence of increased degradation. If most regulation occurs by sequestration, then biochemical purification of P bodies (assuming the development of appropriate techniques) would provide a powerful means both for identifying mirna targets and for assessing levels of regulation [43] Stress Granules: Another class of mrna-containing cytoplasmic aggregates is stress granules (SGs). They form in conditions of global repression of translation initiation in response to stress [39]. SGs are sites where poly(a)+ mrnas bound by stalled 40S ribosomes accumulate when translation initiation is inhibited during stressful conditions like hypoglycemia, viral infection, salt stress, UV irradiation, or hypoxia [305]. SG assembly, usually initiated by the phosphorylation of translation initiation factor eif2α, takes place when different environmental stresses activate distinct eif2α kinases (e.g., PKR, HRI, PERK, and GCN2) [306]. AGO proteins, artificial mirna mimics and repressed reporter mrnas are 39

48 shown to be colocalized in SGs [39]. Remarkably, the localization of Argonaute to SGs requires the presence of mirnas [286], suggesting that Argonaute enters SGs through its association with mirnas [306]. SGs are considered to be sites for mrna sorting and mrnp remodeling upon exposure of cells to stress. This might lead to storage/silencing of certain mrnas at SGs, while the rest transit to P-bodies for degradation [305]. Importantly, localization of mirnp components to SGs might reflect dragging of mrna-associated proteins, but not necessarily an inhibitory mechanism. This scenario might explain why the localization of AGO to SGs, but not to P-bodies, is mirna dependent, since AGO proteins directly interact with other P-body components [237, 259, 307]. SGs are not structurally stable, they can disassemble upon relief of stress [305]. Furthermore, factors localized in SGs are continually moving back and forth between SGs and the cytoplasm (for example, 50% of Argonautes in SGs are replaced within 20 s [286]. Thus, the assembly of these organelles can be rapidly dissolved in response to changing environments [306]. Although many proteins found in P-bodies are absent from SGs and vice versa, they share some component proteins [11]. Furthermore, P-bodies and SGs are frequently located adjacent to each other, possibly exchanging their cargo material [308, 309]. However, it remains to be established whether stress granules indeed play a role in mirna silencing or if enrichment of miriscs in stress granules just reflects a passive dragging of mrna-associated miriscs to these structures under conditions of general translation repression [39] MiRNA-mediated Target Upregulation: It has been thought that mirnas control gene expression, only through negative target regulation. There was no evidence for mirnas directing upregulation of gene expression. These was considered in consistency with the idea that all mirnas act within the silencing complex (RISC) [8]. New data emerged indicating that mirnas are able to mediate translation activation under certain conditions [310]. Recently, several groups have reported that mirnas can activate rather than repress their targets under certain conditions [ ]. Like translational repression, such activation requires base pairing of the targeted message with the seed region of the mirna [7]. First evidence of mirna-mediated translation activation revealed that mirnas oscillate between repression and activation in coordination with the cell cycle (Fig. 9) [310, 311]. Thus, Vasudevan et al. demonstrated that mirnas were able to inhibit translation in actively proliferating mammalian cells, however, they reported the opposite effect in G1/G0 arrest, where mirnas mediate activation 40

49 [310]. The study reports an effect of Human mirna mir369-3 in cells grown under serum starvation, which directs the association of proteins with the AU-rich elements (AREs) in mrna 3 UTRs, and leads to translation activation [310]. The upregulating effect of mirnas has been seen upon serum starvation or treatment with another stress inducing factor, the replication inhibitor aphidicolin [7, 230]. Furthermore, it has been reported that two well-studied micrornas Let-7 and the synthetic microrna mircxcr4 likewise induce translation upregulation of target mrnas on cell cycle arrest and repress translation in proliferating cells [310]. Concluding that activation is a common function of mirnps on cell cycle arrest, and proposing a mechanism by which the translation regulation by mirnps oscillates between repression and activation during the cell cycle [310]. However, another study indicates that this mechanism is probably not universal, since G1-arrested cells in the Drosophila eye use mirna-mediated repression [316]. In addition, conversely to the study by Vasudevan et al. [310] demonstrating the importance of the FXR1 in this mechanism, paralog FMR1 has been reported to contribute to the downregulation of gene expression by RNA interference in mouse and Drosophila cells, possibly by helping sirnas anneal to the messages they target [164, 165, 317, 318]. Figure 9. Dual functions of mirnas. MicroRNAs (mirnas) can boost or block the translation of target mrnas. Physiological conditions affect the recruitment of regulatory proteins, which can alter a mirna s effect. Adapted from Buchan and Parker [319]. Although cells can modulate their gene expression programs in response to stress, transcriptionally by upregulating a subset of mrnas, alternatively, such adaptation can be effected immediately by regulating the existing pool of mrnas without any de novo synthesis. This means translating certain mrnas, while halting translation of the rest (reviewed in [320]). Two proteins have been proposed for the ability of mirnas to activate translation in growth-arrested mammalian cells [7]. One is Ago2, as demonstrated by depletion and tethering experiments. The other is FXR1, an RNA-binding protein homologous to the fragile X mental retardation protein FMR1/FMRP [310, 311]. Reports revealed that upon cell cycle arrest, the ARE in tumor necrosis 41

50 factor-α (TNFα) mrna is transformed into a translation activation signal, recruiting AGO and FXR1, factors that are associated with micrornps [310]. AGO2 is found to associate with FXR1, only in activating, but not repressing mrnp complexes [230]. This regulation occurs on at least two levels. First, recruitment of the mirnp reflects both its expression level and its ability to productively interact with mrna target sites. Second, the AGO2 complex must be subjected to modification because tethered AGO2 differentially regulates translation according to cell growth conditions. Since FXR1 is found exclusively in the activation complex and activation by AGO2 tethering in serum starved conditions requires FXR1 expression, modifications that switch AGO2 from repressor to activator may alter interactions with FXR1. Such modifications upon serum starvation were suggested by changes in the solubility and subcellular localization of the AGO2-FXR1 complex [303]. However, the mechanism by which FXR1 associates with Ago2 to mediate the positive influence of mirnas remains to be known [7]. Another example of the stimulatory effect of mirnas has been reported by Orom et al. [312]. The study revealed that mir-10a interacts with the 5 UTR of many mrnas encoding ribosomal proteins, promoting their translation in response to stress or nutrient shortage. The mirna has been shown to interact immediately downstream of the 5 -terminal oligopyrimidine tract (5 -TOP) motif following the m7gpppn cap, a characteristic of mrnas encoding ribosomal proteins and some translation factors [35]. 5 -TOP motif are present in mrnas encoding proteins involved in translation and are responsible for regulated translation of these mrnas in response to stress or nutrient status [171]. However, base-pairing of mir-10a to the 5 UTR of 5 TOP mrna does not seem to follow classical rules of mirna mrna interactions [6, 45]. The interaction of mirna and target message has been previously reported, not to be exclusively through the classical seed region rule. A report revealed that mir-16 targets the mrna for myt1 kinase and, in conjunction with AGO and FXR1, activates its expression in Xenopus laevis oocytes [321]. Jing et al. [245] proposed that mir-16 might specifically interacts with AU-rich elements in mrna 3 UTR via its central part rather than the seed and causes mrna destabilization. In future, it will be important to establish how common these noncanonical mirna mrna associations are and what rules, if any, apply to them [230]. Further studies showed that upon stress, the endogenous cationic amino acid transporter 1 (CAT-1) mrna in human hepatoma cells is released from the repression mediated by mir-122. Following amino-acid starvation or other types of stress, CAT1 mrna is released from P-bodies and recruited to polysomes [289]. The effect of a mirna-ago complex was demonstrated to be modulated by proteins bound to other sites within the 3 UTR. The increased translation of the CAT-1 mrna in hepatic cells was shown to depend on binding of ELAVL1 (also known as HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, to the CAT1 3 UTR [11, 289]. 42

51 Another example of mir-122-mediating activation is the upregulation of Hepatitis C Virus (HCV) RNA [314, 315]. Addition of mir-122 led to an extremely rapid ( min) increase in 48S initiation complex formation. Interestingly, several studies [ ] reported that the effect of mir- 122 on gene expression is position-dependent. The binding sites present in the HCV RNA 5 UTR had a stimulatory effect, while placement in the 3 UTR made them act as repressors [230]. MiR-122 has been argued to act as a chaperone modifying RNA structure and facilitating ribosome access to the HCV mrna [35]. The activation of mirna-repressed messages might be partially explained by new interactions of mirna/argonaute complexes with RNA-binding proteins that relocate from different subcellular compartments during stress. It has been found that all Argonaute family members (Ago1 4) are relocated within the cell upon stress [286]. Two recently discovered aspects of Argonaute that are changed upon stress: (i) the accessibility and (ii) the adjacent arrangement at mirna-binding sites. (i) Accessibility: In unstressed cells, Argonaute and mirnas mainly localize to the diffuse cytoplasm and a small portion in P-bodies. However, upon exposure to stress stimuli such as oxidative stress, Argonaute and mirnas additionally become localized to newly assembled structures known as stress granules [286, 306]. (ii) Adjacency: Bhattacharyya et al. [289] and Vasudevan and Steitz [311] demonstrated that the modulation of mirna function during stress includes the requirement of an occupied RNA-binding protein site adjacent to the mirna-binding site in targeted mrnas. For example, an ELAV RNA-binding protein family member HuR [289] and the fragile X protein FXR1 [311] are both required for Argonaute-mediated upregulation; knockdown of these components abrogates the upregulation. However, the requirement of neighboring protein-binding sites is not limited to mirna-mediated upregulation [306]. It has been proposed that external stimuli change a normally repressive mirna to an activator by limiting the amount of the nucleocytoplasmic shuttling RNA-binding proteins in the cytoplasm [306]. Such combinatorial requirements for both mirnas and RNA-binding proteins would provide a mechanism for changing the transcriptome and proteome on a global scale. Thus, mirna binding sites would provide the target specificity and other 3 UTR-binding sites could sense the cellular state by coupling with signaling pathways [289, 306, 311]. The mechanism by which the efficiency of translation increases has not yet been determined, nor is clear how changing the growth state of cells causes this striking reversal in the regulatory influence of mirnas and the proteins with which they associate [7]. This recently discovered mechanism raised many questions; what are the mechanisms by which mirnas enhance translation? Does mirna stimulation of translation raise a possible complication, and opportunity, in using mirnas and small interfering RNAs as therapeutics? Assuming mirnas generally stimulate translation in cells exiting 43

52 the cell cycle, what role might mirnas play in developmental and terminal differentiation processes? [7, 319]. 1.5 Functional Aspects of MiRNAs: Models of mirna Function: A substantial fraction of animal mrnas could have their precise level of expression defined by mirna regulation [8]. A model has been suggested, whereby mirna is proposed to function depending on its abundance, differential expression and targeting affinity [200]. The different expression profiles of mirnas in different cell types constitute a mirna milieu, unique to each cell type. Each milieu is able to dampen the expression of thousands of mrnas and provides important context for the evolution of metazoan mrnas. As the UTR sequences drift over the course of evolution, they are continuously sampling matches to coexpressed mirnas. Depending on whether the dampening of protein output is beneficial, inconsequential, or harmful, the sites are selectively conserved, not modulated, or selectively avoided during evolution. The mrnas are thus classified as conserved targets, neutral targets, and antitargets, of the mirna (Fig. 10) [6]. Figure 10. MicroRNA-Mediated Regulatory Effects. Adapted from Bartel D.P. [6]. 44

53 Switch targets: Switch targets are referred to mrnas, with mirnas reducing their protein production to inconsequential levels; the result is equivalent to a discrete off switch [8]. It has been suggested that this mechanism is employed on mrnas that should not be expressed in a particular cell type. Examples include the lin-4 targeting of lin-14 and lin-28, and let-7 targeting of lin-41 [6]. Switch interactions can also include those in which a mirna is already present when the target is first expressed, and is called failsafe interaction. For both types of switch interactions; classical switch and failsafe, the mirna represses protein output to insignificant levels. However, failsafe interactions differ in that protein output falls below functional levels even in the absence of the mirna. In this case, mirna repression adds an additional, functionally redundant layer of repression, helping to ensure that aberrant transcripts do not give rise to a consequential amount of protein [6] Tuning Targets: MiRNA has been suggested to act as a rheostat on these targets. In this case, mirna dampen protein output to a more optimal level, yet one that is still functional in the cell. Hence, mirnas could adjust protein output in a manner that allows for customized expression in different cell types yet a more uniform level within each cell type [8]. For examples; mir-8 and mir-375 that have been shown to reduce protein products of their target to optimal level, but not too low to compromise the function or the viability [322, 323] Neutral Targets: Neutral interactions dampen protein output, but this repression is tolerated or counterbalanced by feedback processes [8]. In this case, the regulatory sites are under no selective pressure to be retained or lost during the course of evolution. Such interactions comprise cases in which targets has no biological function. Nevertheless, it has been suggested that neutral repression might be the most frequent type of biological repression [6] Antitargets: Finally, there are antitargets messages. This type of messages is under selective pressure to avoid fortuitous complementarity to the multitude of mirnas in the cells where they are expressed. This has been suggested as a mechanism by which such complementarity would inappropriately dampen their expression or because it would titrate the mirnas away from their proper targets [8]. Microarray data from mammals showed a mutually exclusive tendency in the expression of messages with nonconserved sites. However, the conserved mrna targets tend to be expressed higher in tissues that lack the mirna. The tendency of conserved targets to be present at low levels in the same tissues as the mirna 45

54 suggests that, rather than performing failsafe functions, mirnas frequently function through a combination of tuning and classical switch targeting [156, 324, 325]. Still, each highly conserved mirna is likely to perform each type of regulatory function, and the proportions of classical switch, tuning, and failsafe interactions could vary widely from one mirna to the next. Moreover, the interaction between each mirna and its target could vary, depending on tissue type or developmental stage [6] Degree of Repression: There is still little known about the influence of endogenous mirnas on the protein output of their many of targets. A larger-scale proteomic analysis [52] revealed that some detected proteins are repressed by 50% 80% by the microrna studied. Nevertheless, the authors proposed more modest effects on its endogenous targets, even those conserved targets, with individual sites usually reducing protein output by less than a half and often by less than a third. It has been presumed that for each highly conserved mirna, a minority of the preferentially conserved targets (much less than 150 for most mirnas) are repressed more than 50% by that mirna, whereas the hundreds of remaining preferentially conserved targets (particularly those with only 6mer sites) are repressed more modestly. These interactions conferring the greatest repression would presumably be enriched in switch interactions (classical or failsafe), whereas those with more modest repression would tend to be tuning interactions [6] Micromanagers of Protein Output: Several factors have been proposed, to explain the subtle effect resulting from disruption of a single mirna:target interaction. It has been shown that more than 90% of the conserved mirna:target interactions involve only a single site for each mirna in a given mrna. Therefore, most of these targets would be expected to be downregulated by less than 50%. But because most messages with a conserved site to one mirna have at least one other conserved site to an unrelated mirna [37, 55, 182], interactions with multiple mirnas might need to be disrupted before the de-repression of that message had perceptible consequences [6]. Another reason that such perturbations are frequently tolerated is the phenomenon of regulatory network buffering. Many mirna:target interactions, presumably fall within complex regulatory networks with bifurcating pathways and feedback control that enable accurate response despite a defective node in the network. With this ability to buffer the effects of losing a node, such networks must be perturbed elsewhere before the lost mirna interaction has discernible phenotypic consequences. Reciprocally, perturbing the mirna node would be expected to sensitize the network to reveal the importance of other regulatory nodes. The fact that 46

55 protein expression levels are so precisely adjusted, with the tight tolerances so often retained through evolution, is one of the most fascinating biological conclusions arising from mirna research of the past few years [6] MiRNAs Physiological Function: The mirna milieu is unique to each cell type, with many targets per mirna productively dampening the translation of thousands of mrnas [200]. In addition, many mirnas can target the same message. It should be considered that the connection is not one mirna one mrna, it is the combination of multiple mirnas that target the same gene under investigation. The individual mirna/mrna interaction may be weak, but the combination of multiple mirnas targeting multiple seed matches may have considerable biological significance [198]. The regulatory capacity of mirnas is increased further by the mirna ability to suppress gene expression using multiple mechanisms that range from translational inhibition to mrna degradation. The high mirna diversity multiplied by the large number of individual mirna targets generates a vast regulatory RNA network than enables flexible control of mrna expression [78]. Interestingly, some mirnas have been predicted to target genes encoding proteins with related functions [154, ]. For example, the vertebrate mirnas of the mir cluster tend to target genes involved in growth control [55], consistent with their oncogenic properties [329]. Another example, the liver-specific mir-122 can regulate multiple genes involved in cholesterol and fatty acid metabolism [244] Intermingled Relation with Transcription Factors: The ability of a single mirna to regulate multiple functionally related mrnas, is analogous to eukaryotic transcription factors that regulate a common set of cellular genes (Fig. 11) [306]. Similar to coding genes, mirna-encoding loci are transcriptionally regulated in a tissue-specific manner, or in response to either developmental or environmental cues [39, 78]. For example, MYC and MYCN both stimulate expression of the mir oncogenic cluster in lymphoma cells [330] and mir-9 in neuroblastoma cells [331], but inhibit expression of several tumour suppressor mirnas (for example, mir-15a), which promote MYC-mediated tumorgenesis [332]. Another example is the RE1 silencing transcription factor (REST). It recruits histone deacetylases and methyl CpG binding protein MeCP2 to the mir-124 gene promoter, preventing its transcription in neuronal progenitors and non-neuronal cells [333]. REST is downregulated upon differentiation, allowing for high mir-124 expression in post-mitotic neurons [39]. 47

56 Control of gene expression by autoregulatory feedback loops is a common mechanism that is particularly important during cell fate determination and development. MiRNAs frequently act in regulatory networks with TFs, which can drive or repress the expression of the mirnas. Unilateral or reciprocal-negative feedback loops result in oscillatory or stable mutually exclusive expression of the TF and mirna components. Many examples described mirnas regulating their own transcription through single-negative or double-negative (or positive) feedback loops with specific transcription factors. For instance, the PITX3 transcription factor and mir-133b form a negative autoregulatory loop that controls dopaminergic neuron differentiation. PITX3 stimulates transcription of mir-133b, which in turn suppresses PITX3 expression [334]. More sophisticated regulation is provided by double negative feedback loops like the one involving mirnas lys-6 and mir-273, and transcription factors DIe-1 and COG-1 in Caenorhabditis elegans. Proper transcriptional activation and/or inactivation is accomplished by spatially controlled mirna expression, and facilitates establishment of the left right asymmetry of ASE chemosensory neurons. The COG-1 TF represses the left ASE (ASEL) cell fate in the right ASE (ASER) neuron and stimulates mir-273 expression. MiR-273 targets the DIE-1 transcription factor in ASER but not in ASEL, in which DIE-1 activates lys-6 expression and promotes the ASEL-specific cell fate. In ASEL, COG-1 is blocked by lys-6 [335]. Figure 11. A schematic visualization of some shared principles of TF and mirna action. Adapted from Hobert O. [336]. 48

57 Methods of Identifying mirna Functions: The rapid increase in the discovery of mirnas species addressed a question regarding their function. Nevertheless, clues of the function and regulatory targets of some of the founder members of mirnas that have been identified by forward genetics, came even before their status as noncoding RNA genes was discovered [19, 20, 109, 183, 263, ]. The breadth and importance of mirna-directed gene regulation are coming into focus as more mirnas and their regulatory targets and functions are discovered [8]. Over the years, a number of methods have been developed to identify mirna targets and the regulation they impose on their targets. These methods range from small-scale genetic studies to computational predictions and high-throughput biochemical approaches to isolate target mrnas or sequences [341]. Using multiple complementary approaches is favorable to confirm mirna targets and their regulatory sequences [184]. Numerous computational approaches are used to predict mirna target sites in silico. These methods rely on algorithms that incorporate diverse criteria for the identification of candidate mirna targets (for examples; conservation of seed region and AU-rich regions) [6]. Nonetheless, most algorithms produce widely divergent predictions of partially complementary mirna target sites with degrees of false positives and false negatives that are difficult to assess [342]. Biochemical methods are often coupled to bioinformatics analyses, to identify mirna targets. These include increased sensitivity and the ability to identify endogenous target mrna transcripts or even the target sequence in the mrna on a large scale [184]. The recently developed CLIP approach aids in identifying endogenous target sites by sequencing those that co-immunoprecipitate with mirisc factors coupled with highthroughput sequencing (CLIP seq) or high-throughput sequencing together with CLIP (HITS CLIP) [196, ]. Genetic methods identify mirna targets through phenotypic suppression tests. Typically, screens are performed to search for candidate genes that rescue an mirna loss-of-function phenotype. An important advantage of this approach is that the genetically identified target is a physiologically relevant gene that is regulated by the mirna. Caveats of these genetic analyses include the inability to distinguish direct and indirect targets of mirnas and the potential difficulty in detecting individual suppressors if many targets contribute to the phenotype [184]. To study target regulation by mirnas, several RNA-expression analyses can be used. Because of the converse relation between mirnas and target mrnas [158], decreased mrna levels could be a result of direct mirna regulation. For analysis of specific targets, northern blotting or quantitative PCR can be used. However, to compare the changes in mrna abundance on a genome-wide scale, microarrays and RNA sequencing are used [157]. Deadenylation is often a characteristic of mirnamediated target regulation, and this can be assessed by poly(a) tail length analysis using RNase-H cleavage of mrna 3 ends followed by northern blotting or PCR-based methods to for compare 49

58 adenylation states [345]. These approaches can show that a predicted target is subjected to regulation at the mrna level. However, failure to detect a change in mrna abundance does not rule it out as a target, as the mirna complex can also use mechanisms to block protein expression that do not involve mrna destabilization[184]. Several direct and indirect approaches have been used to study the influence of mirna on target protein product. This includes western-blot method for measuring changes in protein levels for specific targets and mass spectrometry [52, 53, 346]. A drawback of these methods is that, they are not sensitive enough to detect low abundance proteins that may also be under mirna control. An indirect method for assaying protein production is through ribosome profiling, where the association of mrnas with ribosomes indicates their translation state. In ribosome profiling, the mrna fragment that is associated with individual ribosomes is identified [235]. Not only can a change in ribosome association be detected by ribosome profiling, but the actual positions at which ribosomes are associated with the mrna are revealed. Thus, an accumulation of ribosomes at the 5 end of an mrna can indicate stalled translational initiation. Because mirnas have been reported to regulate translation at both initiation and post-initiation steps, ribosome profiling is a powerful method for determining the mechanism of regulation for individual mrnas [184] Implications in different Physiological Functions: In mammals, mirnas are predicted to control the activity of ~50% of all protein-coding genes [39]. Recently it was demonstrated that the majority of all human genes are under the control of mirnas [56]. Functional studies indicate that mirnas participate in the regulation of almost every cellular process investigated so far and that changes in their expression are associated with many human pathologies [39]. MiRNAs have roles in a broad range of biological processes including cell proliferation, cell death, fat metabolism [183, 340], neuronal patterning [109], and modulation of hematopoietic lineage differentiation [263, 264, 269, 347]. Dysregulation of mirnas have been shown to be implicated in pathological conditions [36]. The alteration of mirna expression and activity can impact human diseases which include infectious diseases, cancers, cardiovascular and neurological disorders [348]. Microarray expression profiles of patients with neurological disorders like Parkinson s disease, Alzheimer s disease and schizophrenia show altered expression in comparison to healthy individuals suggesting their role in regulating these disorders [349, 350]. The emerging picture of mirna regulation in animals is far richer and more complex than the crisp linear pathways of the previous decade, with mirnas participating in executive decisions but also performing much of the work to micromanage protein output [6]. 50

59 Local Protein Translation in Neurons: Some of the most interesting examples of mirna activity regulation are coming from neurons [39]. Each neuron grows a single axon that can extend up to 1m in length in humans and various numbers of dendrites that create a complex three-dimensional dendritic tree. Dendrite processes can spread up to hundreds of mm, contributing to the formation of complex neuronal circuits with different functional outputs. The complexity of information processed in higher centers of nervous system exceeds by far any other system in the human body. It has been estimated that billion neurons in the human brain, each individual neuron being synaptically connected to ~ neurons [78]. Each of these contacts can potentially be modified in an independent manner for long periods of time [ ]. Such complex system has been shown to be orchestrated by highly organized gene expression programs [78]. Messenger RNA translation in neurons can occur at the rough endoplasmic reticulum in a close proximity to the nucleus, additionally, autonomously managed protein translation has been demonstrated to occur in neuronal processes (dendrites and axons) [354, 355]. This implies the presence of the translational machinery in the processes to enable local control of gene expression upon synaptic activity [78]. Bursts of synaptic activity can induce short-term changes in synaptic strength, but more stable modifications typically require modulation of gene expression at the transcriptional and posttranscriptional levels [351, 356]. Through post-transcriptional regulation, synaptic activity may dictate the time and place of neuronal protein synthesis [ ]. Activityinduced protein synthesis has been implicated in neural development as well as the maintenance and plasticity of neural connections [361, 362], and was shown to be subject to tight controls [363]. Selected mrnas and translational machinery, including ribosomes and other noncoding RNAs, are localized to dendritic regions of neurons [364, 365]. Elements in the 3 UTR of some mrnas have been implicated in their localization [ ]. Notably, the percentage of spines that contain polyribosomes increases markedly in CA1 neurons after LTP induction [370]. Furthermore, after the induction of Long Term Potentiation (LTP), the area of the postsynaptic density (PSD) is significantly larger in the spines that contain polyribosomes than in spines that lack them, indicating that the presence of polyribosomes produces structural changes, which, in turn, produce changes in synaptic strength [351]. Some of these mrnas encode proteins such as kinases and translational control factors that are attractive candidates to mediate synaptic changes [362, 371, 372]. Depolarization of neurons results in the translation of somatodendritic, plasticity-related mrnas [362, ]. In addition, within dendrites, and at synapses, the translation of these mrnas may be inhibited until neurons are exposed to appropriate extracellular stimuli such as a neurotrophic factor (for example, brainderived neurotrophic factor (BDNF)) or neurotransmitter release at the synapse [ ]. Localized translation in mammalian dendrites has been proposed to play a role in synaptic plasticity and to 51

60 contribute to the molecular basis for learning and memory [227, ]. Previous study showed that axons of 15-day old cultured hippocampal neurons lack ribosomes and have little if any RNA [379]. On the other hand, other studies demonstrated that axons are capable of localized translation [378, 380, 381]. Taken together, local mrna translation serves many important functions during development or growth of axons and dendrites, as for the regulation of synaptic plasticity. Many excellent works have been done on mrna transport [ ], local translation [358, 385, 386], RNP granule structure and function [283, 305, 387], and ncrna function [387, 388] in neurons [290]. Recently, mirnas have been suggested to be involved in the local translation of neuronal compartments. This has been derived from the observations reporting the presence of mirnas and the protein complexes involved in mirna biogenesis and function in neuronal soma, dendrites, and axons. Interestingly, the profile of suppressed target mrnas as well as the functional outcome of mirna/mrna regulation in individual neurons might differ markedly between the various levels of neuronal activation. The role of mirnas in controlling such complex network might be further evaluated by considering the ability of each mirna to simultaneously target hundreds of targets [56, 157, 182]. Sequencing analysis of Ago2-associated mirnas and mrnas from developing mouse brain confirmed the magnitude of mirna:mrna target regulation in the brain in vivo [342]. MiRNAs can impose effective mechanism to ensure the tight control of neuronal gene expression required for the regulation of neuronal development, function, and survival [78] Expression of mirnas in the Nervous System: Almost 50% of all identified mirnas are expressed in the mammalian brain [16, 142, ]. The expression levels of mirnas in neurons vary significantly. Some mirnas such as let-7, mir-124, and mir-128 are expressed at very high copy numbers (up to copies/neuron) [12, 393], whereas the levels of other brain expressed mirnas can be as low as 1 2 copies/cell [78]. MiRNAs appear to be differentially distributed in distinct brain regions and neuron types [389, ]. The transcription of individual mirna-encoding genes may differ significantly between individual neurons, thus contributing to the neuron-specific pattern and levels of mirna expression. For example; mir-9 in mice revealed significant differences in copy number between a striatal and a magnocellular neuron [397]. Mir-124, mir-7, and mir-9 have been reported as highly abundant neuron-specific mirnas, and mir-128, mir-129, mir-133a, mir-138, mir-153, mir-181a, mir- 181b, mir-218, and mir-219 as brain-enriched mirnas [44, 78]. Importantly, mirnas are reported to be deferentially distributed at the sub-cellular level [ ]. Several mirnas have been reported to be enriched in synapses or dendrites as compared with the perinuclear cell soma [78, 375, ]. Moreover, studies on synaptoneurosomes levels revealed abundant presence of several components of 52

61 mirnas biogenesis pathway and their silencing complex machinery at postsynaptic densities [404]. The mechanisms that contribute to specific enrichment of mirnas within synapses, dendrites, and axons are still unclear [78]. Interestingly, the parallel changes in the mirna and mrna populations are consistent with the hitch-hiking model in which mirnas travel to the dendrites bound to their target mrnas [388]. It has been proposed to involve RNA-binding proteins that deliver or anchor mirnas to particular neuron areas. Several brain-enriched mirnas have been found in association with FMRP, a RNA-binding protein that localized into dendrites and is known for its important role in the regulation of local protein translation [375, 403]. Furthermore, the presence of RISC in the preand postsynaptic neuronal compartments supported the possibility of local mature mirna biogenesis from pre-mirnas [304, 399, ]. Further confirmation of mirna local translational control came from studies demonstrating the effect of mirnas modifications on the synapse function (Fig. 12). Figure 12. Working model for the role of mirnas in local dendritic protein synthesis and spine morphology in CNS physiology and disease MiRNA & Synaptic Plasticity: Recent data on the role of synaptic mirnas and their target gene networks have revealed their importance for synapse development and physiology [388, ]. One of the first studies reporting control of local translation by mirna at synapses upon synaptic activity was by Schratt et al. [375]. MiR-134 has been shown to negatively regulate LimK1 in an activity dependent manner. Limk1 is a protein kinase that regulates actin filament dynamics through inhibition of ADF/cofilin34. The study 53

62 demonstrated the compartmentalization of mir-134 and its target LimK1 mrna at synapto-dendritic area. It has been proposed that mir-134 association with Limk1 mrna keeps the Limk1 mrna in a dormant state while it is being transported within dendrites to synaptic sites. Overexpression of mir- 134 inhibits LimK1 mrna local translation, resulting in a negative regulation of the size of dendritic spines. Exposure of neurons to BDNF relieves mir-134 inhibition of Limk1 mrna translation [375]. Recently, the same group reported another mechanism by which pre-mir-134 is transported to dendritic sites. Dendritic localization of pre-mir-134 is mediated by the DEAH-box helicase DHX36, which directly associates with the pre-mir-134 terminal loop. In accordance with the role of mir134 on spine morphology, functional experiments revealed that DHX36 negatively regulates dendritic spine morphogenesis in hippocampal neurons [414]. Another mirna that showed an important role on dendritic morphology, is mir-138.siegel and colleagues demonstrated that microrna-138 decreases the size of dendritic spines through local downregulation of acyl protein thioesterase 1 (APT1). The authors found that overexpression of mirna 138 decreased spine volume without any effect on other dendrite characteristics, such as spine density and dendrite branching. There was a correlation between the overexpression of mirna 138, a decrease in AMPA (α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid) receptor cluster size and the miniature excitatory postsynaptic currents (mepscs) mediated by these receptors. Palmitoylation has recently been implicated in the regulation of synaptic plasticity [362, 415], and changes in spine structure have been linked to a dynamic balance between the addition of palmitate to, and removal from, synaptic proteins [362]. The authors showed that APT1 negatively regulates the membrane localization of Gα13 (substrate of APT1), suggesting that APT1 effectively depalmitoylates Gα13. In addition, overexpression of mirna-138, which downregulates APT1, increased the membrane localization of Gα13, indicates that the three molecules belong to a common pathway. The authors established a new link between Gα13 and the regulation of spine size, as overexpression of Gα13 counteracted the spine growth observed after inhibition of mirna 138. Although the authors could not directly implicate the palmitoylation of Gα13 by APT1 in the regulation of spine size, they provided evidence that the association of Gα13 with the membrane is necessary to rescue the effect of mirna 138 inhibition [402]. The RISC protein MOV10 has been demonstrated to be a pivotally positioned control element in the regulation of local protein synthesis in dendrites. MOV10 is functionally required to mediate mirnaguided mrna cleavage [288]. Suppression of MOV10 with RNAi, was found to regulate Lypla1 mrnas, a depalmitoylating enzyme, and its mirna silencer, mir-138 [406]. In addition to the depalmitoylating enzyme, Lypla1, two palmitoylating enzymes, Zdhhc2 and Zdhhc17, appeared to be affected upon suppression of MOV10. It has been suggested that the activity-dependent regulation of 54

63 the synthesis of these palmitoylating and depalmitoylating enzymes represents an important facet of the mrna repertoire under the control of the RISC in the context of synaptic stimulation. Among the mrnas found in the screen was the activity-dependent mirna target Limk1 which can modulate structural and functional plasticity by targeting cytoskeletal effectors [375]. Taken together, the set of mrnas that are relieved of their silence represents a coordinately regulated response to synaptic stimulation. The discovery of MOV10 as an ubiquitin-dependent client protein degraded by the proteasome presents a counterintuitive role for degradation. In this case, degradation is linked to increased local mrna translation and thus an increase in the levels of those proteins regulated in the RISC. Degradation of MOV10 and possibly other components of the RISC may underlie observations that both degradation and synthesis are required for synaptic plasticity [405, 406, 416]. MiR-132 is one of the mirnas that has been widely investigated with regard to neuronal function. MiR-132 expression is regulated by neuronal activity due to the presence of CRE elements in its promoter region [356, 417, 418]. Several mirnas, including mir-132, have been shown to regulate the size and number of dendritic spines and alter synaptic activity [403, ]. In vivo and in vitro experiments showed that mir-132 induction led to enhanced dendrite morphogenesis, by inhibiting translation of its target mrna p250gap [422]. Furthermore, experiments showed that the mir132- mediated repression of p250gap and regulation of dendritic growth occur by modulating Rac-family GTPases, a pathway that may contribute to activity-regulated actin remodeling, since other regulators of dendrite and spine growth, such as EphB, Kalirinin, and Tiam1, also show selectivity for Rac in hippocampal neurons [423, 424]. Several downstream effectors of Rac and Cdc42, including Pak, Lim-kinase, and myosin heavy chain IIb, have been proposed to regulate structural or functional dendritic plasticity [425]. The same group has previously shown that BDNF treatment increased mir- 132 levels in immature neurons [417, 422]. MiR-132 has been shown to target methyl CpG-binding protein 2 (MeCP2), a protein involved in dendritic development and synaptogenesis and whose modulation can alter synaptic plasticity [ ]. It has recently been reported that a transgenic mouse overexpressing mir-132 throughout the forebrain showed impairments in recognition memory [430]. This study investigated the effect of mir- 132 on synaptic plasticity associated with memory formation. MiR-132 morphological effects are accompanied by changes in basal synaptic transmission. Although there is some variability between individual studies, mir-132 generally appears to have a positive effect on both the frequency and amplitude of mepscs [403, 419, 420] and also modulates short-term synaptic plasticity [420, 421]. Furthermore, the acquisition of recognition memory depends on CREB-dependent long-lasting changes in synaptic plasticity in the perirhinal cortex (PRh). The mir-132 expression within the PRh impairs short-term recognition memory. This functional deficit was associated with a reduction in both 55

64 long-term depression and long-term potentiation. Indicating a general negative effect of mir-132 on synaptic plasticity mechanisms, which does not shift the balance towards either depression or potentiation [421]. The impairment in LTD would be predicted by the overexpression of mir-132 reducing the actin depolymerisation underlying LTD-associated spine shrinkage [431]. Although, it can be predicted the opposite effect on LTP [432], it is possible that the impairment in LTP is due to some homeostatic adjustment to the chronic overexpression of mir-132. Tognini & Pizzorusso [433] have proposed a model whereby precise control of mir 132 levels is required for regulation of synaptic plasticity. They suggest that if levels are too high then dendritic spines will be excessively stable, impairing plasticity; likewise, if levels are too low then the spines will be very unstable, also hindering plasticity. These deficits in synaptic plasticity have been proposed to be the substrates for the behavioral impairment [421]. MiR-132 expression was shown to occur at the onset of synaptic integration in the olfactory bulb neurons born in the neonatal SVZ. Sequestration of mir-132 led to a reduced dendritic complexity, spine density and the frequency and amplitude of EPSCs, while overexpression had the opposite effects. Data show that mir-132 overexpression in newborn neurons enhances dendrite development resulting in stronger synaptic integration. MiR-132 overexpression in transplanted neurons may thus hold promise for enhancing neuronal survival and improving the outcome of transplant therapies [434]. MiR-124 is shown to be enriched in the nervous system. It has been reported that mir-124 expression is down-regulated by 5-HT, which in turn enhances various components of synaptic plasticity through regulating the expression of camp response element (CRE)-binding protein (CREB) [435]. Besides, the effective component of standardized leaf extract of Bacopa monniera (BESEB CDRI- 08) has been reported to have memory enhancing effect by enhancing serotonin. Bacopa monniera extracts treated rats showed better performance on memory task. There was significant down-regulation of pre-mir- 124, dicer and Ago2 mrna expression in hippocampus, combined with a significant increase in the plasticity related genes, CREB, its phosphorylation and postsynaptic density protein 95. The levels of pre-mir124 were significantly down-regulated in BME, whereas, the level of Creb1 mrna were upregulated [436]. Induction of long-term potentiation (LTP) found to upregulate mir-188 expression. This is accompanied with protein downregulation of its target mrna neuropilin-2 (Nrp-2). Nrp-2 serves as a receptor for semaphorin 3F, which is a negative regulator of spine development and synaptic structure. MiR-188 expression counteracted the decrease in the miniature EPSC frequency and diminished dendritic spine densities induced by Nrp-2 expression in hippocampal neuronal culture. These findings suggest that mir-188 serves to fine-tune synaptic plasticity by regulating Nrp-2 expression [437]. 56

65 Drugs of abuse cause long-lasting changes in the limbic regions of the brain that process reward and addiction, that are viewed as a form of aberrant neuroplasticity. MiRNAs have been implicated in mediating the effects of cocaine [ ], nicotine [442, 443], alcohol [397, 444], and several other classes of drugs of abuse [ ]. Several recent studies investigated the role of AGO2 and mirnas in the biochemical, molecular, and behavioral response to cocaine [ , 448]. For example, qpcr and in situ analysis of specific bioinformatically determined mirnas identified let- 7d, mir-124, and mir-181a as cocaine-regulated in rats [438]. Using next-generation sequencing, a subset of cocaine-regulated mirnas in whole striatum tissue and at the synapse has been identified. Applying gene ontological analysis of the identified target genes revealed that most of the enriched categories are generally linked to synaptic plasticity, learning, and memory [449]. Two studies by the Kenny s group identified induction of mir-132 and mir-212 in dorsal striatum after extended access to cocaine in rats [440, 441]. Studies reported the implication of mir-212 in the behavioral and motivational response to cocaine through CREB, MeCP2, and BDNF signaling [440, 441]. In addition, Schaefer et al. identified an overlapping subset of cocaine-induced and AGO2-knockdown-depleted mirnas in D2 dopamine receptor (Drd2) expressing neurons of the nucleus accumbens, and showed a reduction in cocaine self-administration when AGO2 is depleted from Drd2 neurons [448]. Performing RNA-Seq revealed cocaine-induced increases in Ago2 mrna levels [450]. Synaptically enriched cocaine-regulated mirnas may contribute to long-lasting drug-induced plasticity through fine tuning regulatory pathways that modulate the actin cytoskeleton, neurotransmitter metabolism, and peptide hormone processing. In addition, these studies provide evidence that mirna-mediated gene regulation plays an important role in cocaine-related changes in neurotransmission and behavior [451]. MiRNAs have been shown to be involved in the neuronal plasticity, underling the molecular mechanism of nicotine addiction. Nicotine regulates dynamin 1 gene (Dnm1), which has an essential role in synaptic endocytosis in the central nervous system, through selectively modulating its target mirna expression; mir-140 *[443]. In a study by Lippi el al [446], in vivo chronic drug exposure to psychostimulants (nicotine, cocaine, and amphetamine) found to affect the mir transcriptome in a drug- and region-specific manner. The study further focused on mir-29a/b, identified as regulators of dendritic spine morphology and synaptic connectivity. Indeed, directly target the mrna encoding for Arpc3, a subunit of the ARP2/3 actin nucleation complex [452]. It has been reported that the upregulation mir-29a/b represents an activity-dependent pathway to counterbalance excessive positive cues driving spine formation and reinforcement of synaptic communication. Additionally, mir-29a/b was shown to fine tune structural plasticity via regulating the sensitivity of ARP2/3 to remodeling cues in the spine [446]. Using a chronic intermittent ethanol (CIE) paradigm, a model of alcohol removal, in primary cortical neuronal cultures revealed an alteration in the expression of mirnas in this condition. The predicted 57

66 target of EtOH removal-induced mirnas have been shown to be involved in several cellular functions. This includes; the regulation of gene transcription, neuron differentiation, embryonic development, protein phosphorylation, and synaptic plasticity. These results suggest a potential role of differentially expressed mirnas in mediating EtOH removal-related phenotypes [453]. N-methyl-D-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. A brain-specific mirna mir-219, was found to be downregulated in the prefrontal cortex of mice, upon disruption of NMDA receptor signaling by pharmacological application of dizocilpine, which is associated with behavioral aberrations. MiR-219 targets the CaMKII family of kinases which is considered as core regulators of NMDA signaling that can modulate NMDA-R trafficking and activity state, and can be locally translated in dendrites to promote rapid plasticity [454, 455]. Pretreatment with the antipsychotic drugs prevented dizocilpine-induced effects on mir-219. Proposed mechanism is that silencing of mir-219 in the murine brain releases the translational repression of the mirna on the 3 UTR of the CaMKII_ mrna, thus providing a compensatory mechanism to maintain NMDA-R function during acute antagonism of the receptor and to attenuate associated behavioral manifestations [456]. Saba et al has demonstrated a robust enrichment of mir-181a in the synaptoneurosome of the medium spiny neuron synapses of the nucleus accumbens. This is a region of the brain; that is critical to form drug-seeking habits. MiR-181a was reported to target GluA2 subunit mrna of AMPA-Rs, its overexpression reduced GluA2 surface expression, spine formation, and miniature excitatory postsynaptic current (mepsc) frequency in hippocampal neurons. It has been proposed that mirna effect is likely due to the inhibition of GluA2 mrna translation and/or the promotion of GluA2 mrna degradation at synapses. Moreover, mir-181a expression was induced by dopamine signaling in primary neurons, as well as by cocaine and amphetamines, in a mouse model of chronic drug treatment. Results suggest that the stimulation of dopamine signaling in hippocampus neurons leads to an increase in mature mir-181a, possibly due to increased pre-mir-181a processing [449]. MiR-181a was previously reported to associate with Ago2 in the striatum [448], expressed in a somatodendritic gradient in hippocampal neurons [379], and to be induced by cocaine [438, 448]. Finally, virusmediated overexpression of this mirna in the rat nucleus accumbens was recently shown to enhance conditioned place preference for cocaine [439]. 58

67 The Factors behind Expression Alteration: Neuronal activity plays an essential role in regulating synaptic strength and connectivity of adult neurons [78]. The alteration of mirna-mediated target expression has been referred to different factors. This includes changes on the level of mirna transcription. Similar to the activity-induced activation of protein-coding gene transcription, the transcriptional activation of several neuronal mirnas depends on the neuronal activity-induced transcription factors [418, 419]. Another factor is the post-transcriptional regulation of mirna expression at the level of pre-mirna [ ]. Reports demonstrate the existence of complex regulatory mechanisms involving control of pre-mirna processing and degradation [78]. In addition, differential regulation of mirna stability and turnover rates has been suggested to be a mechanism controlling distinct neuron functions. MiRNA turnover in neurons were reported to be activity dependent [435, 461]. Interestingly, the regulation can impact mirna activity, instead of mirna stability. Recent data revealed the existence of endogenous mirna sponges [ ]. They are a class of long noncoding RNAs that act as mirna sponges, and termed competing endogenous RNAs (cernas) with functions in muscle differentiation and tumor suppression [ ] MiRNAs involvement in the Nervous System Maladaptive States: Neurodegenerative diseases and Psychiatric Disorders: MiRNA has been suggested to be involved in neurological and psychiatric diseases. This has been further supported by the numerous studies showing changes in mirna expression levels and patterns during neurological disorders in humans [78, ]. In the cases of schizophrenia and Autism, mir-16, -30b, and -181b are reported to be upregulated in patients, whereas mir- 132 is found to be downregulated [ ]. MiR-9/9*, -17, -29a/b, -124, -132, -196a, -222, -330, -485, and -486 have been shown to be downregulated in patients and mice suffering from Huntington s disease [146, 476]. MiR-9 and mir-128 were found to be upregulated in the CNS of patients with Alzheimer s disease as compared with their age-matched control samples [477]. The expression levels let-7c, mir-99a, - 125b-2, -155, and -802, were found to be increased in the fetal brain and heart of patients with Down Syndrome (DS) [478]. Furthermore, a mouse model of DiGeorge syndrome revealed an alteration of a specific subset of brain mirnas, including mir-134 [479]. Finally, a single mutation in the 3 UTR of the SLITRK1 gene that leads to a disruption of its 3 UTR mir-189-binding site, was found to be associated with the development of Tourette syndrome and obsessive compulsive disorders [78, 480] Central Nervous System Cancer: Gliomas are the most common form of adult brain tumors, of which glioblastoma the most aggressive is, and has received the most attention in mirna studies. MiR-21 and mir-26a have been shown to be 59

68 upregulated in glioblastomas [481, 482]. While mir-21 is evenly upregulated in all glioma tumors, mir-221 is exceptionally higher in glioblastoma multiforme (grade IV) [483]. MiR-221 and mir-222, which are coexpressed, were found to target and inhibit the cell cycle inhibitor p27kip1 in glioblastoma cells [484]. MiR-21, mir-26a, and mir-221/222 function as oncomirs by suppressing genes that negatively regulate cell growth, survival, and invasion. In contrast, mir-7, mir-34a, mir- 124, mir-137, mir-146b, mir-15b, mir-128, and mir-326 are found to be downregulated in glioblastomas, and proposed to function as tumor suppressors [ ]. Medulloblastoma is the most common form of childhood brain tumors, which may originate from granular cell progenitors (GCP) that fail to differentiate. MiRNA, including mir-125b, mir-326, and mir-324 5p, are reported to be downregulated in MB [491]. These mirnas are both necessary and sufficient for cell proliferation of MB cells. In addition, the mir-17~92a cluster was upregulated [492]. Finally, the Notch receptor pathway also plays a role in MB where it is regulated by mir-199b-5p [493], lower levels of mir- 199b-5p positively correlate with metastasis of MB [482] Pain: The pathophysiological pain states have been shown to be associated with a lot of expression changes in pain-related proteins. This led to the suggestion that mirnas might be involved in pain onset and chronicization by their important function in the regulation of gene expression. Indeed, a differential regulation of mirnas has already been described in initiation and progression of different experimental pain models [494]. Specific mirna mrna interactions might provide a promising approach to modulate pain processing pathways in inflammatory, neuropathic and cancer pain. In the next section, the involvement of mirna in pain conditions will be explained in more details. The general aspects of mirnas biogenesis, target recognition, mode of regulation and the common regulated mirnas in synaptic plasticity and pain conditions, are summarized in the following review. The present work includes 2 functional studies, aimed at investigating the regulatory role of mirnas in synaptic plasticity (hoemoeostatic synaptic plasticity) and in chronic pain condition (bone cancer pain). 60

69 2. MIRNAS REGULATION IN HOMEOSTATIC SYNAPTIC PLASTICITY 2.1 Homeostatic Plasticity Introduction: Central neurons are embedded in complex networks composed of many distinct cell types, including both excitatory neurons and a rich variety of inhibitory neurons with distinct morphologies and functions [495]. The mammalian brain is the most complex system in the known universe, with billions of neurons each forming up to 100,000 synaptic connections. However, this sophisticated system is not hardwired, but is constantly undergoing modifications to store information and adapt to changes in the environment. Such plastic mechanisms, shape the output and function of nervous systems, without compromising the stability and integrity of the underlying circuits that drive behavior [496]. This remarkable feat is accomplished through a set of homeostatic plasticity mechanisms that allow neurons to sense how active they are and to adjust their properties to maintain stable function. Loosely defined, a homeostatic form of plasticity is one that acts to stabilize the activity of a neuron or neuronal circuit in response to perturbations, involving changes in cell size or in synapse number or strength; that alter excitability [ ]. A large number of plasticity phenomena have now been identified in a wide range of systems that conform to this definition of homeostatic plasticity [ ]. Homeostatic plasticity maintains stability in central neural circuits function, by setting excitation and inhibition to the proper levels so that activity can propagate through a network without either dying out or increasing uncontrollably (Fig. 13) [500]. In addition, it stabilizes circuits with plastic synapses, including long-term potentiation (LTP) and long-term depression (LTD) [501, 502], which strengthen synaptic inputs that are effective at depolarizing the postsynaptic neuron and weaken inputs that are not, thus reinforcing useful pathways in the brain. This is illustrated most in learning-related adaptations that require neural networks to detect correlations between events in the environment and store these as changes in synaptic strength or other cellular properties [501]. Homeostatic plasticity acts by preventing the unconstrained positive feedback cycle that might result from synapses that are strengthened become more effective at depolarizing the post-synaptic neuron and will continue to be strengthened, eventually driving neuronal activity to saturation [501, 503, 504]. Hence, by preventing this positive feedback, homeostatic plasticity help in maintaining synapse specificity [496]. To implement homeostatic plasticity neurons need to sense some aspect of 92

70 activity, and when this measure deviates from a target value, a force must be generated to adjust excitability to normalize activity [495]. One type of homeostatic plasticity is the synaptic scaling, which is used by neurons to stabilize their firing rates, contributing to the ability of central neuronal networks to tune themselves up and maintain stable function throughout life [496]. Figure 13 Mechanisms of Synaptic Plasticity Are Potentially Destabilizing (A) Correlated presynaptic and postsynaptic firing induces long-term potentiation (LTP), which then allows the presynaptic neuron to drive the postsynaptic neuron more strongly. This increases the correlation between presynaptic and postsynaptic activation, which drives more LTP, and so on in an unconstrained positive feedback cycle. (B) Unconstrained LTP will lose synapse specificity, because when one input undergoes LTP and drives the postsynaptic neuron more strongly, it makes it easier for other inputs to make the postsynaptic neuron fire, and they begin to undergo LTP as well. (C) Homeostatic synaptic scaling prevents this runaway potentiation. When LTP of one input increases postsynaptic firing, synaptic scaling will reduce the strength of all synaptic inputs until the firing rate returns to control levels. Note that synaptic strengths are reduced proportionally, so that the relative strength of the potentiated synapse remains the same. Adapted from Turrigaiano G. [496] Homeostatic Synaptic Scaling in Excitatory Neurons (In Vitro): Synaptic scaling is a form of homeostatic plasticity that increases or decreases the strength of all of a neuron s synaptic inputs as a function of activity. This mechanism was found to both, globally and locally scale all of a neuron s synapses up or down in strength in the correct direction to stabilize neuronal firing (Fig 14) [505, 506]. Homeostatic synaptic plasticity has been identified in a variety of organisms and systems and appears to be a very general phenomenon [497, 500, 507]. In principle, synaptic strength could be altered via changes in postsynaptic receptor accumulation, presynaptic 93

71 release probability, the number of functional synaptic contacts between pairs of neurons, or all three mechanisms simultaneously. Figure 13. Distinct Mechanisms of Global and Local Changes in Synaptic Strength. (A) Blocking postsynaptic firing while leaving presynaptic and network activity intact scales up synaptic strengths in the dendrites, via a mechanism that results in increased accumulation of both GluR1 and GluR2 subunits of AMPA receptors. (B) When action potential firing is blocked and NMDA receptor activation is locally blocked with the antagonist APV, there is a local increase in synaptic GluR1 accumulation that requires local dendritic protein synthesis [496]. Synaptic scaling was first described a decade ago, in primary culture of rat postnatal visual cortex neurons. It was observed that perturbing network activity could generate compensatory changes in synaptic strength that aim to normalize firing rates back to control values [505]. The authors addressed the question of whether cortex neuron culture could scale the strength of AMPA (aamino-3-hydroxy- 5-methyl-4-isoxazole propionic acid)-mediated synaptic currents up or down as a function of activity. Chronic blockade of cortical culture activity by applying tetrodotoxin (TTX) for 48 hours, which completely abolished firing, was found to increase the amplitude of miniature excitatory postsynaptic currents (mepscs). Besides, there was a progressive increase in mepsc amplitude with treatment duration, indicating that this process is both slow and cumulative [505]. The study tested the involvement of NMDA (N-methyl-D-aspartate) receptors on mepsc amplitude, since some forms of synaptic plasticity require calcium influx through them [508, 509]. In contrast to TTX, application of NMDA antagonist APV (D(-)-amino-7-phosphonovalenic acid) for 48 h did not influence firing rates, 94

72 mepsc amplitude or area. However, blocking AMPA receptors with 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX), which like TTX completely abolished firing, significantly increased mepsc amplitude values [505]. Examining the mepsc kinetics of cultured cortical neurons in homeostatic paradigm, revealed no differences in the rise and decay kinetics [505]. Furthermore, in contrast to LTP and other rapid potentiation protocols [ ], there were no differences in mepsc frequency between conditions. These led to suggestion, in accordance with other studies [513, 514], that altering activity for two days does not significantly influence the placement or number of excitatory synapses onto pyramidal neurons [505]. The regulation of mepsc amplitudes described has been suggested to act on stabilizing firing rates, since raising firing rates decreases the strength of excitatory synaptic inputs, and vice versa. Reports have shown that chronic TTX treatment dramatically increases firing rates when the TTX is removed [515, 516]. The bidirectional regulation of quantal amplitude is likely to contribute significantly to the homeostatic regulation of firing rates [505]. These data lend support to the notion that cortical and hippocampal pyramidal neurons have a target firing rate, and synaptic strengths are regulated to maintain these rates relatively constant to face perturbations in input, providing a robust mechanism for generating stability in network function in in response to developmental or learning- related changes in synaptic input [496]. The activity that regulates mepsc amplitude has been shown, at least partly, to occur through a postsynaptic change, and apparently to affect each synapse in proportion to its initial strength [505]. It has been suggested that scaling might occur through a change in the function of existing AMPA receptors through phosphorylation [517, 518] but also through the conversion of existing receptors between inactive and active states [519, 520]. Another could be changes in the synthesis and insertion of glutamate receptors [521, 522], then receptors must be inserted or converted in proportion to the existing number of functional receptors [505] (Fig. 14). Further experiments, agreed with results obtained from Turrigiano et al. [505], reporting that the changes in excitatory synaptic transmission are accompanied by changes in the postsynaptic sensitivity to glutamate [505, 523] and surface AMPA receptor (AMPAR) accumulation [523, 524]. These data suggest a major postsynaptic contribution to homeostatic synaptic plasticity [505, ]. On the other hand, other studies emerged proposing presynaptic contribution, reporting little or no change in quantal amplitude and an increase in miniature EPSC (mepsc) frequency [526, 527]. This increase in mepsc frequency has been suggested to arise via increased presynaptic release probability, an interpretation supported by the observation that the uptake of the fluorescent styryl dyen-(3-95

73 triethylammoniumpropyl)-4-[4-(dibutylamino)styryl] pyridinium dibromide (FM1-43) in hippocampal cultures increased after activity blockade [528]. The styryl dye FM1-43 was used to probe the possible changes in the presynaptic terminals induced by activity blockade, an increase in the number of functional release sites or an increased release and recycling of vesicles is expected to result in an increased uptake of FM1-43 by the presynaptic terminals during synaptic activity [528, 529]. Synaptic strength is known to be determined by a number of factors in addition to the number of receptors in the postsynaptic membrane. In particular, the number of presynaptic neurotransmitter release sites and the probability that neurotransmitter vesicles will be released after an action potential ( release probability ) are also major determinants of synaptic strength [496]. The question that has been, then, addressed is whether this increase is expressed presynaptically or postsynaptically. In a study by Wierenga et al. [529], both possibilities were examined in sister neurons under same conditions. In contrast to the clear effects on receptor accumulation, activity blockade induced no detectable change in presynaptic parameters of transmission. A thorough analysis of presynaptic function, including short-term plasticity, variability of synaptic transmission, and FM1-43 dye uptake and release, revealed no significant changes in any of these parameters. Together, these data suggest that there are, at best, only subtle change in presynaptic function at these neocortical synapses [529]. Importantly, these differences between studies might reflect differences in culture conditions. In younger cortical cultures (less than 3 weeks in vitro), chronic blockade of activity has no apparent effect on presynaptic function, whereas in older cultures, activity deprivation increases postsynaptic receptor accumulation, synapse number, and the probability of synaptic vesicle release from the presynaptic terminal [530]. Presynaptic expression of homeostatic plasticity in cortical circuits has not yet been reported in vivo [496], whereas postsynaptic forms have [ ]. Hence, it remains to be seen what contribution these presynaptic mechanisms make to experience-dependent plasticity [496]. Nevertheless, it has been hypothesized that changes in postsynaptic glutamate receptor number and in presynaptic release may cooperate to homeostatically regulate synaptic transmission [496]. There is general agreement that synaptic scaling is induced by changes in AMPA receptor accumulation, although its subunit composition of the newly accumulated receptors remains controversial. The study by Wierenga et al. [529], has reported an increase in the synaptic accumulation of GluA1 and GluA2. Results revealed that the intensity of cell-surface GluA1 and GluA2 puncta in cortical pyramidal neurons, increased significantly in neurons that were treated for 2 days with TTX. This increase of synaptic GluA1 and GluA2 puncta wzs more pronounced than in non-synaptic puncta. The authors, furthermore, were able to follow EGFP-GluA2 puncta receptor accumulation in individual neurons, by live imaging distal dendrites. In contrast to control neurons, the intensity of EGFP-GluA2 puncta significantly increased upon treatment with TTX [529]. Obtained 96

74 data, further confirmed a postsynaptic effect on synaptic scaling mechanism. On the other hand, other reports have suggested that in hippocampal cultures, activity blockade leads to preferential increases in GluA1 synthesis and/or synaptic localization without significant changes in GluA2 and altered decay kinetics and pharmacological sensitivity of mepscs in a manner consistent with a greater GluA1 content [527, 534]. It has been suggested that the reported increase in the density of GluA1 puncta after activity blockade is likely to reflect increased GluA1 insertion at existing synapses, because mepsc amplitude increased but frequency was not altered [534]. These subunit-specific differences in receptor accumulation between neocortical and spinal neurons on the one hand, and hippocampal neurons on the other, suggest that there might be different underlying mechanisms controlling the activity-dependent receptor trafficking at these different types of central synapses [529]. The mechanism by which the increased AMPA receptor accumulation at postsynaptic sites occurs remains to be unraveled. Different possibilities have been suggested including: posttranscriptional regulation, posttranslational regulation, or a change in protein turnover perhaps because of interactions with other (synaptic) proteins [529]. An interesting investigation has been carried out by Sutton et al. [535], revisiting different aspects of this phenomenon, including: receptors involved, time course, postsynaptic GluR subunits composition, and AMPA receptor accumulation mechanism. Previous studies reported that, in contrast to TTX, application of NMDA antagonist APV for 48 h did not influence firing rates, mepsc amplitude or area [505]. Another curious feature of this synaptic scaling is its slow time course. An enhancement of mepsc amplitude is not evident until >12 h of Action Potential (AP) blockade. Interestingly, blocking NMDAR minis during the last 1 or 3 h of AP blockade produced a rapid and time-dependent increase in the amplitude of mepscs without altering their frequency. Similar effect has been seen upon blockade of AMPARs with CNQX in the presence of TTX, when CNQX was washed out of the bath. In contrary to other studies, APV delivered in the absence of TTX also produced a selective increase in mepsc amplitude 3 h posttreatment, indicating that AP blockade is not absolutely required for the rapid scaling of mepscs [506]. The slow homeostatic scaling induced by AP blockade is thought to reflect a global cellular response imposed by changes in the overall synaptic drive on the cell [505]. It has been previously reported that during AP blockade, the glutamatergic minis that persist, inhibit ongoing protein synthesis in neuronal dendrites [535]. Thus, it has been hypothesized that miniature synaptic events may exert a stabilizing influence on synaptic function via the tonic inhibition of dendritic protein synthesis that they provide [506]. Interestingly, application of the protein synthesis inhibitors anisomycin or cycloheximide completely prevented accelerated development of scaling by NMDAR mini blockade. These results were the first to suggest that the NMDAR-mediated stabilization of synaptic function is related to the tonic suppression of protein synthesis conferred by 97

75 minis [535]. This has been further confirmed by performing immunoprecipitation, which revealed that either complete or selective NMDAR mini blockade enhanced GluA1 synthesis relative to either blockade of APs alone or untreated controls. However, this was not accompanied by an increase in surface expression of the GluA2 subunit, which is normally present with GluA1 in heteromeric AMPARs in excitatory hippocampal neurons (e.g., [536]). There are certain logical advantages proposed for such global scaling, such as preserving relative differences in synaptic weights while maintaining neuronal firing rates in a dynamic range [500]. The study by Sutton et al. [506], further investigated an alternative form of scaling operating on a local level within dendrites. It has been tested whether NMDAR mini blockade increase GluA1 surface expression in a spatially specific fashion. A dual micropipette perfusion system has been used to locally disrupt miniature synaptic transmission in isolated regions of dendrites. Obtained results were in line with those obtained from whole culture treatment. This is represented by the significant increase in GluA1 surface expression in the perfused region, upon local blockade of NMDAR minis. This effect has been proposed to require new protein synthesis, since brief pretreatment with anisomycin was found to block the localized increase in surface GluA1 expression induced by NMDAR mini blockade. This has been reflected on the surface GluA1 expression, revealing a significant reduction in the perfused region relative to all other dendritic segments, demonstrating a requirement for local dendritic protein synthesis [506]. The study went further, elucidating the postsynaptic AMPA subunit composition involved. Application of Naspm, a specific antagonist of GluA2-lacking AMPARs (e.g., [537]), rapidly reversed the time-dependent enhancement of mepsc amplitude induced by NMDAR mini blockade with little effect on mepsc amplitude in control neurons or those treated with TTX alone. Together, these results demonstrate that NMDAR mini blockade induces a protein synthesis-dependent synaptic insertion of AMPARs that lack the GluA2 subunit. Nevertheless, Naspm showed time-dependent sensitivity, indicating that the synaptic incorporation of GluA2-lacking AMPARs is a transient modification that is slowly reversed by a replacement of these receptors with GlAR2-containing AMPARs [506]. This local mechanism has been proposed to have several advantages that complement those of mechanisms that operate at a global scale. This is including the capability for on demand calibration of synaptic strength, due to the ability of altering synaptic composition through rapid local scaling. In addition, the ability to adjust the magnitude/frequency of miniature synaptic transmission at particular sets of inputs, enables tunable degree of homeostatic control in a spatially specific manner. Additionally, the sensitivity to local synaptic signaling strongly facilitates implementation of the necessary synaptic modifications at the appropriate site. This is an exceptional advantage in pyramidal 98

76 neurons of the hippocampus and cerebral cortex, which receive around 30,000 unique synaptic inputs [506] Homeostatic Synaptic Scaling in Inhibitory Neurons (In Vitro): Neural circuits are composed of many excitatory and inhibitory cell types interconnected in highly specific ways. There is evidence that the rules for scaling excitatory synapses are cell-type specific [495]. Previous studies raised the possibility that inhibitory synaptic strength is regulated homeostatically in the opposite direction from excitatory synapses [495]. In these studies, visual deprivation or inhibition of retinal activity with tetrodotoxin (TTX), decreased GABA immunoreactivity [ ] and reduced inhibition and inhibitory synapse number in cortical and hippocampal cultures [515, 542], leading to a reduction in the amount of functional inhibition [515]. In cultured cortical and hippocampal neurons, excitatory synapses onto pyramidal neurons are scaled up by activity blockade, whereas excitatory synapses onto γ-aminobutyric acid (GABA)-ergic interneurons are either unaffected [543] or reduced [544], an effect that may depend on GABAergic cell type. Conversely, enhancing network activity increases excitatory transmission onto GABAergic interneurons [543, 544] through a process that involves the activity dependent regulation of the immediate early gene Narp [544]. A decade ago, the same study that has first set the notion for synaptic scaling upon activity blockade, investigated as well the same phenomenon, however, upon blocking the inhibition. Application of bicuculline, a GABAA-mediated inhibitor, to cortical pyramidal neurons cell culture, initially raised firing rates. This is accompanied with significant decrease in mepsc amplitude and area. However, over a 48-hour period mespc amplitudes decreased and firing rates returned to close to control values [505]. The same paradigm that scales-up miniature excitatory postsynaptic currents onto pyramidal neurons in culture, scales down the amplitude of miniature inhibitory postsynaptic currents through a mechanism that can involve both changes in accumulation of postsynaptic GABAA receptors and a reduction in presynaptic GABAergic markers, such as GAD65 [545, 546]. Both in vitro and in vivo studies have suggested that homeostatic regulation of inhibition can occur via a constellation of changes in postsynaptic strength, synapse number, and GABA packaging and release in various combinations [532, 545, 546]. The distinct and opposing plasticity rules at excitatory and inhibitory synapses appear to be designed to stabilize the firing of principal neurons (in cortex, and hippocampus, pyramidal), suggesting that, from a network point of view, it is the activity of the principal neurons that is homeostatically constrained [495]. 99

77 2.1.3 Signaling Pathways Underlying Synaptic Scaling: Arguably, the best-understood form of homeostatic plasticity in the central nervous system is synaptic scaling of excitatory synapses, which has been demonstrated both in vitro (in spinal neurons and neocortical and hippocampal pyramidal neurons) and in vivo [500, 505, 523, 524, 547]. Using pharmacological and genetic approaches to disrupt synaptic scaling, by targeting particular molecules or pathways, a number of molecular pathways has now been proposed to mediate synaptic scaling. In addition, an extensive crosstalk might exist between signaling pathways, making it likely that many perturbations could be due to disruptions of molecules that are modulators rather than mediators of synaptic scaling. [496]. Previous studies reported that the neurotrophin BDNF [543], the immediate early gene Arc [548], the cytokine TNFα [549, 550], the immune molecule MHC1 [551], Beta3 integrins [552], and the polo-like kinase 2 (Plk2)-CDK5 signaling pathway [553], among others, are all involved in or essential for synaptic scaling. Several of these molecules are known to regulate AMPA receptor trafficking; for example, Arc interacts with the endocytic machinery that removes AMPAR from the membrane [554], TNFα directly increases synaptic AMPAR accumulation [555, 556], and Beta3 integrins regulate AMPAR surface expression [552]. Some of these molecules are involved in only one branch of synaptic scaling (either scaling up or scaling down), indicating that although some signaling elements (such as CaMKK and CaMKIV) are shared during bidirectional scaling [557, 558], others are not [543, 548, 556]. Many of these signaling molecules are likely to play permissive rather than instructive roles in synaptic scaling, as has recently been shown for TNFα [549]. However, the entire sequence of events that lead from cell-autonomous changes in calcium influx to bidirectional changes in AMPAR abundance is still not fully understood, and a number of parallel signaling pathways, and dozens of molecules, likely contribute to synaptic scaling Evidences for Local Translation and UTR Involvement: Several biochemical signaling pathways that trigger dendritic protein synthesis upon increase in neuronal activity have been identified [385, 559, 560]. Recently, a novel pathway was suggested in activity-induced synaptic scaling [561], which involves retinoic acid (RA) signaling. Aoto et al. [561] reported RA as a potent regulator of synaptic strength in cultured neurons and brain slices. During development, all-trans retinoic acid (RA) regulates gene transcription by binding to retinoic acid receptor (RAR) proteins, which are transcription factors of the nuclear receptor family. In the nervous system, RA signaling is involved in neurogenesis and neuronal differentiation. Deficiencies in retinoid metabolism and signaling cause impaired synaptic plasticity and learning [ ] and may result in neurological diseases [565]. A recent study suggested that RA induces spine formation in cultured neurons by binding to a novel, cell surface-exposed variant of the RA-receptor RARα [566]. According to Aoto et al., the synaptic effect of RA is independent of the formation of new dendritic 100

78 spines, but instead operates by stimulating the synthesis and insertion of new postsynaptic glutamate receptors in existing synapses [561]. These results suggest that RA functions as a synaptic signal that operates via RARα during homeostatic plasticity to upregulate synaptic strength by increasing the size of the postsynaptic glutamate receptor response [561]. RA treatment revealed selective increase of mepsc amplitude but not its frequency, and it has been suggested to act postsynaptically to enhance synaptic strength in existing synapses. Examining surface GluA1 expression levels with surface protein biotinylation, revealed that either RA or TTX + APV treatment alone significantly increased the surface GluA1 level. However, application of both treatments together did not produce additional enhancement. This is in line with the electrophysiological data demonstrating that activity blockade-induced synaptic scaling occludes RA induced increase in synaptic transmission [561]. The RA-induced increase in mepsc amplitude was reversed by bath application of philanthotoxin-433, a blocker of homomeric AMPA receptors lacking the GluA2 subunit [506, 534, 548], in dissociated cultures and in hippocampal slice cultures. Tis latter finding strongly suggests that RA-induced synaptic insertion of homomeric GluA1 receptors is responsible for the observed scaling effect [561]. To examine whether RA-induced synaptic scaling depends on gene transcription or translation, Aoto et al. demonstrated that application of the protein synthesis inhibitor anisomycin blocked the RAinduced increase in mepsc amplitude in both hippocampal slice cultures and dissociated cultures. Moreover, the increase in surface GluA1 expression by RA treatment was also completely prevented by anisomycin or cycloheximide. By contrast, the transcription inhibitor actinomycin D failed to block the effects of RA on mepsc amplitude or on surface GluA1 expression. Thus, and in agreement with previous reports [506, 534, 535], RA was proposed to regulate synaptic transmission by a translationdependent but transcription-independent mechanism [561]. Combining in situ hybridization and immunocytochemistry, Grooms et al. found that GluA1 mrna was present in neuronal dendrites, consistent with previous reports [567]. Interestingly, brief treatment of synaptoneurosomes with RA specifically increased GluA1 protein level, but not other synaptic proteins such as GluA2 and PSD-95. This effect that was blocked by the protein synthesis inhibitors anisomycin and cycloheximide, but was not affected by the transcription inhibitor actinomycin D. Taken together, these results indicate that RA acts in neuronal dendrites to activate local translation of GluA1 protein in a transcription-independent manner [561]. The study by Aoto et al. [561] established that dendritic RA signaling plays an important role in homeostatic synaptic plasticity, which occurs through RARα-mediated translational regulation in dendrites [561, 568]. As RNA binding proteins are crucial for the intracellular sorting and translational 101

79 control of mrnas in dendrites [359], it has been speculated that RARα could directly associate with mrnas. Other studies suggested the implication of RARα in directly regulating translation [569]. RARα consists of six modular domains. The A/B domain, or the N-terminal activation domain, plays a role in transcriptional regulation. The C domain functions as the DNA binding domain and is adjacent to the hinge region (the D domain), which contains nuclear localization signals (NLS). The E domain contains ligand binding and C-terminal activation domains, and the function of the F domain is largely unknown [570]. RARα was shown to be transported by active nuclear export into neuronal dendrites and binds to a subset of dendritically localized mrnas, including the mrna encoding the glutamate receptor subunit, GluA1. Reporter assay of GFP flanked by the 5 and 3 UTRs of Glu1 (A1-UTR-GFP), revealed that when the GluA1 5 and 3 UTRs were not included, RARα-mediated repression was not observed, suggesting that the GluA1 UTRs participate in RARα-regulated protein translation. It has been suggest that RARα regulates GluA1 translation by UTR binding. The RA-induced increase in A1-UTR-GFP expression was blocked by the translational inhibitor anisomycin, but not by the transcriptional inhibitor actinomycin D. Together, these data indicate that RARα-mediated translational repression of GluA1 is both UTR-dependent and RA-sensitive [569] Homeostatic Synaptic Scaling (In Vivo): Activity is globally scaling quantal amplitudes in a multiplicative manner, which preserves relative differences between inputs, while allowing a neuron to adjust the total amount of synaptic excitation it receives. This process is suggested to be important, especially during development, by helping neurons to remain responsive to inputs when the number of synaptic inputs is small (early development), and later as the number rises [571]. In addition, by contributing to stabilization in firing rates, synaptic scaling may help to counteract the destabilizing effects of Hebbian synaptic modifications (Fig. 13) [503, 504, 572]. The instability arises with synaptic potentiation increases postsynaptic firing rates, leading to an increase in the correlation with presynaptic activity and resulting into a positive feedback loop that eventually saturates even weakly correlated inputs. Furthermore, synaptic scaling was suggested to contribute to the processes of synaptic competition and elimination [505, 573, 574]. Homeostatic plasticity appears to stabilize circuit function in vivo in different brain areas [497, 499, 575]. Synaptic scaling has been most thoroughly studied in vivo in the visual system, using standard visual deprivation paradigms to generate an analog of activity blockade in culture. This is exemplified in the study reporting that during the second and third postnatal weeks, as the number of excitatory synapses rises (therefore increasing mepsc frequency) in specific layers of primary visual cortex, and 102

80 visual input increases, synaptic strength is reduced through an activity-dependent homeostatic mechanism. This has been further confirmed by the observation that raising animals in the dark prevents the developmental decrease in mepsc amplitude [531]. Interestingly, the plasticity of synaptic scaling mechanism is further illustrated in observations revealing that synaptic scaling was turned off and on in spatiotemporal pattern [531, 533, 576]. It is believed that in order to understand the functional consequences of synaptic scaling, it is important to understand its underlying mechanism(s) [529]. Understanding when, where, and how homeostatic plasticity operates in the central nervous system is likely to generate important insights into how circuits adapt during experience-dependent plasticity, as well as the genesis of aberrant states, such as addiction or epilepsy, that involve adaptive plasticity or imbalances in synaptic excitation and inhibition [496] MiRNAs Regulation in Homeostatic Plasticity: Homeostatic mechanisms are required to control formation and maintenance of synaptic connections to maintain the general level of neural impulse activity within normal limits. However, the mechanisms involved in genes controlling during homeostatic synaptic plasticity are unknown. MiRNAs control of mrna stability and translation is well documented. MiRNAs rapidly and coordinately regulate stability and translation of sets of mrnas mediating specific processes [236, 388], suggesting that mirnas could have an important role in homeostatic synaptic plasticity. This is supported by previous reports revealing deregulation of mirnas and their targeted genes upon activity changes Homeostatic Synaptic Plasticity & MiRNAs (In Vitro): To our knowledge, only one study has been done investigating the role of mirnas regulation upon homeostatic synaptic plasticity [577]. The findings reveal a role for mir-485 and the presynaptic protein SV2A in homeostatic plasticity [577]. To identify mrnas that are rapidly reduced by posttranscriptional mechanisms, hippocampal neurons in culture were treated with BiC/4-AP for 5 min in the presence of a transcriptional blocker. Microarray analysis showed that the abundance of several mrna transcripts was rapidly decreased in hippocampal neurons in cell culture after increasing activity. They used a bioinformatics screen to identify mirnas sequence motifs enriched in the 3 UTR of rapidly destabilized mrnas, upon synaptic activity. This approach revealed that many of these rapidly down-regulated transcripts shared sequences in their 3 UTR for several mirnas, however, the study focused only on one of these motifs for a rare brain-enriched mirna (mir-485). 103

81 In culture, both mir-485 and the precursor mirna increased in an activity-dependent manner, after a 1-h treatment with BiC/4-AP. Authors identified that this developmentally and activity-regulated mirna (mir-485), controls dendritic spine number and synapse formation in an activity-dependent homeostatic manner. Many plasticity-associated genes containing predicted mir-485 binding sites were reported, whereas the study mainly focused on the presynaptic protein SV2A as a target of mir MiR-485 negatively regulated dendritic spine density, postsynaptic density 95 (PSD-95) clustering, and surface expression of GluA2. Furthermore, mir-485 overexpression reduced spontaneous synaptic responses and transmitter release, as measured by miniature excitatory postsynaptic current (EPSC) analysis and FM 1 43 staining. SV2A knockdown mimicked the effects of mir-485, and these effects were reversed by SV2A overexpression. Moreover, 5 days of increased synaptic activity induced homeostatic changes in synaptic specializations that were blocked by a mir- 485 inhibitor [577] Homeostatic Plasticity & MiRNAs (In Vivo): Two studies from different groups revealed in vivo mirnas regulation upon natural neuronal activity, in what might be considered as homeostatic plasticity [461, 578]. Barmack et al. [578] investigated mirna regulation in naturally evoked synaptic activity at the climbing fiber-purkinje cell synapse in the mouse cerebellar flocculus. The authors used prolonged horizontal optokinetic stimulation (HOKS) of the visual climbing fiber pathway to the cerebellar flocculus to control climbing fiber synaptic activity in a restricted region of the flocculus. Mice received 24 h of binocular horizontal optokinetic stimulation (HOKS) evoking sustained increases in climbing fiber activity to Purkinje cells in one flocculus and decreases to Purkinje cells in the other. Prolonged binocular HOKS evoked increased transcription of several mirnas. MiRNA microarray analysis was used to compare between RNA samples extracted from the right flocculi (increased climbing fiber activity) with the samples extracted from the left flocculi (decreased climbing fiber activity). Increased climbing fiber activity induced an increase of the transcription of 12 mirnas in the flocculus. Three of these mirnas (mir126, mir335 and mir361) had significant values, however, the study focused on one of them; mir-335. MiR-335 regulation was proportional to the duration of the stimulation, increasing 18-fold after 24 h of HOKS. A question has been further addressed; whether the differential transcription of mir-335 was induced not by increased climbing fiber activity in the right flocculus, but by the reduced climbing fiber activity in the left flocculus. Testing this possibility showed that monocular HOKS increased the transcription of mir-335 in the right flocculus relative to the non-stimulated left flocculus. Together the mirna microarray and the qrt-pcr data established that enhanced climbing fiber activity evoked by HOKS increases the transcription of mir-335 in the flocculus. Transcripts of mir-335 decayed to baseline within 3 h after HOKS was stopped. The authors identified mrna 104

82 targets for mir-335 using multiple screens: sequence analysis, microinjection of mir-335 inhibitors and identification of mrnas whose transcription decreased during HOKS. From 149 predicted targets, 2 genes; calbindin and θ passed these screens. This study suggests that mirna transcription could provide an important synaptic or homeostatic mechanism for the regulation of proteins that contribute to Purkinje cell plasticity [578]. The other elegant study has been performed by Krol et al. [461]. The first steps of vertebrate visual processing occur in the retina [579]. Light is converted to neural signals by photoreceptors, the more sensitive rods and the less sensitive cones, which can adapt to the changes in intensity. Information flows from photoreceptors to bipolar cells and then to ganglion cells, which then communicate with higher brain centers. Adaptation to different light levels in the retina occurs on a timescale ranging from milliseconds to hours depending on the mechanism involved. The authors investigated whether the process of light-dark adaptation in the mouse retina involves mirnas. The layered organization of the retina and the fact that retinal cells activity can be controlled in vivo by light, the physiological input, make the retina a good model to study mirnas regulation in neural circuits [461]. To obtain a global picture of small RNAs expressed in these two conditions, cdna libraries of gelpurified small RNAs were subjected to deep sequencing. Analysis of reads identified 253 retinal mirnas expressed in either dark-adapted (DA) or light-adapted (LA) states. Using combinatorial approaches, the study reported mirnas, whose levels were deregulated in LA retina. These mirnas included those encoded by the intergenic sensory neuron-specific mir-183/96/182 cluster and intronic mirnas, such as mir-204 and -211, identified previously as being expressed in the retina [ ]. Candidate mirnas showed to be downregulated during dark adaptation and upregulated in light, with rapid mirna decay and increased transcription being responsible for the changes. To gain an insight into the biological role of mirnas undergoing light-induced changes in the retina, the authors compiled a list of potential targets of mirnas from the highly expressed mir-183/96/182 cluster, using different computational target prediction algorithms. The gene encoding the voltage-dependent glutamate transporter, SLC1A1 was selected, with a potential fine-tune synaptic function in different light-adaptation states. Western blot analysis performed with different antibodies recognizing distinct sequences of SLC1A1, revealed that the level of the protein increased by up to 3.8-fold following 2- or 3 h adaptation to dark, an effect expected to accompany the decrease in mir183/96/182 levels in the dark. The decay of mirnas in retinal neurons was found to be much faster than in nonneuronal cells. Following transfer of mice to the dark, levels of mir-183/96/182, and mir-204 and -211, reached their minimum after approximately 90 min. However, upon return to light following dark adaptation for 3 h, the mirnas reached maximal levels after only 30 min. The kinetic data indicated that the more prolonged decrease in mirna levels might be due to mirna decay, while the rapid increase could result from augmented transcription and RNA processing. The high turnover is also characteristic of 105

83 mirnas in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro. Blocking activity reduced turnover of mirnas in neuronal cells while stimulation with glutamate accelerated it. These results demonstrate that mirna metabolism in neurons is higher than in most other cells types and linked to neuronal activity [461]. 106

84 2.3 MiRNA Regulation in Homeostatic Synaptic Scaling Discussion Given the complexity of most central neural circuits, maintaining all functions stable is a problem that permeates nearly every aspect of circuit development and plasticity. This remarkable feature is accomplished through a set of homeostatic plasticity mechanisms that allow neurons to sense how active they are and to adjust their properties to maintain stable function [ ]. Loosely defined, a homeostatic form of plasticity is one that acts to stabilize the activity of a neuron or neuronal circuit in response to perturbations, such as changes in cell size or in synapse number or strength, that alter excitability [496]. The best-understood form of homeostatic plasticity in the central nervous system is the synaptic scaling of excitatory synapses, which has been demonstrated both in vitro and in vivo, in spinal neurons, neocortical and hippocampal pyramidal neurons [500, 505, 523, 524, 547]. The consensus across studies from cultured hippocampal, cortical, and spinal neurons is that the main mechanism of synaptic scaling involves changes in the accumulation of postsynaptic AMPA receptors [505, 523, 524, 529, 548, 550] and NMDA receptors [496, 525, 584, 585]. Most of the work on synaptic scaling has focused on the changes in AMPA receptor expression. AMPA receptors are heterotetramers composed of subunits GluA1-4 [586], and each of these subunits confers different properties to the receptor [587] and can be regulated differently by interactions with scaffolding and signaling molecules [588]. There is general agreement that synaptic scaling is induced by changes in AMPA receptor accumulation, whereas there is less agreement on the subunit composition of the newly accumulated receptors. Several studies have reported that synaptic scaling operates on GluA2-containing AMPA receptors [523, 529, 552], but several others have reported enhanced GluA1 accumulation with smaller or absent changes in GluA2 [506, 534, 547]. This discrepancy between studies likely reflects the existence of two mechanistically distinct forms of homeostatic synaptic plasticity, a global mechanism (synaptic scaling) and a local mechanism, triggered by different modes of activity deprivation [496]. In a well-characterized paradigm, a local enhancement of homeostatic plasticity is induced when postsynaptic firing and NMDA receptors are blocked simultaneously [506]. Treatment with tetrodotoxin and NMDA receptor blockers together induces relatively selective increases in GluA1 [506, 534, 547]. Interestingly, this local enhancement of AMPA receptor accumulation has been reported to require local protein synthesis in the dendrites [506, 589], suggesting that miniature 122

85 synaptic transmission acts as a local modulator of the AMPA receptors accumulation when action potentials are also blocked [496]. MiRNAs role in neuronal gene expression has been well documented. There is growing body of data revealing involvement of mirnas in neuronal plasticity [411, 412, 470, ]. Recently, the importance of mirnas in synaptic plasticity is attracting the attention of many scientists. This is supported by the observations reporting the presence of mirnas and translational machinery in the close synaptic environment, suggesting a role of mirnas on local protein translation [227, , ]. In the present study, we hypothesized that local regulation of AMPA receptor translation in dendrites upon homeostatic synaptic plasticity may involve mirnas The Presence of mrna in Dendrites: Several studies have shown that dendrites possess the machinery required for the synthesis and trafficking of membrane proteins [ ]. MessengerRNAs have been reported to have selective subcellular localizations into the dendrites, and were shown to be involved in local protein synthesis [598]. Growing evidence shows that these mechanisms are involved in a wide range of synaptic plasticity phenomena [359, 599]. In the present study, we were able to detect the presence of GluA1 mrna in both; the hippocampal neurons soma and dendrites. This is in agreement with previous reports revealing the presence of mrnas for AMPA glutamate receptors in dendrites [355, 371, 534, 567, 598, 600, 601]. Interestingly, a recent study has reported the presence of all AMPAR GluA1-4 mrnas in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. Using several biochemical and molecular approaches, in vitro and in vivo, the authors reported that AMPAR splicing isoforms could be differentially targeted to the dendrites. Indeed, there was a difference in the presence of edited and unedited mrna variants between the cell soma and the synaptic terminal [600]. The spatial restriction of gene expression within specific cell compartments, such as synaptic spines, confers the capacity to regulate morphology and neurotransmission at the subcellular level in response to specific stimuli in neurons [359]. Further confirmation came from Ju et al. [534], who provided strong evidence for mrna presence and local dendritic synthesis of AMPARs, in isolated dendrites. GluA1 mrna deleted of its 3 UTR showed little or no dendritic translation in isolated dendrites. This indicated that the 3 UTR of GluA1 mrna contains specific targeting sequences that are required for its transport to and/or stabilization within dendrites [534]. These results came in agreement with previous reports demonstrating that the 3 UTR contain key sequences responsible for mrnas targeting and/or stability within dendrites [362, 371]. MiRNAs bind to the seed regions of targeted mrnas and interfere with their translation, by several mechanisms [8]. Endogenous mirnas have their targeted sites on UTRs and ORF of mrnas. However, experimental data revealed an enrichment of mirna binding sites in 3 UTR of mrna 123

86 coding for downregulated proteins [52, 55, 156, 157]. One reason why 5 UTRs and ORFs may be less hospitable for targeting is that the physical presence of the microribonucleoprotein (mirnp) complex bound to these regions would be displaced by the translation machinery with ribosome scanning during initiation and/or with reading of the message [8, 189]. To explore whether mirnas could regulate GluA1 mrna translation in response to activity blockade, we used 3 UTR of rat fused to a GFP cassette as a translation reporter. In contrast to untreated neurons, the GFP intensity measured in live cells increased steadily upon TTX/APV treatment and became statistically significant after 45 min exposure. This increase suggested de novo protein synthesis in dendrites, further supporting our ISH results identifying the presence of GluA1 mrna in the dendrites. In parallel, our results show an increase in surface GluA1 immunostaining after 4 hours of TTX/APV treatment. Together, these experiments suggested that mirnas binding to GluA1 3 UTR could be implicated in the homeostatic increase of GluA1 level in response to TTX/APV treatment. Some investigators raised concerns regarding the mechanism, arguing that an experiment that unequivocally demonstrates that local, rather than somatic, translation of a specific mrna is required for synapse remodeling is still missed [602]. It has been proposed that the observed GluA1 postsynaptic insertion upon activity blockade is due to newly synthesized protein that is required to bring GluA1 to the membrane. Below, we provide evidences from previous reports and from our own observations, supporting local protein translation of postsynaptic AMPARs upon activity blockade Experiments indicating Local Translation: The local synthesis and membrane insertion of glutamate receptors in dendrites has implications for many aspects of the neuronal function. For example, it has been postulated that long-term potentiation (LTP) requires postsynaptic neuronal exocytosis [603]. One mechanism that would be consistent with this is the transport of glutamate receptors to the membrane through the secretory pathway. Additionally, the work of Kang and Schuman [376], suggesting that stimulated protein synthesis in dendrites is necessary to develop LTP, is consistent with and supported by the idea that functional ionotropic glutamate receptors can be made in dendrites and function locally to modulate the responsiveness of individual synapses [598]. Smith et al. [604] have previously investigated the molecular mechanism by which dopamine influences synaptic function, in the hippocampus. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, they were able to show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. The GluA1 subunit of AMPA receptors has been identified as one of the proteins upregulated by dopamine receptor activation, with increased incorporation of surface GluA1 at synaptic sites. However, the study did not provide concrete evidence that the increase in 124

87 GluA1 protein induced by dopamine agonists is the result of local, dendritic translation [604]. Nevertheless, using quantitative immunocytochemistry, they revealed a rapid increase in dendritic GluA1 that was detected within 15 min of agonist exposure. Examining the different (e.g., proximal versus distal) regions of the dendrites, the authors found that the increase in GluA1 in the most distal region of the dendrite was equal to that observed in more proximal regions; this observation is consistent with a local source of GluA1 [604] Experiments confirming Local Translation: In a study investigating the potential role of postsynaptic sites in the modulation of cell cell communication, tagged GluA2 mrna was transfected into isolated hippocampal neuronal dendrites [598]. Data showed that tagged GluA2 subunit mrna can indeed be translated in response to pharmacologic activation of metabotropic glutamate receptors in the dendrites of neurons from primary cell cultures of rat hippocampi, and these fusion proteins can be inserted into the plasma membrane. The requirement for pharmacologic stimulation of dendritic protein synthesis suggests that mrnas localized in dendrites await appropriate stimulation as a condition for translation to occur. The localization of mrnas in dendrites and their translation response to pharmacologic or synaptic stimulation can result in proteins that can act locally or distal to the dendrites [598, 605]. A method for directly examining and comparing the local trafficking of preexisting and recently synthesized proteins in specific subcellular domains uses biarsenical dyes that bind to short sequences containing four cysteine residues. These dyes are nonfluorescent until they bind to the tetracysteine motif, at which point they become strongly green (FlAsH-EDT 2 ) or red (ReAsH-EDT 2 ) fluorescent [606]. Using this methodology, Ju et al. [534] examined and compared the dendritic trafficking of preexisting and recently synthesized AMPAR subunits. To determine how activity modifies dendritic AMPAR trafficking, the authors examined the consequences of chronic blockade of synaptic activity. This paradigm has been shown to enhance synaptic strength due, at least in part, to the accumulation of AMPARs at synapses [607] (also known as homeostatic synaptic plasticity). Activity blockade of rat cultured neurons increased dendritic GluA1, but not GluA2, levels. Examination of transected dendrites revealed that both AMPAR subunits were locally synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluA1 but not GluA2. In addition the study provided evidence that locally synthesized AMPARs could be delivered to synapses, a process that can be regulated by activity and thereby likely contributes to activity-dependent changes in synaptic strength [534]. 125

88 Two studies emerged from the same group showing the importance of retinoic acid (RA) in mediating activity blockade-induced synaptic scaling by activating dendritic GluA1 synthesis and that this process requires RARα, a member of the nuclear RA receptor family [561, 568]. The authors reported that activity blockade or RA treatment in neurons enhances the concentration of RARα in the dendritic RNA granules and activates local GluA1 synthesis in these RNA granules. Blocking neuronal activity promotes RA synthesis in neurons [561], blocking neuronal activity with TTX and APV for 30 min also significantly increased GluA1 protein level in these RNA granules, and the increase persisted for at least 2 h. The slower time course of GluA1 accumulation in RNA granules after activity blockade in comparison with direct RA treatment has been suggested to likely reflect the time required for the production and local accumulation of RA triggered by reduced activity. A translation inhibitor, anisomycin, blocked the observed changes induced by activity blockade or RA, indicating that the increase in GluA1 protein level in RNA granules is due to local synthesis of GluA1. The exposure to the transcriptional inhibitor, actinomycin D, did not affect the RA-induced increase [568]. Finally, in an elegant study investigating the ability of dendrites for local synthesis, a dual micropipette perfusion system was used to locally disrupt miniature synaptic transmission in isolated regions of dendrites. Dishes of cultured neurons were continuously perfused with saline containing TTX. A delivery micropipette was loaded with the same solution plus APV and a fluorescent dye to visualize the perfused area. A nearby suction micropipette was used to draw a stream of perfusate from the delivery pipette across isolated regions of selected dendrites and to remove the treatment perfusate from the bath. Local blockade of NMDAR minis induced a significant increase in GluA1 surface expression in the perfused region, indicating the local surface addition of GluA1. Interestingly, examining surface GluA1 expression at equidistant locations (relative to the soma) in untreated dendrites from those same cells, showed no change. Brief pretreatment with anisomycin (30 min prior to local APV perfusion) blocked the localized increase in surface GluA1 expression induced by NMDAR mini blockade. To investigate the possibility that this translation requirement is also local, dendrites were locally perfused with either anisomycin or emetine, two protein-synthesis inhibitors, during bath application of TTX + APV. Imaging of surface GluA1 expression revealed a significant reduction in the perfused region relative to all other dendritic segments, demonstrating a requirement for local dendritic protein synthesis. Taken together, these experiments suggest that the control of local protein synthesis by ongoing miniature synaptic transmission regulates the surface population of GluA1 subunits. Together, these results demonstrate that NMDAR mini blockade induces a protein synthesis-dependent synaptic insertion of AMPARs that lack the GluA2 subunit [506]. Importantly, the authors were able to reproduce the data from acute hippocampal slices, where the intrinsic hippocampal circuitry is largely preserved [506]. 126

89 Previous work showed that dendrites often contain the core elements of the secretory pathway necessary for the synthesis and transport of integral membrane proteins, specifically ER and Golgi [362, 371, 594, 595, 608, 609]. Furthermore, isolated dendrites can incorporate sugar precursors indicative of Golgi function [597], and surface AMPARs can be detected after transfection of isolated dendrites with GluA2 mrna [598]. These results as well as direct imaging of ER-to-Golgi transport in dendrites [594] suggest that integral membrane proteins, and not merely cytoplasmic proteins, can be locally synthesized in dendrites [534] GluA1 mrna Regulation upon Activity Blockade: MiRNAs regulatory mechanism was shown to be determined by the degree of complementary between loaded mirna and its target [38, 166, 168, 179, 211, 212]. Once incorporated into a cytoplasmic RISC, the mirna will specify cleavage if the mrna has sufficient complementarity to the mirna, or it will repress productive translation if the mrna is imperfectly complementarity to mirna [166, 179, 211]. Here, we investigated the regulatory mechanism by which mirnas exert their control, on GulA1 mrna. It is noteworthy that the rat GluA1 mrna 3 UTR was not available on databases and thus, we used combinatory approaches including degenerated primers to successfully amplify the rat GluA1 mrna 3 UTR from rat genomic DNA. Amplified region was further sequenced and aligned, allowing us to have closer look at rat GluA1 mrna 3 UTR. Applying qrt- PCR, we did not observe any change in the relative expression of GluA1 mrna levels in neurons treated with TTX/APV compared to untreated neurons. This indicated that mirna(s) regulate GluA1 mrna through translational repression, but not degradation Messenger GluA1 mirna(s) Targeting Prediction and Candidate MiRNAs: We then use computational approaches to identify potential mirnas targeting the 3 UTR of GluA1 mrna. Target prediction sets are typically ranked, with the assertion that the better scoring predictions are more likely to be authentic or effective [52]. When considering current predictions from mirbase Targets [610], miranda [181, 611], PicTar [182, 327], PITA [185] and TargetScan [55, 171], all of which use site conservation as a prediction criterion, TargetScan was reported as one of those performing the best. TargetScan predictions are ranked by total context score, which is based on site type, site number and site context [52]. In addition, when assessed with available proteomics results, the higher-ranked predictions of several tools trend toward better performance, with the most robust discrimination observed for TargetScan rankings [52]. When evaluated independently, each of the parameters used to rank TargetScan predictions site conservation, site number, site type (with 127

90 8mer > 7mer-m8 > 7mer-A1), and site context correlate with targeting efficacy [6, 52, 53, 56, 171, 172]. Using TargetScan, 9 mirnas were predicted to target GluA1 mrna (Gria1 gene). However, upon applying qrt-pcr, we were able to detect only four mirnas (mir-92a, -92b, -128 and -182) in the brain. To identify which mirnas varied upon homeostatic scaling, we quantified the levels of mirnas extracted from cultures treated or not with TTX/APV. Results revealed that mir-92a, -92b and -182 levels were decreased upon TTX/APV application, whereas no change was detected in mir-128 level. Recently, mir-96/182 regulation on targeted mrna encoding Glypican-3, a heparan-sulfate proteoglycan has been investigated [612]. MiR-96 and mir-182 belong to the mir-182/183 cluster; their seed region (5 -UUGGCA-3, nucleotides 2 7) is identical suggesting potential common properties in mrna target recognition and cellular functions. Surprisingly, although mir-96 and mir- 182 carry the same seed, mir-182 had no effect on GPC3 protein expression, nor mrna level. In silico, mir-96:gpc3 3 -UTR pairing is defined as an 8-mer site by Targetscan, whereas mir- 182:GPC3 3 -UTR interaction is defined as 7-mer-1A [171]. The only difference between these two types of site is that in mir-96, the nucleotide at position 8 immediately downstream to the seed can pair the GPC3 3 -UTR (G C pair), whereas that of mir-182 cannot. To assess mir-182 functioning, the authors studied its regulatory effect on Adenylate cyclase type 6 (ADCY6), one of its validated target [583]. MiR-182 downregulated expression of ADCY6 mrna and protein. Noticeably ADCY6 3 -UTR is predicted to contain one 8-mer site for mir-182. Hence, the hypothesis that pairing of the target with the seed+nucleotide 8 might be a prerequisite to yield a stable mirna:mrna complex has been tested. The guanosine (G) of GPC3 3 -UTR, which normally matches a C at position 8 of mir- 96, was mutated in U. Results showed that mutation abrogated the regulation of egfp-gpc3 by mir- 96. In addition, it allowed mir-182 to target the GPC3 3 -UTR and control egfp-gpc3 expression. By site-directed mutagenesis, they demonstrated that the mirna nucleotide 8, immediately downstream the UUGGCA seed, plays a critical role in target recognition by mir-96 and mir-182. This differential regulation was confirmed on two other targets, FOXO1 and FN1 [612]. Since mir- 182 seed region in the sequence of rat GluA1 3 UTR obtained from sequenced rat genome revealed to be similar to that of GPC3, we decided to exclude this mirna from further analysis. Comparing the effect of TTX/APV treatment on the 2 candidate mirnas, we observeda more pronounced decrease on mir-92a than on mir-92b. MiR-92a and mir-92b are members of the same family. Several families of mirnas have been identified, whose members have common 5 sequences but differ in their 3 ends. Brennecke et al. [37] proposed a model where 5 dominant canonical and seed sites should respond to all members of a given mirna family, whereas 3 compensatory sites should differ in their sensitivity to different mirna family members depending on the degree of 3 128

91 complementarity. The study used assay with 3 UTR reporter transgenes and overexpression constructs for various mirna family members. The study examined the 3 UTR reporters of the pro-apoptotic genes grim [154], containing sites in their 3 UTRs that are complementary to the 5 ends of the mir-2, mir-6, and mir-11 mirna family [153, 154]. These mirnas share residues 2 8 but differ considerably in their 3 regions. The site in the grim 3 UTR is predicted to form a 6mer seed match with all three mirnas, but only mir-2 shows the extensive 3 complementarity that was predicted to be needed for a 3 compensatory site with a 6mer seed to function ( 19.1 kcal/mol, 63% maximum 3 pairing, versus 10.9 kcal/mol, 46% maximum, for mir-11 and 8.7 kcal/mol, 37% maximum, for mir-6). Indeed, only mir-2 was able to regulate the grim 3 UTR reporter, whereas mir-6 and mir-11 were non-functional. Taken together, these experiments indicate that transcripts with 5 dominant canonical and seed sites are likely to be regulated by all members of a mirna family. However, transcripts with 3 compensatory sites can discriminate between mirna family members [37]. Here, the interaction between GluA1 mrna and mir-92a or mir-92b was predicted using intrna tool (v1.2.5). Thermodynamic parameters are moderately in favor of a mir-92a-glua1 interaction (lower hybridization energy compared to mir-92b). In addition, we used MiRTif online free software ( that is designed as a post-processing filter that takes mirna:target interactions predicted by other target prediction softwares such as TargetScan, PicTar and miranda as inputs, and determines how likely the given interaction is a real or a pseudo one [613]. Testing sequence obtained from the rat GluA1 3 UTR, with each of mir-92a and mir-92b gave real interaction. However checking the support vector machine (SVM) scores revealed to be higher for mir-92a predicted interaction than for mir-92b ( and ; respectively). SVM produces prediction scores that have no units. The discriminant score is proportional to the sample's distance from the hyperplane. So a large positive value implies high confidence that the sample lies in the positive class. Taken together, we decided to focus further on mir-92a regulatory on GluA1 expression. We were able to confirm mir-92a binding affinity to GluA1 3 UTR mrna using reporter assay. MiR-92a significantly decreased the luciferase signal compared to mutated vector. It is worth to note that we verify mir-92a/glua1 mrna interaction, yet, we do not reject the possibility of mir- 182 and mir-92b regulation on GluA1 mrna Regulatory Role of mir-92a on GluA1 upon Activity Blockade: Then, we addressed the question of whether mir-92a could modulate endogenous AMPAR. In untreated neurons, we found that the expression of mir-92a slightly but not significantly reduced the surface GluA1 staining. This might indicate that mir-92a does not alter the basal level of AMPAR expression. In agreement with previous studies, treatment of neurons expressing empty vector with 129

92 TTX/APV, increased GluA1 synaptic staining. Strikingly, treatment of mir-92a-transfected neurons with TTX/APVdid not induced any increase in synaptic GluA1 staining. These results suggest that the effects of mir-92a on GluA1 regulation are complex, and likely influenced by yet undetermined variables such as mirna to target-mrna stoichiometry. Thus, one could hypothesize that in basal condition mir-92a level is already high to control GluA1 translation and that additional mir-92a would have no effect. Indeed, it is well accepted that mirnas exert more a fine tuning of gene expression than a switch on / switch off regulation. In contrast, when the neuronal system is challenged, like in the homeostatic plasticity paradigm, and if mir-92a is decrease, then exogenous mir-92a supply could affect strongly GluA1 translation. Although within different context, the ability of mirna to exert its regulation without alteration of the basal level has been previously reported. MiRNA-132 is a neurotrophin-induced mirna that has been demonstrated to affect neuronal characteristics such as neurite outgrowth and cell excitability. Because of its documented ability to regulate cellular characteristics in an activity dependent manner [417, 418, 422, 614], a role for mir-132 in synaptic function was investigated [420]. Overexpression of mir-132 in cultured hippocampal neurons led to selective changes in short-term synaptic plasticity. Interestingly, overexpression of mir-132 increased the paired-pulse ratio and decreases synaptic depression in cultured mouse hippocampal neurons without affecting the initial probability of neurotransmitter release, the calcium sensitivity of release, the amplitude of excitatory postsynaptic currents or the size of the readily releasable pool of synaptic vesicles. The authors suggested that mir-132 can modulate the computational properties of hippocampal neurons by regulating short-term plasticity in ways that promote facilitation and/or reduce synaptic depression without affecting basal synaptic transmission. The molecular mechanism of this effect remains elusive, and future studies will be aimed at evaluating candidate mrna targets of mirna-132 that might mediate this phenotype [420]. In parallel to live GluA1 immunostaining we did patch-clamp recordings of AMPAR-mediated miniature EPSCs (AMPARs-mEPSCs). In untreated neurons, expression of mir-92a had no significant effect on the amplitude of AMPARs-mEPSCs, compared to neurons expressing empty vector. However, TTX/APV treatment of neurons expressing empty vector increased AMPARmEPSCs amplitude but not the frequency. This is in line with previous results [505, 523, 607], where mepsc amplitude (but not frequency) was significantly increased in activity-blockaded cultures [506, 534]. Interestingly, TTX/APV treatment was also accompanied by a decrease in mepsc rise-time and decay-time constants, which has previously been linked to the synaptic incorporation of GluA1 homomers [506, 534, 535, 547, 568, 615],. Importantly, rescued expression of mir-92a blocked the increases in mepsc amplitude, as well as the kinetic changes induced by TTX/APV treatment. 130

93 Homeostatic adjustments in excitatory synaptic strength require that neurons sense some aspect of activity and translate these changes into compensatory changes in synaptic strength. The standard paradigms to induce synaptic scaling postsynaptically include blockade or enhancement of network activity in culture [505, 523, 524, 547, 548] and sensory deprivation in the intact animal [ ]. Below, we discuss two reports investigated the modulation of mirnas in intact animals upon activity deprivation in two different sensory systems [461, 578]. These observations indicate that increased activity was associated with upregulation of mirnas, while there was a high mirna turnover upon activity blockade. The first study [578] investigated the deregulation of mirna upon naturally evoked synaptic activity at the climbing fiber-purkinje cell synapse in the mouse cerebellar flocculus. Mice received binocular horizontal optokinetic stimulation (HOKS) evoking sustained increases in climbing fiber activity to Purkinje cells in one flocculus and decreases to Purkinje cells in the other. Increased climbing fiber activity increased transcription of 12 mirnas in the flocculus [578]. The second study demonstrated that levels of the mir-183/96/182 cluster, mir-204, and mir-211 are regulated by different light levels in the mouse retina. Concentrations of these mirnas were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes. The study also provided evidence for a faster decay of mirnas in retinal neurons than mirnas in nonneuronal cells. The high turnover is also characteristic of mirnas in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro [461]. Our data demonstrate that mir-92a selectively regulates GluA1 translation and is necessary for synaptic scaling associated with incorporation of new GluA1 receptors during activity blockade in hippocampal neurons. This novel type of regulation may cooperate with other mechanisms underlying synaptic scaling, including retinoic acid signaling [561, 569], β-camkii activation [616], and the mir- 485 presynaptic target SVA [577]. 131

94 3. MIRNAS REGULATION IN BONE CANCER PAIN 3.1 Pain Introduction: Pain has been previously described by Charles Darwin as a homeostatic emotion, essential for the survival of species [617]. Later on, it has been defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage (IASP, 1986) (Fig. 14) [618]. The sensation of pain alerts us to real or impending injury and triggers appropriate protective responses [619]. The nervous system is able to detect different range of stimuli; including thermal, mechanical, and chemical irritants [620]. Pain sensation does not only involve the detection of noxious stimuli, it includes as well cognitive and emotional processing by the brain [619]. The perceptions and interpretations of pain sensation are markedly affected by the subject s mood, attentiveness, personality, and past experience [621]. Fig. 14. Descartes s depiction of nerve transmission in a man perceiving the heat of an open fire via the sensory nerves of his toe [622]. De L Homme. Paris: Charles Angot) [Mackie Family Collection in the History of Neuroscience, University of Calgary]. However, pain might outlive its usefulness as an acute warning system and instead becomes debilitating [619, 620]. Upon intense noxious stimuli, the nervous system pathway underlying pain sensation might exhibit maladaptive plasticity that leads to enhancing pain signals and producing hypersensitivity [620]. In such cases, pain can take on a disease character in pathological states such as 132

95 inflammation, neuropathy, cancer, viral infections, chemotherapy and diabetes [623]. In the nervous system, pain syndromes can be initiated or maintained at peripheral (primary sensory neurons) and/or central loci (spinal cord and brain) [620]. Pain can be acute or chronic, each with different clinical nature. Acute pain is provoked by a specific disease or injury, serves a useful biologic purpose, is associated with skeletal muscle spasm and sympathetic nervous system activation, and is self-limited. Chronic pain, in contrast, may be considered a disease state. It is pain that outlasts the normal time of healing, if associated with a disease or injury. Chronic pain may arise from psychological states, serves no biologic purpose, and has no recognizable end-point [624]. Other types of pain are characterized by spontaneous ongoing or shooting pain and evoked amplified pain responses after noxious or non-noxious stimuli [623, ]. The profound differences between pain types, demonstrate that pain is not generated by an immutable, hardwired system, but rather results from the engagement of highly plastic molecules and circuits [620] Nociception: The somatosensory system functions can be divided into Exteroceptive functions, Propriceptive functions, and Interoceptive functions. The sensation of wide range of stimuli (touch, temperature, and pain) is included in the Exteroceptive functions. Exteroceptive functions have been further divided into (1) mechanoreception, through which all non-painful mechanical stimuli are sensed; (2) thermoreception, composed of heat and cold; and (3) nociception, the sensation of both burning pain and sharp pain [628]. All sensory systems must convert environmental stimuli into electrochemical signals. In the case of vision or olfaction, primary sensory neurons need only detect one type of stimulus (light or chemical odorants). In this regard, nociception is unique because individual primary sensory neurons of the pain pathway have the remarkable ability to detect a wide range of stimulus modalities (Fig 14) [619, 629]. Nociception is the activation of specialized primary afferent neurons that activate specific regions of the CNS devoted to pain processing, are called nociceptors [621]. Compared with sensory neurons of other systems, nociceptors must therefore be equipped with a diverse repertoire of transduction devices [619, 629]. These receptors are adapted for rapid response upon stimulus of sufficient intensity to cause or indicate existing tissue damage [630]. They are not localized in a particular anatomical structure; where their terminals that detect painful stimuli are found dispersed over the body (innervating skin, muscle, joints and internal organs) [619]. Furthermore, their cell bodies are located in the dorsal root ganglia (DRG) for the body and the trigeminal ganglion for the face [620]. All DRG neurons have a common feature, they do not give rise to dendrites and do not receive synaptic input [621]. They have two processes, a peripheral and central axonal branch that 133

96 innervates their target organ and the spinal cord, respectively [620]. Upon activation, they transmit the information from their peripheral terminals to their central terminals in the spinal cord or brain stem, via action potentials and neurotransmitter release [621, 630]. Figure 15. Nociceptive, inflammatory and neuropathic pain. (a) Noxious stimuli are transduced into electrical activity at the peripheral terminals of unmyelinated C-fiber and thinly myelinated Aδ-fiber nociceptors by specific receptors or ion channels sensitive to heat, mechanical stimuli, protons and cold. This activity is conducted to the spinal cord and, after transmission in central pathways, to the cortex, where the sensation of pain is experienced. (b) Damaged tissue, inflammatory and tumor cells release chemical mediators creating an 'inflammatory soup' that activates or modifies the stimulus response properties of nociceptor afferents. This, in turn, sets up changes in the responsiveness of neurons in the CNS. (c) Neuropathic pain arises from lesions to or dysfunction of the nervous system. Conditions affecting the peripheral nervous system, as in carpal tunnel syndrome, the spinal cord after traumatic injuries or the brain after stroke, can all cause neuropathic pain, which is characterized by a combination of neurological deficits and pain. Adapted from Scolz and Woolf [631] 134

97 3.1.2 Classification of Primary of Afferents: Primary afferent nerve fibers are highly specialized with regard to the stimulus modality and stimulus intensity that elicits a response (Fig 16). Afferents that respond to low modality and stimulus intensities are thought to encode innocuous percepts such as touch, cooling, or warmth. Afferents that respond at high stimulus intensities are thought to encode pain sensation [630]. Mechanoreceptors are those with low threshold, they are myelinated afferents that are sensitive to slight deformation of the skin. They have been differentiated by their response to stepped indentations of the skin and the end-organ structure associated with their terminal [630]. Another subpopulation of afferents is unmyelinated afferents. They are exquisitely sensitive to gentle warming of their punctate receptive fields, and called warm fibers. Warm fibers are thought to encode the quality and intensity of warmth sensation [ ]. Similarly, there is a subpopulation of the thinly myelinated Aδ-fibres [635] or unmyelinated C-fibres afferents [636] that respond selectively to gentle cooling stimuli and are thought to encode cooling perception [630]. Nociceptors have a high threshold for activation preferentially to intense, noxious stimuli, and considered polymodel, because of their ability to respond to multiple stimulus modalities [630]. Nociceptors can be classified depending on physiological criteria: 1. The conduction velocity of their parent axon (i.e. unmyelinated C-fibres afferents versus myelinated A-fibres). 2. The stimulus modalities that evoke a response (i.e. mechanical, heat, or chemical). 3. The temporal characteristics of their response to a stimulus modality (rapid versus slow response). 135

98 Figure 1 Different nociceptors detect different types of pain. a, Peripheral nerves include small-diameter (Aδ) and medium- to large-diameter (Aδ,β) myelinated afferent fibres, as well as small-diameter unmyelinated afferent fibres (C). b, The fact that conduction velocity is directly related to fibre diameter is highlighted in the compound action potential recording from a peripheral nerve. Most nociceptors are either Aδ or C fibres, and their different conduction velocities (6 25 and ~1.0 m s 1, respectively) account for the first (fast) and second (slow) pain responses to injury. Panel a adapted from [619], b from [637]. (1) C-fibres mechano-heat nociceptors: Unmylinated afferents responsive to mechanical and heat stimuli C-fibres mechano-heat nociceptors (CMHs) are probably the most commonly described class of cutaneous nociceptors. They have high mechanical and heat thresholds [630, 638]. CMHs adapt markedly to heat stimulus. Most CMHs also respond to chemical stimuli, such as acid or capsaicin, the pungent ingredient in hot chilli peppers, and can therefore be considered polymodal [630, 639]. Activity in CMHs is thought to lead to the perception of burning pain [630]. However, not all C fibers are nociceptors, with some responding to cooling, and innocuous stroking of the hairy skin. Yet, they do not respond to heat or chemical stimulation. These latter fibers appear to mediate pleasant touch [640]. (2) A-fibers nociceptors: A-fibers nociceptors are thought to sense pricking pain, sharpness, and perhaps aching pain. Three distinct types of A-fibers nociceptors exist [641]. Type I and type II A-fibers nociceptors are typically responsive to heat, mechanical, and chemical stimuli and may therefore be referred to as AMHs or polymodel nociceptors [630, 639]. Another group of A-fiber nociceptors are unresponsive to heat stimuli and have been called High Threshold Mechanoreceptors (HTMs) by many investigators [642, 643]. The heat thresholds for type I A-fiber nociceptors are high (typically >53 C) for short duration (less than 1 sec.) [630]. In addition, if the heat stimulus is maintained, these afferents will respond at lower temperatures [620]. However, they have relatively low mechanical thresholds. Type I are seen in hairy and glabrous skin [630]. Type II A-fibres nociceptors respond well to short duration (less than 1sec) heat stimuli with heat thresholds of around 47 C [641]. In contrast to their low temperature thresholds, they have a relatively higher mechanical threshold and many do not respond to mechanical stimuli. Type II nociceptors are seen only in hairy skin [630]. These myelinated afferents differ considerably from the larger diameter and rapidly conducting Aβ fibers that respond to innocuous mechanical stimulation (i.e., light touch) [620]. It has long been assumed that Aδ and C nociceptors mediate first and second pain, respectively, namely the rapid, acute, sharp pain and the slowly conducted, more diffuse, dull pain evoked by noxious stimuli [629]. 136

99 (3) Mechanically insensitive afferents: These Mechanically Insensitive Afferents (MIAs) have a very high mechanical threshold [ ]. They have also been called silent or sleeping nociceptors due to their propensity to become sensitized after injury [644, 647]. The majority of type II A-fibers nociceptors are MIAs, whereas the majority of type I A-fiber nociceptors are Mechanically Sensitive Nociceptors (MSAs). C-fibers MIAs are more responsive to chemical stimuli than C-fibers MSAs [648] and may account for some forms of chemogenice pain [649]. Recent evidence indicates that this population of afferents is responsive to prolonged application of intense mechanical stimuli [650] indicating that MIAs are not necessarily unresponsive to mechanical stimuli [630] The Neurochemistry of Nociceptors: Nociceptive neurons express diverse molecular mechanisms, by which noxious stimuli that impinge upon their terminals are transduced into membrane depolarization [630]. Components involved in the detection of noxious chemical stimuli: Tissue injury triggers the production and liberation of an array of diverse ions, nucleotides, lipids, amino acid derivates, and proteins capable of activating nociceptors or augmenting nociceptor responses to mechanical and thermal stimuli. Certain agents (e.g. protons and capsaicin) directly depolarize nociceptive neurons by triggering the opening of cation channels permeable to sodium and/or calcium. In contrast, agents such as bradykinin and nerve growth factors act on G proteincoupled receptors and tyrosine kinase receptor, respectively, to trigger intracellular signaling cascades that in term sensitize depolarizing cation channels to their respective physical or chemical regulators. Still, other agents (e.g. glutamate, acetycholine, and adenosintriphosphate) activate both ion channels and G protein-coupled receptors to produce a spectrum of direct and indirect effects on nocicepptor membrane potential [630]. Ion channels gated by vanilloid compounds: An ion channel activated directly by capsaicin (the hydrophobic compound that lends hot peppers their pungency) and other vanilloid compounds (TRPV1, previously known as VR1) has been identified and found to be selectively overexpressed in a subset of small to medium diameter nociceptive neurons [651]. Blockade of the vanilloid receptor with the relatively selective antagonist, capsazepine [652], or targeted deletion of the TRPV1 gene in mice [653, 654], eliminates vanilloid sensitivity in vitro and in vivo [630]. 137

100 Ion channels gated by acid: Nociceptive neurons can also be activated by reduction in extracellular ph, a circumstance often observed in the context of tissue injury, inflammation, or ischemia [655]. One group of ion channels implicated in acid-evoked nociception is the acid-sensing ion channel (ASIC) family of proteins [656]. At least three homologus isoforms ASIC1 (aka ASIC, BNaC2), ASIC2 (aka BNC1, mdeg, BNaC1), and ASIC3 (aka DRASIC)- are expressed in nociceptive neurons, and multiple splice variants of ASIC1 and ASIC2 have been identified [657, 658]. Another potential site of proton action is the vanilloid receptor, which can be activated directly by protons and whose capsaicin sensitivity can also be augmented by modest reduction in ph [659, 660]. G protein-coupled receptors (GPCRs) involved in nociceptive signal transduction: Many of the chemical substances that bath the nociceptor terminal following tissue injury or inflammation modulate neuronal activity through their actions on metabotropic receptors coupled to intracellular, heterotrimeric, guanyl nucleotide regulatory proteins (G proteins). Examples of such substances include the peptide, bradykinin [661]; the lipid, prostaglandin E2 [662]; and even certain proteases [663]. Occupancy of GPCRs triggers a diverse array of downstream target proteins that include ion channels, enzymes such as adenylyl cyclase and phospholipase C, and other signaling molecules. These can exert profound effects on the ion channels expressed at nociceptor terminals. Furthermore, it should be noted that while many substances (e.g. bradykinin, PGE2) increase nociceptor excitability when they bind their respective GPCRs, other substances that act on GPCRs (e.g. opiate peptides, cannabinoids) can inhibit nociceptor excitability and blunt pain sensation [664, 665]. Neurotrophin and cytokine signaling at the nociceptor terminal: Among the most important regulators of nociceptor excitability are the neurotrophin proteins. These polypeptides fall into two main classes- the nerve growth factor (NGF) family (NGF, BDNF, NT3, NT4/5) and the glial cell line-derived neurotrophin factors (GDNF) family (glial cell line-derived neurotrophic factor, artemein, persphrin, neurturin) [666, 667]. Each of the two classes of neurotrophins binds to a corresponding class of multi-subunit cell-surface neurotrophin receptor proteins. The resulting activation of tyrosine kinase domains on these receptors leads to the coordinate stimulation of several intracellular enzymes, including phospholipase C and phosphatidylinositol 3- kinase. Through these signaling pathways, neurotrophins play critical roles in the development and survival of nociceptive neurons. In addition, however, neurotrophins can acutely enhance nociceptor excitability and nociceptive signal transduction [668]. Components involved in transduction of painful thermal stimuli: Ion channel gated by heat: there are two populations of non-selective cationic channels in a subset of neurons (C-fiber nociceptors and the type II A-fiber nocicptors) that can be activated by increasing 138

101 ambient temperature to >43 C and >52 C, respectively [ ]. These include vanilloid receptors TRPV1, that can be activated not only by capsaicin or protons, but alternatively by heat at temperature greater than 43 C [651, 660]. Another protein that might be responsible for some of the TRPV1- independent heat transduction is TRPV2 (previously known as VRL-1 or GRC) a TRPV1 homolog highly expressed by subset of medium- to large-diameter neurons that can be activated by temperatures exceeding 52 C [672]. Two other TRPV1 homologs TRPV3 [ ] and TRPV4 [676] can also be activated by heat, both exhibit a threshold for activation (~34 C) below the noxious range, making their potential contribution to thermal nociception unclear [630]. Ion channels gated by cold: Several ion channels expressed in sensory neurons are gated by decreases in temperature. Among these amiloride-sensitive sodium channels of the Epithelial Sodium Channel (ENaC) family, which can be activated by cold [677, 678]; potassium leak channels such as TREK1, whose opening is inhibited by cold [679]; and two non-selective cation channels of the TRP family, TRPM8 [Cold- and Menthol-sensitive Receptor (CMR-1)] [680] and TRPA1 [Ankyrin-like with Transmembrane domains Protein 1 (ANKTM1)] [681]. Components involved in transduction of painful mechanical stimuli: Intense mechanical stimuli such as pinching or pressing the skin are examples of perhaps the most familiar cause of pain. A number of candidate mechanosensory ion channels have been identified, including members of the ASIC [682, 683], TRP [684], and TREK [685] families. After conversion of the noxious stimuli into an electrical signal (i.e. membrane depolarization), this information is then transmitted to the spinal cord. In a process that includes additional steps, each of them requires a distinct set of ion channels precisely positioned at discrete sites throughout the plasma membrane. The interrelationship between cytoarchitecture and ion channel composition or properties of nociceptors results in an extraordinary capacity to encode the spatial, temporal, and intensity properties of noxious stimuli [630] Central Projections of the Nociceptor: Somatosensory information reaches the spinal cord and areas of the brainstem through primary afferent sensory fibres (Fig 17) [ ]. The dorsal horn contains four different neuronal components [630]: 1. The central terminals of primary afferents 2. Neurons with long ascending axons that project to the brain (projection neurons) 3. Intrinsic spinal neurons (interneurons, many of which have axons that terminate locally) 139

102 4. Axons that descend from various parts of the brain, including certain monoaminergic nuclei in the brainstem. Primary afferent nerve fibers project to the dorsal horn of the spinal cord, which is organized into anatomically and electrophysiological distinct laminae [629]. They terminate within the ten designated laminae (I-X) of the spinal cord in a highly reproducible and characteristic fashion depending upon their diameter, biochemical composition, and receptive field properties. The superficial dorsal horn, is known to be the most involved in nociceptive transmission, with relatively good knowledge about its neuronal organization. However, the deeper laminae of the dorsal horn are also important in pain: many cells in the deeper laminae (including projection neurons) respond to acute noxious stimuli, and the low-threshold mechanoreceptive afferents which terminate in this region are responsible for some of the symptoms of neuropathic pain [630]. Figure 17. Connections between Primary Afferent Fibers and the Spinal Cord. There is a very precise laminar organization of the dorsal horn of the spinal cord; subsets of primary afferent fibers target spinal neurons within discrete laminae. The unmyelinated, peptidergic C (red) and myelinated Aδ nociceptors (purple) terminate most superficially, synapsing upon large projection neurons (red) located in lamina I and interneurons (green) located in outer lamina II. The unmyelinated, nonpeptidergic nociceptors (blue) target interneurons (blue) in the inner part of lamina II. By contrast, innocuous input carried by myelinated Aβ fibers (orange) terminates on PKCγ expressing interneurons in the ventral half of the inner lamina II. A second set of projection neurons within lamina V (purple) receive convergent input from Aδ and Aβ fibers. Adapted from Basbaum et al [620]. Lamina I of the dorsal horn is also known as the marginal layer [620, 630], neurons within this lamina are generally responsive to noxious stimulation (via Aδ and C fibers) [620]. Lamina II is known as the substantia gelatinosa. Those two laminae are collectively referred to as the superficial part of the dorsal horn [630]. Nociceptive information is relayed to spinal cord neurons throughout the dorsal horn, particularly laminae V-VII, in addition to laminae I-II, but largely avoids populations of neurons within laminae III and IV [620, 630]. Neurons in laminae III and IV are primarily responsive to 140

103 innocuous stimulation (via Aβ), and neurons in lamina V receive a convergent nonnoxious and noxious input via direct (monosynaptic) Aδ and Aβ inputs and indirect (polysynaptic) C fiber inputs. The latter are called wide dynamic range (WDR) neurons, in that they respond to a broad range of stimulus intensities [620]. Neurons within laminae I and II and V and VI receive nociceptive information through unmylinated C fibers and finely myelinated Aδ sensory afferents [620, 630]. C afferents can be divided into two major neurochemical types: those which contain neuropeptides (e.g. substance P, somatostatin, and calcitonin gene-related peptide) and those which do not [688]. There are differences between the termination regions of these two types of unmyelinated afferent: the peptidergic ones arborize mainly in lamina I and the outer part of lamina II (lamina II0), whereas those that lack peptides innervate the central part of lamina II [690]. In contrast, projection neurons in lamina I receive almost exclusively peptidergic C-fibres input from the body. Notably, both lamina I and lamina V neurons, potentially receive all types of nociceptive input [620, 630]. The great majority of neurons in each lamina of the dorsal horn are interneurons, with axons that remain in the spinal cord. Many of these cells have axons that are restricted to the spinal segment which contains the cell body, and these sometimes referred to as local circuit neurons. However, there is also evidence that a significant number of dorsal horn interneurons have longer intraspinal connections [630]. A basic functional distinction can be made between excitatory and inhibitory interneurons. The major inhibitory transmitters in the dorsal horn are GABA and glycine, and there is evidence from studies in various parts of the central nervous system that these amino acids are highly enriched in neurons that use them as a neurotransmitter. Glutamate is the main transmitter used by excitatory interneurons in the spinal cord, as well as by primary afferents and projection neurons, and it is likely that most dorsal horn neurons that do not use GABA or glycine as their transmitter are glutamatergic [630] Neuronal Transmission and Receptors: The last critical step for afferents in the rapid transmission of nociceptive information is the release of neurotransmitter at central synapses. Release of transmitter from neuronal terminals requires an increase in the concentration of free cytosolic Ca+². Voltage-gated Ca+² channels are a major source of calcium in many neurons, including sensory neurons. Functionally, this translates to four main currents that can be distinguished on the basis of pharmacological sensitivity and biophysical properties; the conotoxin sensitive (or N-type) current, dihydropyridine sensitive (or L-type) currents, 141

104 currents sensitive to agitoxin (P-type), and currents insensitive to any of the three primary channel blocker (R/Q type). Each of these channel types is differentially distributed among subpopulations of sensory neurons [630]. Several receptors have been identified on primary afferent terminals in the spinal dorsal horn. These include neurotransmitter/modulator receptors as well as receptors for neurotrophic factors. These include; ionotrpic glutamate receptors of the AMPA, metabotropic glutamate receptors of NMDA receptors, GABAA receptors, GABAB receptor 1, opioid receptors, neurokinin 1 (NK1), both nicotinic and muscarinic cholinergic receptors, Alpa-2-adrenergic receptors, vanilloid receptor (VR1), the purinergec receptor P2X3, the three high-affinity receptors for the neurotrophin family trka, trkb, trkc, the low affinity neurotrophin receptor p75 [630] The Ascending Pain Pathways: Ascending tract include axons of the central processes of dorsal root ganglion cells, as well as axons of subset of spinal neurons (Fig 18). These projected axons transmit pain messages to higher brain centres, including the reticular formation, thalamus and ultimately the cerebral cortex [621, 629]. They make synapses in several nuclei in a pattern that is characteristic for each tract [621]. 142

105 Figure 18. Anatomy of the Pain Pathway. Primary afferent nociceptors convey noxious information to projection neurons within the dorsal horn of the spinal cord. A subset of these projection neurons transmits information to the somatosensory cortex via the thalamus, providing information about the location and intensity of the painful stimulus. Other projection neurons engage the cingulate and insular cortices via connections in the brainstem (parabrachial nucleus) and amygdala, contributing to the affective component of the pain experience. This ascending information also accesses neurons of the rostral ventral medulla and midbrain periaqueductal gray to engage descending feedback systems that regulate the output from the spinal cord. Adapted from Basbaum et al [620] Neurons that project to the brain are not uniformly distributed within the dorsal horn. There is a relatively high concentration in lamina I and scattered projection cells are present throughout the deeper lamina (III-VI) [630]. Projection neurons within laminae I and V constitute the major output from the dorsal horn to the brain [629]. These neurons are at the origin of multiple ascending pathways, including the spino-thalamic and spino-reticulothalamic tracts, which carry pain messages to the thalamus and brainstem, respectively. The former is particularly relevant to the sensorydiscriminative aspects of the pain experience (that is, where is the stimulus and how intense is it?), whereas the latter may be more relevant to poorly localized pains [620]. In the lumbar segments, very few lamina II neurons have supraspinal projections. Dorsal horn projection neurons send their axons to several brain regions, including the thalamus and hypothalamus, the midbrain periaqueductal grey matter, the lateral parabrachial area in the pons, various parts of the medullary reticular formation, and the nucleus of the tractus solitaries [691]. More recently, attention has focused on spinal cord projections to the parabrachial region of the dorsolateral pons, because the output of this region provides for a very rapid connection with the amygdala, a region generally considered to process information relevant to the aversive properties of the pain experience [620]. The lateral spino-thalamic tract projects multimodal sensory inputs from spinal Wide-Dynamic Range (WDR) neurons to the lateral thalamus and has been implicated in processing sensory and discriminative aspects of pain. By contrast, the medial part of the spino-thalamic tract and the spino-parabrachial tract project to the medial thalamus and limbic structures and are believed to mediate the emotional and aversive components of pain. The experience of pain is perceived in the cortex, and information is accordingly sent to the spinal cord to enable withdrawal from the noxious stimulus [623]. The projections have common features in terms of the lamina location of their cells of origin (laminae I and III-VI) and the course of their axons, which generally cross the midline and ascend in the ventral and lateral funiculi of the spinal white matter. In addition, many neurons send their axons to more than one of these supraspinal targets. This group of tracts is therefore sometimes referred to as the anterolateral system [630]. From these brainstem and thalamic loci, information reaches cortical structures. There is no single brain area essential for pain [692]. Rather, pain results from activation of a distributed group of 143

106 structures, some of which are more associated with the sensory-discriminative properties (such as the somatosensory cortex) and others with the emotional aspects (such as the anterior cingulate gyrus and insular cortex). Recently, imaging studies demonstrated activation of prefrontal cortical areas, as well as regions not generally associated with pain processing (such as the basal ganglia and cerebellum). Whether the extent of the activation of these regions is more related to the response of the individual to the stimulus, or to the perception of the pain is not clear [620]. Other ascending pathways from the dorsal horn: are ascending tracts from the dorsal horn that are independent of the anterolateral system. These include the postsynaptic dorsal column (PSDC) pathway and spino-cervical tract, with axons that travel to the dorsal column nuclei and the lateral cervical nucleus, respectively. Both have been forum to originate mainly from cell bodies in laminae III-IV [630] Descending Inputs: Descending tracts are those that carry regulatory influences from the brain to spinal neurons (Fig 18). Descending axons originate in the cerebral cortex and in subcortical regions, and terminate on neurons of the spinal cord [621]. Several pathways project from the brain to the dorsal horn of the spinal cord, and of these, the ralphe-spinal serotoninergic system and the noradrenergic projection from the pons have received particular attention because of their involvement in pain mechanisms. Serotonincontaining axons originating from cells in the nucleus raphe magnus are found throughout the dorsal horn, but are concentrated in lamina I and the outer part of lamina II [630] Plasticity of Nociception: Plasticity at the level of neurons in nociceptive pathways is seen as an increase in the magnitude of responses to a sensory stimulus, an increase in spontaneous activity or after discharges activity. These might represent continued activity after the termination of a nociceptive stimulus, leading to central amplification of pain (central sensitization) [693, 694]. Furthermore, the peripheral receptive fields of neurons can expand, allowing hyperalgesia to spread to uninjured regions [623]. Hyperalgesia is characterized by a leftward shift of the stimulus-response function that relates magnitude of pain to stimulus intensity [620, 630]. In other words, normally painful stimuli elicit pain of greater intensity [620]. Allodynia corresponds to the lowering of pain threshold such that normally innocuous stimuli (e.g. light touch or warmth) become painful [620, 630]. The neurophysiological correlate of hyperalgesia and allodynia [619] is sensitization. Peripheral sensitization is a lowering of 144

107 primary afferent nociceptor activation thresholds that occur at and near the site of injury. Central sensitization is the increase in responsiveness of spinal cord pain transmission neurons [619, 630]. The processes involved in this mechanism will be discussed in more details below. Figure 19. Central Amplification of Pain (Sensitization). Adapted from Kuner R. [623]. Central sensitization phenomenon differs from windup; central sensitization is concerned with the facilitation that manifested after the end of the conditioning stimuli, and that once triggered remained autonomous for some time, or only required a very low level of nociceptor input to sustain it [695]. The net effect of multiple processes in dorsal horn nociceptive transmission neurons is a progressive increase in the action potential discharge elicited by each successive stimulus in a train of lowfrequency stimuli - a phenomenon known as windup [696]. Windup is thus a form of activitydependent plasticity that manifests over the course of a stimulus and terminates with the end of stimulation [630]. Results from in vivo experiments suggested that windup also depends on the amplification properties of spinal neurons, the triggering of which requires previous activation of NMDA receptors. In contrast to windup, sensitization of central pain pathway neurons outlasts, by up to many hours, the duration of the nociceptor inputs that initiates it [697]. These inputs cause the engagement of multiple intracellular signaling cascades that were dormant before activation, leading to an orchestrated modification of neuronal behavior consisting of enhanced excitatory postsynaptic responses and depressed inhibition, equivalent to increased excitability or a facilitation of the neurons [630] Peripheral Mechanisms of Plasticity: Peripheral Nociceptive Plasticity following Inflammation: Injury heightens our pain experience by increasing the sensitivity of nociceptors to both thermal and mechanical stimuli [619]. Tissue damage is often accompanied by the accumulation of endogenous 145

108 factors released from activated nociceptors or nonneural cells that reside within or infiltrate into the injured area (including mast cells, basophils, platelets, macrophages, neutrophils, endothelial cells, keratinocytes, and fibroblasts) (Fig. 20) [620, 698]. Resulting inflammation leads to the dynamic production, release, and destruction of numerous chemical substances capable of producing spontaneous nociception firing, shifting the thresholds for nociceptor activation and increasing the responsiveness of these neurons to normally noxious thermal and mechanical stimuli [630]. Collectively, these factors, referred to as the inflammatory soup, [620]. Inflammatory soup products represent a wide array of signaling molecules, including neurotransmitters, peptides (substance P, CGRP, bradykinin), eicosinoids and related lipids (prostaglandins, thromboxanes, leukotrienes, endocannabinoids), neurotrophins, cytokines, and chemokines, as well as extracellular proteases and protons [619, 620]. Importantly, nociceptors sensitivity to temperature or touch is heightened, through their ability to express one or more cell-surface receptors, capable of recognizing and responding to these proinflammatory or proalgesic agents [620]. In addition, the ability of nociceptors to release peptides and neurotransmitters (for example, substance P, calcitonin-gene-related peptide and ATP) from their peripheral terminals upon activation, facilitate inflammatory soup production, by promoting the release of factors from neighboring non-neuronal cells and vascular tissue, a phenomenon known as neurogenic inflammation [629]. Below, they are discussed in more details. Figure 20. Peripheral Mediators of Inflammation Tissue damage leads to the release of inflammatory mediators by activated nociceptors or nonneural cells that reside within or infiltrate into the injured area, including mast cells, basophils, platelets, macrophages, neutrophils, endothelial cells, keratinocytes, and fibroblasts. This inflammatory soup of signaling molecules includes serotonin, histamine, glutamate, ATP, adenosine, substance P, calcitonin-gene related peptide (CGRP), bradykinin, eicosinoids prostaglandins, thromboxanes, leukotrienes, endocannabinoids, nerve growth factor (NGF), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), extracellular proteases, and protons. These factors act directly on the nociceptor by binding to one or more cell surface receptors, including G proteincoupled receptors (GPCR), TRP channels, acid-sensitive ion channels (ASIC), two-pore potassium channels (K2P), and receptor tyrosine kinases (RTK), as depicted on the peripheral nociceptor terminal. Adapted from [620]. 146

109 1) Extracellular protons and tissue acidosis: Local tissue acidosis is an hallmark physiological response to injury, and the degree of associated pain or discomfort is well correlated with the magnitude of acidification [699]. A number of proton-sensitive channels are found on sensory neurons, two main candidates have emerged VR1 and a family of ASICs [619]. 2) Peptides and growth factors: Tissue damage promotes the release or production of bioactive peptides from non-neural cells and plasma proteins at the site of injury [700], especially, nonpeptide bradykinin [701]. In contrast to its roles in embryonic neurons, in the adult, NGF acts on primary sensory nerve terminals to promote thermal hypersensitivity [702]. NGF binds to TrkA tyrosine kinase receptors on peptidergic sensory neurons to activate mitogen-activated protein (MAP) kinase and Phospholipase C (PLC)-signaling pathways [703]. Injury promotes the release of numerous cytokines, chief among them interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) [704]. Although there is evidence to support a direct action of these cytokines on nociceptors, their primary contribution to pain hypersensitivity results from potentiation of the inflammatory response and increased production of proalgesic agents (such as prostaglandins, NGF, bradykinin, and extracellular protons) [620]. 3) Sensitization and activation by lipids: Cyclooxygenase (COX) enzymes that convert arachidonic acid, a lipid messenger, into proinflammatory prostanoid products, notably prostaglandin E2 (PGE2). Most studies indicate that PGE2 contributes to peripheral sensitization by binding to G-protein-coupled receptors that increase levels of cyclic AMP within nociceptors [700]. However, it now seems likely that cyclooxygenase products are also present in the spinal cord, where they could interact with receptors on the central terminals of nociceptors [705]. This idea has aroused great interest because it suggests that COX inhibitors may exert their pain-relieving effects by modulating nociception at both peripheral and central sites [706]. Nociceptor plasticity includes mainly two types of mechanisms; the first, rapid onset peripheral sensitization (or desensitization) of nociceptive terminals, this type includes above mentioned processes. A second form of nociceptor plasticity includes the slower onset of phenotypic changes in nociceptor properties as a consequence of regulation of the gene expression [630]. Such regulation affects many aspects of nociceptor function including: genes coding for neurotransmitters that are released with activity from the central terminals of nociceptors; genes coding for receptors which are transported to both the peripheral and central terminals of nociceptors; genes coding for ion channels expressed throughout the neuron and potentially affecting its sensitivity. Genes regulating structural proteins in nociceptors are also affected and this may alter anatomical features of these neurons [630, 707]. 147

110 There are several potentially important signals for this plasticity of gene expression. NGF was found to be up-regulated in several experimental models of inflammation and in some clinical inflammatory disorders [ ]. It has been suggested that the NGF/trkA complex maintains autophosphorylation and activates transcription factors such as CREB and Oct-2 that control gene expression [714, 715]. Furthermore, other inflammatory mediators might drive abnormal gene expression in nociceptors. The upregulation of cytokines LIF, TNFα, and IL1β in some injury states, alter nociceptor phynotype and may be necessary for some inflammation-induced plasticity [ ]. Another possible mechanism for transcriptional regulation of nociceptors is neuronal activity itself. It has been reported that electrical stimulation of peripheral axons nociceptors at high frequency led to increased expression of BDNF mrna in DRG cells [719]. The expression of substance P and BDNF has been found to dramatically increase in sensory neurons innervating tissues, subjected to experimental inflammation with either carrageenan or Freund s adjuvant [709, 711, ]. A variety of receptors have been shown to up-regulate in primary sensory neurons, upon inflammation. These include G-protein coupled receptors such as bradykinin B1 and B2 receptors and the mu opioid receptor [ ]. Some ionotropic receptors such as TRPV1 and P2X3 are also up-regulated in inflammatory conditions [727, 728]. VR1 is down-regulated after axotonomy [729]. The up-regulation of these receptors may increase the sensitivity of peripheral terminals to appropriate ligands, and may establish a positive feed-back circuit to maintain this upnormal state. These receptors are also transported to the central terminals of nociceptive neurons, where their presynaptic activation may regulate nociceptor transmitter release. TTX-resistent sodium channels can regulate the excitability of afferent nerve terminals [730, 731] and, since these channels are largely restricted to nociceptors, they act as an important peripheral determinant of pain thresholds to all forms of stimulation. They are found to be regulated in inflammatory as well as neuropathic pain [630] Peripheral Nociceptor Plasticity following Nerve Injury: Nerve injury is frequently associated with abnormal pain sensations, neuropathic pain states. In humans, nerve injury may be metabolic (e.g. in diabetes), infectious (e.g. following HIV), druginduced (e.g. some anti-cancer drugs such as taxol), or traumatic. In animal models there are two different populations of sensory neurons that need to be considered. First, the neurons whose axons are damaged and that are therefore disconnected from peripheral targets. Secondly, the neurons that remain intact but have their axons intermingling with those degenerating in the distal nerve [630]. 148

111 One cause of plasticity is the altered availability of target-derived neurotrophic factors. In adulthood, the survival of sensory neurons does not depend upon these peripheral factors, but trophic factors such as NGF and GDNF continue to exert powerful effects on nociceptor gene expression. When the axon of a sensory neuron degenerates following injury, the cell loses contact with its peripheral target and loses supply of target-derived factors such as NGF [630]. Schwann cells in the damaged nerve start to produce some of these factors, but this surrogate supply is insufficient to replace that normally derived from target [732, 733]. It is perhaps therefore not surprising that many of the changes in gene expression in damaged nociceptors are the exact opposite of those occurring with tissue inflammation (when target-derived factors are up-regulated). Many of the neurotransmitters/modulators constitutively expressed by nociceptors (such as subsntance P, CGRP and BDNF) are down-regulated in damaged neurons [702, 729, 734]. Associated with this down-regulation, there is a reduced release of these agents in response to nociceptor activity [735]. Similarly, there is a down-regulation of TRPV1 and P2X3 receptors in damaged nociceptors. There is also a down-regulation of TTX-transient sodium channel subunits Nav1.8 and Nav 1.9 [736]. There are, however, some increases in nociceptor gene expression that are rather unexpected on the basis of altered trophic factor availability. One of these is the dramatic up-regulation of the peptide, galanin, in a great number of the damaged afferents, including most of the nociceptive population and many of the large cells [737]. Injury leads to an associated up-regulation of the transcription factor ATF3 [738]. It is possible that in addition to negative factors (i.e. reduced neurotrophic factor support), positive factors (i.e. increased cytokines) may contribute to nociceptor plasticity after damage. Such a mechanism may explain the dynamic pattern of up-regulation exhibited by the shwan cell mitogen, Reg-2, after nerve injury [739]. Several cytokines are known to be released in damaged nerves [740, 741] and may act to induce peptide expression in particular subpopulations of DRG cells. One transcriptional change that may be of functional importance is the altered expression of Nav1.3 in damaged neurons. It is not normally expressed in adult DRG but rapidly up-regulated following nerve damage [736]. Several changes in gene expression have been observed in intact nociceptors after partial nerve lesions. These include the up-regulation of substance P, TRPV1 [742], BDNF [743], and P2X3 [744]. One report [736] demonstrates an increase in Nav1.8 mrna by PCR in spared afferents, but another recent study found no change in the number of Nav1.8 immunopositive cells in intact axons in several models of partial nerve injury [745]. The ability of ERK1 and ERK2 to phosphorylate ion channels probably mediates only the acute component of ERK-induced hyperalgesia. However, ERK1 and ERK2 can also induce long-lasting changes in pain sensitivity by migrating to the cell nucleus and acting on gene transcription. Moreover, camp and CamKIV are also synapse-to-nucleus communicators and thereby recruit a chronic memory component for pathological pain [746]. In the cell nucleus, camp and ERK trigger the activation of the camp response element (CREB), which drives the expression of a variety of 149

112 pain-related proteins, such as COX-2, transient receptor potential vanilloid-1 (TRPV1) and calcium channels. Another transcriptional repressor, DREAM, acts constitutively to suppress prodynorphin expression in spinal cord neurons [623, 747]. Knocking out DREAM results in sufficient dynorphin expression to produce a strong reduction in generalized pain behavior, highlighting the role that intracellular molecules play in modulating pain gating in the spinal cord [748] Central Mechanisms of Plasticity: Pain can be produced by activity in non-nociceptive primary sensory fibers, which is the case in central sensitization [619]. Central sensitization is the process through which a state of hyperexcitability is established in the central nervous system, leading to enhanced processing of nociceptive messages [694] (Fig 21). Figure 21. Central Sensitization. (1) Glutamate/NMDA receptor-mediated sensitization. After intense stimulation or persistent injury, activated C and Aδ nociceptors release a variety of neurotransmitters, including glutamate, substance P, calcitonin-gene related peptide (CGRP), and ATP, onto output neurons in lamina I of the superficial dorsal horn (red). As a consequence, normally silent NMDA glutamate receptors located in the postsynaptic neuron can now signal, increase intracellular calcium, and activate a host of calcium-dependent signaling pathways and second messengers including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and Src. This cascade of events will increase the excitability of the output neuron and facilitate the transmission of pain messages to the brain. (2) Disinhibition. Under normal circumstances, inhibitory interneurons (blue) continuously release GABA and/or glycine (Gly) to decrease the excitability of lamina I output neurons and modulate pain transmission (inhibitory tone). However, in the setting of injury, this inhibition can be lost, resulting in hyperalgesia. Additionally, disinhibition can enable nonnociceptive myelinated Aβ primary afferents to engage the pain transmission circuitry such that normally innocuous stimuli are now perceived as painful. This occurs, in part, through the disinhibition of excitatory PKCγ expressing interneurons in inner lamina II. (3) Microglial activation. Peripheral nerve injury promotes release of ATP and the chemokine fractalkine that will stimulate microglial cells. In particular, activation of purinergic P2-R receptors, CX3CR1, and Toll-like receptors on microglia (purple) results in the release of brain-derived neurotrophic factor (BDNF), which through activation of TrkB receptors expressed by lamina I output neurons, promotes increased excitability and enhanced pain in response to both noxious and innocuous stimulation (that is, hyperalgesia and allodynia). Activated microglia also release a host of cytokines, such as tumor necrosis factor α (TNFα), interleukin-1β and 6 (IL-1β, IL-6), and other factors that contribute to central sensitization. Adapted from Basbaum et al. [620]. 150

113 There has been great deal of interest in changes of the neurochemical organization of the dorsal horn which occur in pathological conditions, since they may contribute to chronic pain states. Many neurochemical changes have been demonstrated following nerve injury and in inflammatory pain states. Some of these affect primary afferents, while others involve neurons within spinal dorsal horn. 1) Neuropeptides: The expression of neuropeptides in the central terminals of dorsal horn, are differently regulated following peripheral nerve injury [734]. This includes downregulation of most of the peptides that are normally expressed in fine-diameter afferents, such as substance P, CGRP, and Somatostatin, and up-regulation of galanin. However, the levels of mrnas for CGRP, substance P, and galanin in dorsal root ganglion cells are shown to be increased in inflammatory conditions. These results suggest that there is an increase in the rate of synthesis of these peptides by primary afferents. Furthermore, vasoactive intestinal polypeptide (VIP) and NPY that are either not expressed by intact primary afferents or are present only in a small number, appear de novo after nerve injury. However, NPY and VIP are apparently not up-regulated in primary afferents in inflammatory model of tissue inflammation [ ]. In addition, in peripheral inflammation conditions, there is dramatic increase in the level of preprodynorphin mrna in spinal cord neurons of lamina I and V, as well as more modest increases in the mrnas for encephalin, galanin, and substance P [ ]. 2) Neuropeptide receptors: NK1 receptor has been shown to increase in the dorsal horn, as a result of inflammation or nerve injury [ ]. Changes have also been reported for the levels of Mu-Opioid Receptor (MOR-1) and Y1 receptor in the dorsal horn. MOR-1has been found to increase in the ipsilateral dorsal horn upon inflammation and neuropathic models, however, reduced in another neuropathic model consisting in a tight ligation of the sciatic nerve [757]. MOR-1 expression was found to increase in dorsal root ganglia [758] upon inflammation. Also, Y2 receptor expression in the dorsal horn found to be up-regulated in response to inflammation and nociception [759]. Although numerous mechanisms involve in central sensitization, several studies focused on the alteration in glutamatergic excitatory system, loss of inhibitory controls, and glialneuronal interactions [620]. 3) Inhibitory Controls: The net output of the spinal dorsal horn represents a balance between diverse excitatory processes and spinal inhibitory interneurons, which can be disrupted in pathological states [623]. The basic scientific background of the initial spinal cord stimulation 151

114 trials was based on the gate control theory of Melzack and Wall [760]. It has been demonstrated in multiple studies that dorsal horn neuronal activity caused by peripheral noxious stimuli could be inhibited by concomitant stimulation of the dorsal columns [761]. GABAergic or glycinergic inhibitory interneurons are highly expressed in the superficial dorsal horn [620]. They have been shown to significantly contribute to pain inhibition; they represent the basis of the longstanding gate control theory of pain [760]. Both the synthetic enzyme GAD, and GABA are increased in the dorsal horn ipsilateral, in response to a peripheral inflammation [762, 763]. It has been suggested that the increase of GABA (the major inhibitory transmitter), may partially compensate for the increases activation of dorsal horn neurons that results from the inflammation [630]. However, it has been shown that peripheral injury leads to a decrease in inhibitory postsynaptic currents, leading to enhanced depolarization and excitation of projection neurons. This disinhibition enhances spinal cord output in response to painful and nonpainful stimulation, contributing to mechanical allodynia [764, 765]. Glycinergic signaling modulation can also lead to disinhibition of neurons in the spinal cord. Upon tissue injury, the activation of EP2 receptors expressed by excitatory interneurons and projection neurons; result in stimulation of the camp-pka pathway. This pathway phosphorylates GlyRa3 glycine receptor subunits, rendering the neurons unresponsive to the inhibitory effects of glycine. Accordingly, mice lacking the GlyRa3 gene have decreased heat and mechanical hypersensitivity in models of tissue injury [620]. 4) Glutamatergic excitatory system: in the CCI model of neuropathic condition, AMPA receptors have been found increased in the medial part of the dorsal horn on the ipsilateral side [766]. The increase of neurotransmitters release from nociceptors will sufficiently depolarize postsynaptic neurons to activate quiescent NMDA receptors. This will lead to exacerbate responses to noxious stimuli (that is, generate hyperalgesia), as a consequence to strengthened synaptic connections between nociceptors and dorsal horn pain through the increase in calcium influx [620]. Furthermore, this mechanism contributes to the condition in which innocuous stimulation of areas surrounding the injury site can produce pain. This is involving Aβ afferents in pain transmission circuits, resulting in mechanical allodynia [620]. 5) Other compounds: The level of nitric oxide synthase is upregulated in the spinal following nerve injury, contributing to the maintenance of pain [767]. However, it does not alter in peripheral inflammatory conditions [757]. PKCγ in the dorsal horn is up-regulated in inflammatory conditions [768]. Although, it has been stated that there is no change in its expression as a results of nerve injury [630], an earlier report demonstrating deletion of the 152

115 gene encoding PKCγ in the mouse leads to a marked decrease in its expression in sensory nerve fibers. Furthermore, changes in the projection neurons might contribute to the disinhibitory process. Peripheral nerve injury downregulates the K+-Cl cotransporter KCC2, which is expressed in lamina I projection neurons, resulting in a shift in the Cl gradient. Hence, leading to activation of GABA-A receptors depolarizes, rather than hyperpolarizes these neurons, resulting in enhancing excitability and increase pain transmission [620, 769]. Importantly, complexity is added by the fact that plasticity not only occurs at a functional level, but can also take place at a structural level. Examples of this include an increase or a decrease in the density of synaptic spines, degeneration or regeneration of axons leading to aberrant connectivity, degeneration of neurons and proliferation of astrocytes and microglia, which influence nociceptive processing by releasing modulatory substances. Importantly, structural plasticity can account for the long term persistence of changes that arise in pathological pain states [623] In addition, modulation can be achieved by structural modifications of nociceptive pathways. Structural plasticity can occur at various anatomical and temporal scales [623] Glial-Neuronal Interactions: Glia outnumbers neurons in the CNS by about 10:1. Recently, a growing body of evidence is changing the previous view of glia as just a supportive element in the CNS. They have been shown to be involved in physiological and pathophysiological processes in the CNS [770, 771]. Under normal conditions, microglia function as resident macrophages of the central nervous system, and was found to be homogeneously distributed within the gray matter of the spinal cord [620]. Microglia in the dorsal horn, are shown to be activated with peripheral nociceptive stimulation [630]. They are found to accumulate in the superficial dorsal horn within the termination zone of injured peripheral nerve fibers [620]. Their activation leads to the release of several cytokines (such as TNF-α and IL-1β and -6) which enhances neuronal central sensitization and nerve injury induced persistent pain [620]. Furthermore, activated microglia was found to be cellular intermediators in mechanical alloynia following peripheral nerve injury. Their activation appeared to be sufficient to trigger the persistent pain condition [772]. Since microglia are activated after nerve injury, but not inflammatory pain, it has been suggested that physical damage of the peripheral afferents is inducing the release of specific signals that are detected by microglia. Chief among these is ATP, which targets microglial P2-type purinergic receptors. Of particular interest are P2X4 [772], P2X7 [773], and P2Y12 [774, 775] receptor subtypes [620]. It has 153

116 been proposed that ATP/P2X4- mediated activation of microglia leads to the release of the brainderived neurotrophic factor (BDNF) [776], which triggers a mechanism of disinhibition [620]. Activated microglia, like peripheral macrophages, are shown to release and respond to numerous chemokines and cytokines, contributing to central sensitization. The chemokine fractalkine (CX3CL1) is expressed by both primary afferents and spinal cord neurons [ ]. However, their receptor (CX3CR1) is expressed on microglial cells and upregulated after peripheral nerve injury [777, 779]. Furthermore, Fractalkine is cleaved from the neuronal surface prior to signaling, an action that is carried out by the microglial-derived protease, cathepsin S [780]. Blockade of CX3CR1 or inhibition of Fractalkine cleavage are shown to reduce injury induced persistent pain [ ]. Several members of the Toll-like receptor (TLRs) family have also been implicated in the activation of microglia following nerve injury. TLRs are transmembrane signaling proteins expressed in peripheral immune cells and glia. Genetic or pharmacological inhibition of TLR2, TLR3, or TLR4 function, results in decreased microglial activation and reduction of hypersensitivity triggered by peripheral nerve injury [ ]. Moreover, peripheral injury activates glia not only in the spinal cord, but also in the brainstem, where glia contribute to supraspinal facilitatory influences on the processing of pain messages in the spinal cord, a phenomenon named descending facilitation [785, 786]. Nociceptive stimulation has been shown to activate astrocytes in the dorsal horn [630, 785]. Astrocyte activation is represented by an enhancement of the expression of astrocyte-specific intermediate filament, GFAP [787, 788]. Counteracting astrocyte activation was found to suppress the enhancement of nociceptive behaviours in models of inflammatory [789, 790] and neuropathic [791]. In contrast to microglia, astrocyte activation is generally delayed and persists much longer. It has been proposed that astrocytes are more critical to the maintenance, rather than to the induction of central sensitization and persistent pain [620]. 154

117 3.2 Bone Cancer Pain Introduction: Pain is the cancer-related event that is the most disruptive to cancer patient s quality of life. In 20 50% of all cancer patients, pain is reported as the first symptom of disease. Furthermore, 75 90% of advanced or terminal cancer patients must cope with chronic pain syndromes related to failed treatment and/or tumor progression [792]. There are two types of cancer pains that are considered the most difficult to control; those related to (i) invasion of peripheral nerves and (ii) destruction of bone. Both of them are found to be accompanied in bone cancer pain, and accounting for 75% of all chronic cancer pain [ ]. Bone cancer pain can develop in patients with primary bone cancer (sarcoma or hematologic malignancies), or from metastatic tumors that are mostly originating from breast, lung, ovarian, and prostate cancers [798, 799]. Although there is quite an important variation in the type, severity, and evolution of bone cancer pain, between patients, there are two common types of pain that are generally recognized [800]. The first type is usually described as a dull aching or throbbing pain that increases in severity over time, known as ongoing pain [801]. With increased tumor progression and bone destruction, the pain intensifies resulting in the second type of cancer pain, known as breakthrough [802]. Breakthrough pain is also known as movement-evoked or episodic pain, which emerges frequently over time, and which is more acute in nature. It has been defined as a transitory exacerbation of pain experienced by the patient who has relatively stable and adequately controlled baseline pain [801, 803, 804]. This type of pain has been divided into spontaneous pain at rest and incident pain (either volitional or non-volitional) [618, 804]. Breakthrough pain has been reported to be present in 75% of cases of cancer-induced bone pain [805], and is frequently difficult to effectively manage [806]. This pain is so depleting in a sense that it greatly interferes with important aspects of patients life; such as mood, relationships, sleep, activity, walking ability, work, and quality of life [805]. The negative impact that cancer pain has on the quality of life cannot be overestimated. As advances in the disease detection and treatment are extending the life expectancy of cancer patients, increasing attention has been directed toward improving patients quality of life [807] Models of Bone Cancer Pain: Although the significant efforts in cancer therapy and diagnosis, the basic neurobiology of cancer pain is still poorly understood [807]. Previously, the majority of gained knowledge about the neurochemistry of cancer was obtained from clinical studies and aimed at a better management of pain in patients with cancer [802]. Recently, developed animal models are contributing in understanding 155

118 bone cancer pain underlying mechanisms [618, 807]. These models were found to accurately replicate the symptoms of cancer-induced bone pain in humans [808], in addition to their plastic nervous system, which respond to varying stimuli [809]. All these contributed in giving validity to the comparisons of cancer-induced bone pain in animal models and humans [808]. There are several commonly used in vivo mouse models to study tumor-induced bone destruction [810]. Intracardiac injection of cancer cells has been used as a model for bone cancer pain. In this model, tumor cells are injected into the left ventricle of the heart and then spread to multiple sites including the bone marrow where they grow and induce remodeling of the surrounding bone [811, 812]. Although the advances of this model in replicating the observations of bone of metastatic cancer, a major problem with this model is the variability in the sites, size, and extent of the metastasis between animals. Furthermore, assessing the nociceptive state of bone pain in this model is difficult, because of the ability of the tumors to metastasize to vital organs of the animal, such as the lung or liver, which sustain the general health of the animal. Given these problems, intracardiac injection of cancer cells as a model for bone cancer pain has proven to be difficult [810]. Another bone cancer pain model has been developed, based on canine prostate tumor cells (ACE-1) [813]. Prostate cancer is unique in that bone is often the only clinically detectable site of metastasis, and frequently inducing bone pain. This pain can be difficult to fully control as it seems to be driven simultaneously by inflammatory, neuropathic, and tumorigenic mechanisms. This model includes injection and confining the ACE-1 to the intramedullary space in the femur of nude mice [813]. Cancer cells, then induce significant formation of new woven bone at the proximal head, distal end, and the diaphysis of the bone. This marked bone formation is also found to be accompanied by bone destruction, giving the tumor-bearing femur a unique scalloped appearance. This appearance is consistent with what is observed in human patients with prostate tumor metastases [810]. There is well accepted animal model of bone cancer pain that has been developed by Schwei et al [802]. This murine model of bone cancer pain share many features of human bone cancer-induced pain. The development of the model involves intramedullary injection of the osteolytic sarcoma cell line cells into the femur. A crucial component of this model is that the tumour cells are confined to the marrow space of the injected femur and do not invade adjacent soft tissues [802]. These cells aggressively destroy bone, with the developed tumor invading the periosteum, providing localized pathological findings found in human osteolytic bone cancer [ ]. An important feature of that original experimental model is that sarcoma cells that had destroyed bone and extended into adjacent soft tissue caused pain, whereas sarcoma cells confined to the musculature of the thigh did not [817]. The animal model is accompanied by painful behavior that has been shown to correlate with the extent of bone destruction [802]. Although osteosarcoma cells were the first cells used in this model, other 156

119 animal and human tumor cells, including prostate, breast, melanoma, colon, and lung tumors, have now been used in the closed femur model of bone cancer pain [818] Pain Development and Maintenance: Bone is densely innervated by both sensory and sympathetic nerve fibers within bone marrow, mineralized bone, and periosteum [819, 820], and these fibers are mainly associated with blood vessels [619, 821, 822]. Since sensory and sympathetic neurons are present within all three structures, all are potentially impacted by fractures, ischemia, or the presence of tumor cells and may play a unique but coordinated role in the generation of bone cancer pain [800]. However, this sensory innervation is found occasional in the tumor, and when present, it is usually associated with the blood vessels that vascularize the tumor [823] (Fig 22). 157

120 Figure 22. Schematic showing factors in bone (A) and receptors/channels expressed by nociceptors that innervate the skeleton (B) that drive bone cancer pain. A variety of cells (tumor cells and stromal cells including inflammatory/immune cells, osteoclasts, and osteoblasts) drive bone cancer pain (A). Nociceptors that innervate the bone use several different types of receptors to detect and transmit noxious stimuli that are produced by cancer cells (yellow), tumor-associated immune cells (blue), or other aspects of the tumor microenvironment. There are multiple factors that may contribute to the pain associated with cancer (B). The transient receptor potential vanilloid receptor-1 (TRPV1) and acid sensing ion channels (ASICs) detect extracellular protons produced by tumor induced tissue damage or abnormal osteoclast-mediated bone resorption. Tumor cells and associated inflammatory (immune) cells produce a variety of chemical mediators including prostaglandins (PGE2), nerve growth factor (NGF), endothelins (ET-1) and bradykinin (BK). Several of these proinflammatory mediators have receptors on peripheral terminals and can directly activate or sensitize nociceptors. It is suggested thatmovement-evoked breakthrough pain in cancer patients is partially due to the tumor-induced loss of the mechanical strength and stability of the tumor-bearing bone so that normally innocuousmechanical stress can now produce distortion of the putative mechanotransducers (TRPV1, TRPV4, and TRPA1) that innervate the bone. Adapted from Jimenez-Andrade et al. [810]. Although, the prevailing opinion is that bone pain arises predominately, if not exclusively, from the periosteum [ ], this does not take into account the pain perceived by patients in whom the pathology is confined principally to the bone marrow or mineralized bone, where there is no significant periosteal involvement [827] Inflammatory Point of View: Most osteolytic tumors release a variety of factors that induce excessive osteoclast activity [ , 828, 829]. In addition, in most of developed cancers, tumour mass is made up of many cell types other than cancer cells, including immune-system cells such as macrophages, neutrophils, T-lymphocytes, fibroblasts, and endothelial cells [807, 810]. Tumor and/or tumor stromal cells are known to secrete various factors that sensitize or directly excite primary afferent neurons, and include prostaglandins [830, 831], bradykinin [810], tumour necrosis factor-α (TNF-α) [ ], endothelins [836, 837], interleukin (IL)-1 and IL-6 [832, 838, 839], epidermal growth factor [840], transforming growth factor-β [841, 842], platelet derived growth factor [810, ]. In addition to nerve growth factor (NGF) [846, 847], with many of the these factors receptors expressed by primary afferent neurons [807]. NGF gained a significant interest in the etiology of bone cancer pain, because of their direct sensory neurons activation, expressing their cognate TrkA receptor [810, 848]. All these secreted factors may contribute to the pain associated with cancer. Both the intracellular and extracellular ph of solid tumours are lower than that of surrounding normal tissues [849]. Osteoclasts resorb bone by maintaining an extracellular microenvironment of low ph ( ) at the bone site [802, 850]. There are several mechanisms by which tumours could cause a decrease in ph, including the release of protons by invading inflammatory cells, and the apoptosis occurring in the tumour environment as apoptotic cells release intracellular ions to create an acidic 158

121 environment [807]. With the progression of bone resorption, the stromal cells might progressively become in contact with the sensory nerve fibers innervating the bone. Many of these fibers are shown to express acid-sensing ion channels [851], that can be directly sensitized and/or excited in these conditions [802, 849, 850, 852]. The two main classes of acidsensing ion channels, expressed by nociceptors, are vanilloid receptor subtype 1 (VR1) [651, 660] and the acid-sensing ion channel-3 (ASIC3) [851, 853, 854]. Although both channels are sensitized and excited by a decrease in ph, VR1 is activated when the ph falls below 6.0, whereas the ph that activates ASIC3 seems to be highly dependent on the co-expression of other ASIC channels in the same nociceptor [657] Neuropathic Point of View: Tumour cells growth in the bone marrow space leads to the compression and distortion of both the haematopoietic cells that normally comprise the marrow, and the sensory fibers that normally innervate the marrow and mineralized bone [807]. Myelinated and unmyelinated primary afferent sensory fibers are present at and within the leading edge of the tumor [800]. However, in the deep stromal regions of the tumor, sensory nerve fibers display a discontinuous and fragmented appearance and ultimately are undetectable by microscopy [800]. All these might result in nerves injures, causing mechanical injury, compression, ischaemia or direct proteolysis [801]. Nevertheless, tumours themselves are not highly innervated by sensory neurons [823, 855, 856]. Proteolytic enzymes that are produced by the tumour cells can also cause injury to sensory and sympathetic fibers, causing neuropathic pain [807]. In addition, tumor-induced injury of sensory nerve fibers is accompanied by an upregulation of activating transcription factor 3 (ATF3) and galanin by sensory neurons that innervate the tumor-bearing femur [857]. In addition to stromal released factors, as the tumor continues to induce excessive osteoclast-mediated bone resorption, over time the bone is weakened, becomes mechanically compromised and ultimately will fracture [817]. Nociceptors also express both mechanically gated channels which activate a signaling cascade upon excessive stretch [858] and several purinergic receptors, which are activated by adenosine triphosphate (ATP) released from cells during excessive mechanical stimulation [728, 859]. 159

122 Neurochemical Changes in the Spinal Cord: Cancer pain is induced and maintained by peripheral and central sensitization [802, 807, 817, ]. Studies involving the mouse model of bone cancer pain show extensive neurochemical reorganization in the spinal cord segments that receive input from primary afferent neurons that innervate the tumour-bearing bone [817, 820, 860]. This profound reorganization of the spinal cord that may be reflective of a central sensitization is frequently observed in persistent pain states [802]. The alterations in the neurochemistry of the spinal cord and the sensitization of primary afferents are positively correlated with the extent of bone destruction and the growth of the tumor. Interestingly, the magnitude and localized nature of these changes reported in the spinal cord segments that are almost exclusively ipsilateral to the side of bone destruction. This neurochemical signature of bone cancer pain appears unique when compared to changes that occur in inflammatory or neuropathic pain states [802]. 160

123 Figure 23. Neurochemical changes in the dorsal horn of the spinal cord 21 d after unilateral injection of an osteolytic sarcoma in the intramedullary space of the femur. Confocal images of coronal sections of the L4 spinal cord illustrate the distribution of the astrocyte marker GFAP (A); DYN with arrows indicating the cell bodies expressing this pro-hyperalgesic peptide (B); c-fos protein in the basal unstimulated state (C); c-fos protein at 1 hr after normally non-noxious palpation of the knee (D); SP (E); PKCg isoform (F). Note that the major changes occur in the spinal cord ipsilateral to the cancer-bearing femur and include an increase in GFAP (A), DYN (B), basal c-fos expression (C), and increased expression of c-fos in neurons located in laminae I and II after normally non-noxious palpation (D). In contrast, levels of SP (E), a peptide contained in primary afferent neurons that is frequently upregulated in persistent pain states, and PKCg (F), a kinase that is expressed in a subset of spinal neurons in lamina II and that is frequently upregulated in neuropathic pain states, remained unchanged. Adapted from Schwei et al. [802]. Substance P: Non-noxious palpation of the bone with cancer was found to induce behaviors indicative of pain, accompanied by internalization of the substance P receptor in lamina I spinal cord neurons (Fig 23). Furthermore, substance P receptors internalization was observed only in lumbar segments L3-L5 ipsilateral to palpation. There was no similar effect in deeper laminae neurons. This internalization was found to significantly correlate with extent of bone destruction [802]. It has been previously reported that, in the normal animal, only noxious stimulation results in the release of SP and the subsequent internalization of the SPR in lamina I neurons. In contrast in animals with either persistent inflammatory or neuropathic pain, normally nonnoxious or noxious somatosensory stimulation both induce SPR internalization in lamina I neurons [756, 802, ]. Dynorphin: In tumour-bearing animals, an upregulation of the pro-hyperalgesic peptide dynorphin was also observed in the spinal cord neurons located in laminae III-VI (Fig 23). These dynorphinoverexpressing neurons are only observed in spinal cord segments L3-L5 ipsilateral to cancerous femur, and correlated with the extent of bone destruction. In addition, they are located in close proximity to the astrocytes showing marked hypertrophy [802], and as a result, spinal-cord neurons that would normally be activated only by noxious stimuli can be activated by stimuli that would normally be non-noxious [807]. c-fos: In unstimulated bone cancer bearing animals, there is a significant increase in the number of spinal cord neurons expressing c-fos protein in laminae V-VI (Fig 23). As the case for dynorphin, this accumulation has been observed only in spinal cord segments L3-L5, ipsilateral to cancerous bone. This increase is found to significantly correlate with the extent of bone destruction [802]. In the normal animal, noxious stimulation is required to induce c-fos expression in lamina I neurons [ ]. However, non-noxious mechanical stimulation (palpation) of the distal femur, induces an increase in the number of c-fos-expressing laminae I neurons in cancer bearing animals. Induction of 161

124 c-fos expression was found to principally occur in the lateral part of laminae I-II in lumbar segments L3-L5, and to significantly correlate with the extent of bone destruction. These data suggest that there is sensitization of primary afferent neurons in animals with bone cancer, and that this sensitization is correlated with the extent of bone destruction and growth of the tumor [802]. Astrocyte Hypertrophy: In the segments of the spinal cord that receive primary afferent input from the bone with cancer, and only in these segments, there is a massive astrocyte hypertrophy without neuronal loss [802] (Fig 23 and 24). Hypertrophy of spinal astrocytes has previously been reported after sciatic nerve injury [ ], yet examination of the sciatic nerve that innervates the femur with cancer revealed no sign of direct physical injury [789]. Astrocytes are reported to express glutamate aspartate transporters and thus are intimately involved in regulating the extracellular levels of excitatory amino acids [ ]. Upon hypertrophy, this is accompanied by a decreased expression of glutamate re-uptake transporters [881, 882]. This results in increased extracellular levels of the excitatory neurotransmitter glutamate and concomitant excitotoxicity within the central nervous system [807]. Additionally, astrocytes that have undergone hypertrophy have been shown to release a variety of cytokines and growth factors that can dramatically alter the surrounding neurochemical environment [ ]. 162