EXAMINATION OF NMDA RECEPTOR SUBUNIT PREVALENCE AND DISTRIBUTION IN CRUDE SYNAPTIC MEMBRANES PURIFIED FROM A MOUSE MODEL OF RETT SYNDROME.

Transcription

1 EXAMINATION OF NMDA RECEPTOR SUBUNIT PREVALENCE AND DISTRIBUTION IN CRUDE SYNAPTIC MEMBRANES PURIFIED FROM A MOUSE MODEL OF RETT SYNDROME. by Ewelina Maliszewska-Cyna A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto Copyright by Ewelina Maliszewska-Cyna September 2009

2 II EXAMINATION OF NMDA RECEPTOR SUBUNIT PREVALENCE AND DISTRIBUTION IN CRUDE SYNAPTIC MEMBRANES PURIFIED FROM A MOUSE MODEL OF RETT SYNDROME. Ewelina Maliszewska-Cyna Master of Science Graduate Department of Physiology University of Toronto Graduation date: November 13, 2009 ABSTRACT In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

3 III ACKNOWLEDGEMENTS I am thankful to many great individuals who have provided advice, encouragement, and support throughout this project. I am particularly indebted to my supervisor Dr. James Eubanks, who gave me a chance to participate in this interesting research project and who sometimes believed in me more than I did myself. He supported me with his encouragements and many fruitful discussions and he certainly made a great impact in my entire life. I am grateful to my committee members, Dr. James Gurd, Dr. Michelle Aarts, and Dr. Joanne Nash, for their advice and guidance throughout this research. They certainly recommended a number of important improvements, and by genuinely sharing their invaluable experience they helped me in more ways that I can describe. I would like to thank the members of my laboratory, Richard Logan and Guangming Zhang, for their advice and support during my study as well as for their patience and understanding. They graciously helped me figure out my way around the lab and made this project an enjoyable and exciting experience. I am thankful to Damanpreet Bawa for teaching me the techniques used in my project. I am also grateful to all students in my lab, Andreea, Lidia, Elena, Jennifer, and Tea, for creating a pleasant work environment. I would also like to express my gratitude to the Ontario Rett Syndrome Association for giving me an opportunity to meet Rett girls and their families. They became my inspiration and motivation, and showed me what is truly important in life. I am grateful to Ms. Julie Wan and Ms. Poonam Cardoso for their secretarial assistance and to the staff at Animal Care Facility for my training and the maintenance of my animals. At the end, I wish to thank my parents and my family for their support and understanding. Finally, I wish to express my deepest gratitude to my husband, who offered both advice and encouragement during the past years. I dedicate my work to them.

4 IV TABLE OF CONTENT Title Page...I Abstract...II Acknowledgements...III Table of Content...IV List of Tables and Figures...VIII List of Abbreviations...IX 1. Prologue Introduction Rett syndrome Clinical features Genetic basis of Rett syndrome MeCP2 function and gene targets Neuropathology and altered morphology Phenotypic variability Rett syndrome in males Animal models Four mouse models of Rett syndrome Male vs. Female mouse models of Rett syndrome Glutamate receptors Classification...23

5 V Ionotropic glutamate receptors taxonomy and general structure NMDA glutamate receptors NMDAR subunit assembly Developmental switch of NMDAR subunits NMDAR trafficking to the cell surface NMDAR functions BDNF regulation of NMDARs The postsynaptic components of excitatory synapse Lipid rafts Postsynaptic densities Extra-synaptic membranes Biochemical purification Detergents and Critical Micelle Concentration Detergent extraction of synaptic microdomains Rationale, Hypothesis and Objectives Materials and Methods Animal subjects Tissue harvesting Genotyping Genomic DNA extraction Polymerase Chain Reaction Biochemical Purification Preparation of Synaptic Membranes Protein Concentration Solubilization and Sucrose Density Gradient Centrifugation...66

6 VI 4.3. Characterization of Synaptic Membranes Sucrose Refractive Index SDS-PAGE Western Immunoblotting and antibodies Densitometric analysis Statistical analysis Results The mass of the MeCP2-deficient mouse brain is less than that of the wild-type Total protein yield in the MeCP2-null mouse whole brain homogenate and S1 fraction is greater than in wild-type control mouse Separation by gradient centrifugation Total protein and sucrose concentrations across the gradient are similar in mutant and wild type mice Characterizing the membrane microdomains Preservation of the detergent-resistant, detergent-soluble, and postsynaptic density membranes in the MeCP2-deficient mouse brain Characterizing the prevalence and distribution of Flo-1, GP50, and PSD Total protein yield in detergent-resistant, detergent-soluble membranes and postsynaptic density membranes is conserved in the Rett mouse brain Characterizing the prevalence and distribution of NMDA receptor subunits in membrane microdomains The localization of the NR1, NR2A, and NR2B NMDA receptor subunits is preserved in the MeCP2-null synaptic membranes The overall prevalence of the NR1 and NR2A subunits is decreased, while NR2B prevalence is preserved in the MeCP2-null synaptic membranes Ratio of NR2A-to-NR2B is decreased in the MeCP2 null brain NR2A and PSD-95 distributions to the synaptic microdomains parallel each other in the MeCP2-deficient synaptic membranes...109

7 VII 6. Discussion Smaller brain mass and increased protein yield in MeCP2-deficient mice Synaptic membranes from the MeCP2-deficient mouse brain distribute across the gradient similarly to the control Synaptic membrane microdomains are preserved in MeCP2-deficient brain The prevalence of PSD-95 is decreased in the MeCP2-deficient mouse brain PSD-95 in mice and rats exhibit different distribution patterns Preservation of NMDAR subunits localization Mouse-to-rat differences in NMDAR subunits distribution Decreased prevalence of NR1 and NR2A subunits of NMDAR complex in the MeCP2- null brain Ratio of NR2A-to-NR2B is decreased in the MeCP2-null brain PSD-95 and NR2A co-associate to the same synaptic membranes in the MeCP2- deficient mouse brain Pathological significance Future directions References...135

8 VIII LIST OF FIGURES AND TABLES Figures Introduction Figure 1: Rett syndrome progression of symptoms...5 Figure 2: Structure and mutations of MeCP2...9 Figure 3: Glutamate receptors classification...24 Figure 4: NMDAR structure (subunit and complex)...28 Figure 5: Synaptic membrane microdomains...39 Figure 6: Lipid Rafts as a protein and cholesterol-rich microdomain...42 Materials and Methods Figure 7: PCR genotyping...58 Figure 8: Flowchart A purification of synaptic membranes...64 Figure 9: Flowchart B separation on a sucrose density gradient...67 Figure 10: Densitometric analysis example...74 Results Figure 11: Brain mass...78 Figure 12: Protein yield in fractions from the synaptic preparation...81 Figure 13: Sucrose Refractory Index and protein yield across the gradient...85 Figure 14: Immunoblots of Flo-1, GP50, and PSD Figure 15: Distribution of Flo-1, GP50, and PSD-95 proteins across synaptic membranes...91 Figure 16: Flo-1, GP50, and PSD-95 prevalence...93 Figure 17: Protein yield in DRM, DSM, and PSD...96 Figure 18: Immunoblots of NR1, NR2A, and NR2B...99 Figure 19: Distribution of NR1, NR2A, and NR2B proteins across synaptic membranes Figure 20: NR1, NR2A, and NR2B prevalence Figure 21: Ratio of NR2A-to-NR2B Figure 22: NR2A and PSD-95, NR2B and PSD-95 distribution comparison Figure 23: NR2A-to-PSD95 and NR2B-to-PSD95 ratios Discussion Figure 24: Illustration of results Figure 25: Model of Rett syndrome pathophysiology Tables Materials and Methods Table 1: Solutions used for the biochemical purification...62 Table 2: Antibodies...71

9 IX LIST OF ABBREVIATIONS AMPA alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid AMPAR(s) alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor(s) BDNF Brain-Derived Neurotrophic Factor CaMKII calcium/calmodulin dependent protein kinase II CDKL5 cyclin-dependent kinase-like 5 CMC Critical Micelle Concentration CNS Central nervous system CREB cyclic AMP response element-binding protein CSGE conformation-sensitive gel electrophoresis C-terminal carboxy terminal DNMT1 maintenance DNA methyl transferase 1 DRM Detergent Resistant Membrane DSM Detergent Soluble Membrane ER Endoplasmic Reticulum ERK Extracellular Signal-Regulated Kinase fepsp field excitatory postsynaptic potential FOXG1 Forkhead box protein G1 (formerly brain factor 1 (BF-1)) GluR1-4 AMPA receptor subunit 1-4 GluR5-8 Kainate Receptor subunit 5-8 GP50 glycoprotein of 50 kda H homogenate HDAC Histone Deacetylase iglur Ionotropic glutamate receptor IUPHAR International Union of Basic and Clinical Pharmacology JARID1B Jumonji AT rich interactive domain 1B KAR Kainate Receptor KA1-2 Kainate Receptor subunit 1-2 Ld liquid-disordered phase Lo liquid-ordered phase LTD long-term depression LTP long-term potentiation MAP-2 microtubule-associated protein 2 MAPK mitogen-activated protein kinase MBD Methyl Binding Domain MCD methyl-β-cyclodextrin MeCP2 methyl-cpg-binding domain protein 2 MECP2 human gene coding for MeCP2 protein Mecp2 mouse gene coding for MeCP2 protein MeCP2_e1 isoform 1 of MeCP2 protein MeCP2_e2 isoform 2 of MeCP2 protein

10 X mglur Metabotropic glutamate receptor NCAM-120 neuronal cell adhesion molecule of 120 kda NMDA N-methyl-D-Aspartate NMDAR(s) N-methyl-D-Aspartate Receptor complex(es) NR1 N-methyl-D-Aspartate Receptor subunit 1 NR2A-D N-methyl-D-Aspartate Receptor subunit 2A-D NR3A-B N-methyl-D-Aspartate Receptor subunit 2A-B N-terminal amino terminal PCR Polymerase Chain Reaction PDZ PSD-95, Disc-Large, Zonula-Occludens 1 PKA Protein Kinase A PKC Protein Kinase C PMSF phenylmethansulfonyl fluoride PSD post-synaptic density PSD-95 post-synaptic density protein of 95 kda RI Refractive Index ROD Relative Optical Density SAP-97 synapse-associated protein of 97 kda SAP-102 synapse-associated protein of 102 kda SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Sin3A Syntaxin 1A TRD Transcriptional Repression Domain

11 1 CHAPTER 1 PROLOGUE Dr. Andreas Rett ( ) Source: In the year 1954 in Vienna, Austria, two young girls were sitting in a waiting room to see their physician. The two patients had unusually similar repetitive hand-washing movements. This coincidence did not go unnoticed by the physician, who soon discovered that he had six other patients with very similar behaviour, and their clinical, as well as developmental histories, were very much alike. A few years later, in 1966, Dr. Andreas Rett reported for the first time an unusual clinical occurrence that now bears his name (Rett, 1966). Unfortunately, post-war Europe did not pay much attention to the mentally disabled citizens, and as a result, Rett s work went unnoticed. He continued to care for mentally challenged children and created a first in the world facility, named Rosenhugel (Rose Hill)

12 2 (IRSF, 2008), which provided medical help for those who were then considered social outcasts. The building assigned for this facility was a 70 year old structure belonging to an institution for aged people, with very few doctors and nurses, and no water faucets in the sick bays; patients in wheelchairs had to be hand carried down the many flights of steps. Dr. Rett s dedication and charisma led to several improvements, and soon it became a sophisticated centre for the handicapped, with the first EEG and the first clinical psychologist in a state hospital in Austria. It was not until 28 years later in 1983, when the disorder was reported in an English journal, Annals of Neurology, by Dr. Bengt Hagberg of Sweden (Hagberg, et al. 1983), that Rett syndrome was finally recognized internationally. Shortly thereafter, a number of girls were diagnosed with the syndrome in a host of countries. Over several years that followed, physicians and researchers alike focused their efforts on expanding their knowledge of the pathophysiology of this condition. A major breakthrough came in 1999, when the genetic cause of Rett was discovered by a group of researchers led by Carolyn Schanen from Uta Francke and Huda Zoghbi research group. Mutations in a gene coding for the MeCP2 protein was found to cause the disease (Amir, et al. 1999). Its location on the X chromosome provided further proof that Rett syndrome is an X-linked condition, and offered an explanation as to why it occurs predominantly in female patients. In addition to expansive research efforts, several organizations formed which continue to provide assistance and support to families, organize meetings and conferences, and solicit funds from private and institutional donors in order to foster new research. Among

13 3 them, the International Rett Syndrome Association started in 1984 on Kathy Hunter s kitchen table, when her daughter Stacie became the first child in the United States to be diagnosed with Rett syndrome (Hunter, 1998). Today, operating as a non-profit corporation, IRSF is the largest private source of funds for biomedical and clinical research on Rett syndrome (IRSF, 2008). During the 50 years that spanned his career, Dr. Andreas Rett received many awards and distinctions, including the Grand Medal for Special Earnings from the Republic of Austria, and the National Association for Retarded Citizens 1989 Distinguished Research Award, in which he was recognized as a pioneer in the care of mentally and physically handicapped children. Dr. Rett never gave up on his dream to find answers to the puzzling disorder that took his family name. During a conference address he stated: We are aware of the fact that many mysteries of this syndrome still remain undisclosed, and therefore, for the time being, we have no option but to live with it. However, the children with their very special ways give us enough impulse to share their lives. (...) Their appearance and the sparkle in their eyes make it easy to love them. Daily care for them and working with them gives us grownups strength, enabling us to learn the special treatment required, thus furthering our own development. To live with them, to love them and to learn from them are the rudimentary principles of our work." 25, Dr. Andreas Rett died at the age of 73 in his hometown of Vienna, Austria on April

14 4 CHAPTER 2 INTRODUCTION 2.1 Rett syndrome Clinical features The incidence of Rett syndrome in the female population is estimated to be between 1 in 10,000 to 1 in 15,000 cases (Percy, et al. 2007). Rett syndrome is a postnatal neurodevelopmental disorder in which patients appear to develop normally for the first few months and meet critical developmental milestones (Hagberg and Witt-Engerström, 1986), including the ability to walk, and some even begin to use few words (for review see Chahrour and Zoghbi, 2007; Nomura, 2005). After that initial 6-18 month period of normal development, there is an onset of developmental stagnation, observed initially by the deceleration of head growth (Hagberg, et al. 2000). In some cases, there also is a reversal of previously acquired skills, such as speech and the ability to walk (Figure 1). As a consequence of head growth deceleration, microencephaly is seen in many patients, followed by growth retardation, weight loss, and muscle hypotonia.

15 5 Figure 1 : Rett syndrome progression of disease. After initial 6-18 months of normal development, the clinical symptoms begin to be apparent in the female patients. The disease progresses through four stages, with initial rapid regression of development, followed by the plateau stage. In general, Rett patients have normal life span and are fertile.

16 months Initial normal development meet critical developmental milestones Begin to walk and speak 1 3 years Developmental Stagnation Phase Deceleration of head growth Loss of previously acquired skills 1 6 years Rapid Regression Phase Autistic features (loss of speech, social interaction) Stereotypic hand wringing Impaired motor abilities Mental retardation Seizures Respiratory abnormalities 3 years - adult Stationary Phase / Post-regression Stage No worsening of symptoms Loss of autistic features and increased social interest (intense gaze) Motor impairments Anxiety Scoliosis adult Plateau Stage Muscle dystonia Parkinsonian features Late motor deterioration

17 7 The next step in the disease progression is a rapid regression of development and an appearance of autistic features: hypersensitivity, indifference to surrounding environment, and loss of social interaction abilities. This stage is also characterized by the loss of purposeful hand movement and development of stereotypic hand wringing or washing movement features initially noticed by Dr. Andreas Rett. Concurrently with developmental stagnation, there usually is a loss of motor abilities, lack of coordination, ataxia, and gait apraxia. Another feature of the rapid regression phase is the occurrence of seizures, which can also vary in severity. The seizure episodes are most frequent during the early years of a patient s life, and tend to decrease in severity into adulthood (Jian, et al. 2006). During the rapid regression stage, when communication and social skills are being impaired, the child might be diagnosed with autism. However, in contrast to autism, Rett patients appear to progress from this autistic-like stage and soon develop increased social interest, exemplified by the intense gaze. After the rapid regression phase, there is a stationary phase, where symptoms begin to plateau, and there is little or no further worsening of the symptoms. Patients continue to suffer from continuous motor abnormalities, muscle dystonia, rigidity, and worsening scoliosis with most patients loosing the ability to walk, becoming wheel-chair bound. Later into adulthood, patients develop Parkinsonian features and reach a plateau stage (Hagberg, 2005; Roze, et al. 2007). Usually, female patients have a normal life span and are fertile.

18 Genetic basis of Rett syndrome It is now well established that Rett syndrome is caused by mutations in a gene coding for the methyl-cpg-binding domain protein 2 (MeCP2). Using information from rare familial cases, Amir et al. (1999) was the first to identify by exclusion mapping Xq28 region as a candidate for bearing the genetic cause of Rett syndrome. Conformation-sensitive gel electrophoresis (CSGE) within that region revealed that the great majority of patients carry a mutation in a region coding for MeCP2 protein. Additional screening of the parental gene revealed no abnormalities, demonstrating that these are de novo mutations. It has been estimated that 95% of Rett cases test positive for a mutated MECP2 gene (Amir, et al. 1999). Most mutations arise in the paternal germline during spermatogenesis and often involve a C to T mutation (Trappe, et al. 2001; Wan, et al. 1999). The great majority (>95%, Trappe, et al. 2001) of mutations in MECP2 from the paternal chromosome can be explained by the higher levels of methylation in male germ cells at early stages of gametogenesis, and by greater number of mitotic divisions in the male germ line (Driscoll and Migeon, 1990; El-Maarri, et al. 1998). There have been over 300 mutations reported within this gene (Christodoulou, et al. 2003), resulting in different phenotypes that lead to a whole spectrum of neurophysiological and clinical features of the syndrome (Figure 2). These mutations include missense, nonsense, and frameshift mutations, as well as deletions encompassing several nucleotides or even whole exons (Ravn, et al. 2005). Several of these mutations introduce a premature stop codon resulting in truncated, and therefore, unstable and non-functional

19 9 Figure 2 : Structure and mutations frequency of MeCP2. A) MeCP2 protein exists in two isoforms, e1 and e2, with the e1 isoform being more prevalent in the brain. Isoform e1 excludes exon 1, whereas e2 is translated from a start codon in exon 2 excluding exon 1. Figure adapted from Chahrour and Zoghbi, B) There are over 300 different mutations in the MECP2 that contribute to a wide spectrum of disease presentation. These mutations include missense, nonsense, and frameshift mutations, and several of them introduce a stop codon causing the formation of a truncated and nonfunctional protein. Source: C) Recent data suggest that MeCP2 acts as both transcriptional activator and repressor and its function depends on the type of proteins it attracts to the transcriptional complex. Figure modified from: Chahrour and Zoghbi, 2007.

20 10 A) MeCP2_e1 noncoding sequence MeCP2_e2 coding sequence B) C) Sin3A HDAC MeCP2 M CpG X CREB1 Coactivator MeCP2 M CpG

21 11 protein. The great majority of MECP2 mutations causing Rett syndrome occur in exons 3 and 4 which are excluded from alternate splicing, while mutations on exon 1 are rare and cause an atypical form of Rett syndrome. There are two isoforms of the MeCP2 protein: MeCP2_e1 isoform excludes exon 2 and has an alternative N-terminus, translated from exon 1, whereas the MeCP2_e2 isoform is translated from a start codon in exon 2 and excludes exon 1 (Figure 2A) (Mnatzakanian, et al. 2004). Although exon one is a component of the more prevalent isoform of the MeCP2 protein in the brain, Amir et al. (2008) found that mutations in this exon account for only 0.33% of all classic Rett cases. Mutations in exon 3 or 4 affect both isoforms, whereas exon 1 mutations are specific for the MeCP2_e1 isoform only. No mutation on exon 2 has been reported to cause Rett syndrome. An explanation of this intriguing phenomenon may be that MeCP2_e2 is not predominant in the brain, and as such is present in small amounts when compared to exon one-containing MeCP2_e1 isoform. Consequently, it may be that mutations in exon 2 would not result in the Rett phenotype because of a high abundance of the MeCP2_e1 isoform and the fact that MeCP2_e2 levels are very low in the central nervous system. The MECP2 mutation is the leading, but not the only cause of Rett-like pathology. It has been recently reported that patients with mutations on another X-chromosome linked gene, the cyclin-dependent kinase-like 5 gene (CDKL5), have clinical features resembling Rett syndrome (Evans, et al. 2005). Unlike patients with the MECP2 mutation, patients with a mutated CDKL5 develop a congenital Rett-like disorder with an early-onset epilepsy, infantile spasms, and severe mental retardation.

22 12 Another cause of the Rett syndrome phenotype is a deletion encompassing several genes in the long arm of chromosome 14 (Ariani, et al. 2008; Jacob, et al. 2009). Within that region is FOXG1, coding for a brain-specific transcriptional repressor known to be important in the early development of telencephalon. The interaction of FoxG1 with transcriptional repressors, such as JARID1B and the Groucho family members, is of functional importance for early brain development. In this regard, the function of FoxG1 parallels the role of MeCP2 in controlling normal brain development. However, the redundancy of the two systems is unlikely as it is hypothesized that the two transcriptional regulators act on different stages of the process that leads to proper cortical development (Ariani, et al. 2008) MeCP2 function and gene targets Methyl CpG-binding domain protein 2 is expressed in many tissues, with the highest levels being detected in the brain. Studies using tissue from different developmental stages revealed that MeCP2 expression is very low or absent in nestin-positive progenitor cells (Jung, et al. 2003; Kishi and Macklis, 2004). This in vitro culture study suggests that MeCP2 is expressed in more differentiated neurons rather than in less differentiated neuroblasts. Under in vitro conditions using neural precursor cultures, Kishi and Macklis showed that MeCP2 mutant neural precursors differentiate into morphologically mature neurons and glia. This study also revealed that the levels of MeCP2 increase during neuronal maturation, and are highest in post-mitotic, post-migratory mature neurons. This suggests that MeCP2 is involved both in neuronal plasticity and maturation, as well as in dendritic arborisation and maintenance, but not in neurogenesis or neuronal migration.

23 13 MeCP2 is a member of a large family of proteins that bind to methylated CpG dinucleotides via their respective Methyl Binding Domains (MBDs) (Hendrich and Bird, 1998) and it is believed to selectively regulate the expression of a vast array of genes (for review see: Chahrour and Zoghbi, 2007; Shahbazian and Zoghbi 2002; Francke, 2006; Moretti and Zoghbi 2006). Upon binding to a methyl-cpg (mgpg) dinucleotide in a target gene, MeCP2 recruits other proteins to form a complex, which then changes the chromatin conformation. Among the proteins that MeCP2 recruits to the complex through its TRD are the corepressor Sin3A and histone deacetylases HDAC1 and 2 (Nan, et al. 1997). Because MeCP2 appears to be a common link between DNA methylation, histone deacetylation and transcriptional corepressors, it has been proposed that it acts as a transcriptional repressor by compacting the chromatin and by promoting nucleosome compacting. MeCP2 has also been shown to recruit CREB1 to the complex (Chahrour, et al. 2008). CREB1 is a potent transcriptional activator and its association with MeCP2 suggests that MeCP2 also has the ability to activate transcription. Until very recently, it was commonly accepted that MeCP2 acts predominantly as a transcriptional repressor. However, the latest evidence presented by the Zoghbi research group suggests that it may in fact possess activating as well as repressing abilities (Chahrour, et al. 2008). Adding to the complexity of this issue is the fact that, at least in vitro, MeCP2 binds to the transcriptional modulators in a very weak mode (Ballestar, et al. 2000). This suggests that MeCP2 effects are transient and the whole complex can quickly disintegrate. These dual properties of the protein may explain some inconsistencies in the literature pertaining to its ascribed function.

24 14 For example, if MeCP2 is indeed a transcriptional repressor, one might predict that a loss-offunction of the protein would result in the abnormal expression of a large number of genes, because they would be released from a repressive mechanism. However, large-scale gene expression studies using microarray analysis of both human and mouse Mecp2-deficient brains have failed to prove this hypothesis (Tudor, et al. 2002). Although a small number of differentially expressed genes have been reproducibly identified, the changes are low in magnitude and occur in a smaller than expected number of genes. Since discovery of the involvement of MeCP2 in Rett syndrome pathology, several new protein targets of MeCP2 protein-protein interactions continue to be identified. For example, Kimura et al. found that MeCP2 interacts with the maintenance DNA methyl transferase 1 (DNMT1), which in turn acts on hemimethylated DNA during the replication stage of the cell cycle (Kimura and Shiota, 2003). Because this interaction was found to occur independently of the Sin3A/HDAC complex, it was proposed that MeCP2 guides DNMT1 to the sites that require methylation after replication. In addition, DNMT1 is known to be expressed in neurons, but not in glia (Goto, et al. 1994), and a conditional deletion of Dnmt1 in E9-E10 neuronal precursors adversely affects neuronal survival after birth, but does not affect their early development and survival (Fan, et al. 2001). Such function, together with confirmed binding of MeCP2 to Dnmt1, makes it a likely candidate in downstream effectors of MeCP2-mediated pathology of Rett syndrome. One verified target gene of MeCP2 is a brain-derived neurotrophic factor (BDNF). Chen et al. found that in the absence of neuronal activity, MeCP2 binds to its promoter III and functions as a negative regulator of BDNF expression (Chen, et al. 2003). Upon the

25 15 influx of calcium ions induced by neuronal activity, however, MeCP2 becomes phosphorylated and is released from the promoter III, thereby removing the repression of transcription. Interestingly, a study with Mecp2 knockout mice showed a decrease, not an increase, in BDNF levels (Martinowich, et al. 2003). This finding is surprising, as the in vitro data from Chen et al. would suggest that by removing a repressor (as is the case in Mecp2 k/o) the transcription of BDNF should proceed uninterrupted. These seemingly contradictory results may be reconciled with the observation that Mecp2 knockout mice exhibit diminished neuronal activity, which by itself is known to decrease BDNF expression. Thus, neuronal inactivity in vivo might counteract the direct effect of a loss of Mecp2 function on BDNF transcription (showed in vitro by Chen et al.), with the net effect of reducing BDNF levels (see also section ) Neuropathology and altered morphology Despite severe clinical features and a deteriorating course of Rett syndrome, the pathology of the Rett brain reveals only subtle abnormalities. Imaging studies, as well as post mortem examinations, display a typical 15 35% decrease in brain growth in Rett patients (Jellinger and Seitelberg, 1986). Reduced brain size of Rett patients results in prominent neuropathological features including: a reduction in cortical thickness, reduced neuronal size, and diminished dendritic arborisation (Jellinger and Seitelberg, 1986; Armstrong, 1997). At the same time, the number of neurons remains normal, further suggesting that Rett syndrome is a neurodevelopmental, and not a neurodegenerative, disease (Armstrong, 2001). Additionally, a lack of atrophy or inflammation in the Rett brain further suggests a

26 16 neurodevelopmental etiology. It has also been reported that neuronal cell packing density is increased, consistent with the model of stalled development of axons and dendrites. For example, Bauman et al. found that neurons in several brain regions, including the cerebral cortex, thalamus, hippocampus, amygdala, and substantia nigra, were smaller and were more densely packed than in control subjects. In addition, observation of substantia nigra s reduced pigmentation by Jellinger and Seitelberg (1986) suggests impaired neuronal development, as melanin pigment normally continues to accumulate until the age of 15 years (Fenichel and Bazelon, 1968; Spence and Gilles, 1971). The morphology of neurons is also affected in the Rett brain. It has been reported previously that there are fewer dendritic branches in the Rett neurons of the somatosensory cortex and the ones that are present are shorter, have elongated spine necks, as well as a reduced area of spine heads. These dendritic alterations may be related to the observed reduction in levels of microtubule-associated protein 2 (MAP-2), which is an important cytoskeletal component of dendrites (Kaufmann, et al. 2000). During neuronal development, interactions between MAP-2 and tubulin were shown to be important for the extension of neuronal growth cones and for normal dendritic growth (Kaech, et al. 1996). Also, consistent with this neuropathology is a reduction of substance P a neuropeptide stimulating neurite extension in cultured neuroblastoma cells - in the Rett brain (Deguchi, et al. 2000; Whitty, et al. 1993). These studies suggest that reduced brain growth in Rett syndrome is, at least in part, due to reduced development of axonodendtritic connections. However, it remains to be established whether these molecular alterations are a cause or a consequence of abnormal neuronal development. This morphological and neuropathological profile suggests that Rett

27 17 neurons undergo relatively normal initial development, but fail to form proper synaptic networks preventing normal signal transmission Phenotypic variability Due to the fact that there are over 300 different mutations within the MECP2 gene, it is of no surprise that different mutations can elicit a different phenotype (Christodoulou, et al. 2003). In general, early-truncating mutations cause a more severe diseased phenotype than mutations associated with C-terminal deletions (Smeets, et al. 2005). However, it has been reported that R133C mutations cause an overall milder phenotype (Neul, et al. 2008), whereas the R270X mutation is associated with increased mortality (Bienvenu and Chelly, 2006). The most severe phenotype is associated with R168X, R255X, and R270X nonsense mutations, which truncate the protein within its TRD and disrupt its Nuclear Localization Signal (NLS) (Bebbington, et al. 2008). By contrast, mutations at the carboxy terminal lead to disruption of the reading frame, causing the formation of the truncated form but leaving the Transcriptional Repression and Methyl Binding Domains intact. As a result, these mutations manifest in milder phenotype (Smeets, et al. 2005). There also are several atypical forms of Rett syndrome that deviate from the classical clinical presentation. For example, there are milder forms with later disease onset on one side of the spectrum, and more severe forms with an earlier onset on the other side of the spectrum (Christodoulou, et al. 2003). Both classical and atypical phenotypes vary between patients in severity of the phenotype and time of disease onset.

28 18 A major source of phenotypic variability associated with different MECP2 mutations is the pattern of X chromosome inactivation (Zoghbi, et al. 1990). In females, only one of two X chromosomes is active in each cell, and an inactivation of maternal or paternal chromosome occurs at random. As a result, roughly half of all cells (with the exception of the germline cells) have the maternal X chromosome active whereas the other half has the paternal X chromosome active. Therefore, a female with a MECP2 mutation is actually a mosaic with half the cells expressing the wild-type MECP2 allele and the other half expressing the mutated MECP2 allele. Reports of females carrying mutations in MECP2 describe them as asymptomatic, or only having a mild learning disability, although rare, support this hypothesis (Hoffbuhr, et al. 2001; Amir, et al. 2000). The most dramatic example of the influence of X-chromosome inactivation on phenotypic severity comes from studies of monozygotic twins, in which the same genetic makeup carrying an identical mutation in the MECP2 gene, results in a very different disease phenotype (Ogawa, et al. 1997; Ishii, et al. 2001). Nevertheless, these are rare occurrences, as the majority of females appear to have a balanced X-chromosome inactivation pattern in both peripheral as well as in brain tissue (Amir, et al. 2000; Shahbazian, et al. 2002). This in turn suggests that skewed X- chromosome inactivation is rare and usually occurs due to chance Rett syndrome in males Rett syndrome has been reported to occur occasionally in male patients (Jan, et al. 1999). Because of the fact that males have only one X chromosome, all cells express the maternal allele. If the maternal allele has a mutated MECP2 gene, then all cells express an

29 19 aberrant protein, resulting in a very severe phenotype (Hoffbuhr, et al. 2001). MECP2 mutations in males cause a host of neurological aberrations, ranging from mental retardation to severe encephalopathy. The same mutations that cause Rett in females result in a more severe phenotype in males. For example, mutations that cause classical Rett syndrome in females, cause severe encephalopathy in males, and death in the first year of life. Furthermore, mutations that do not cause sufficient changes in MeCP2 function to elicit the disease in females can cause moderate to profound mental retardation or neurological disorders in males. However, mutations causing classic Rett syndrome in females result in a classic Rett phenotype in males with Klinefelter syndrome (47, XXY), because of the presence of a second X chromosome in males (Armstrong, et al. 2001; Hoffbuhr, et al. 2001) Animal models Four mouse models of Rett syndrome Mice have become an animal of choice to model human disorders involving genetic defects, because the mouse genome has been fully sequenced, and mouse genes can be easily manipulated (Lodish, et al. 2004). The use of genetically altered mice, through either loss- or gain-of-function manipulations, has provided an excellent opportunity to study the genetic basis of disease pathogenesis, behavioural consequences, and potential therapeutic targets. Development of animal models mimicking the pathology of Rett syndrome made possible studies on consequences of mutated Mecp2 on brain morphology and function.

30 20 There are currently four mouse models of Rett syndrome (for review see Chahrour and Zoghbi, 2007). Three of those models have complete loss-of-function deletions of MeCP2 and one has a truncated form of MeCP2. The loss of MeCP2 function in those mice results from conditional deletion of either exon 3 of the Mecp2 gene (MeCP2 1lox ) (Chen, et al. 2001) or from conditional deletions of both exons 3 and 4 (MeCP2 tm1-1 ) (Guy, et al. 2001). Conditional deletion in both models was achieved by using the Cre-loxP recombination system. These models successfully recapitulate several pathophysiological and behavioural features of Rett syndrome. MeCP2-deficient male mice undergo initial stages of normal development for the first 3-5 weeks and then begin to develop Rett-like symptoms. These impairments include: hypoactivity, tremor, hindlimb clasping, increased anxiety, impaired balance and grip strength, diminished cue and contextual memory, and impaired social interactive skills. Both mouse models are also smaller in size and weigh less than their wildtype littermates. The symptoms worsen over time, leading to severe progressive dysfunction, and to death at 8-10 weeks of age. Another Mecp2-null mouse was generated by the targeted deletion of the Methyl Binding Domain coding region and a disruption of the mrna splicing site resulting in the complete loss of the Mecp2 transcript and protein (Pelka, et al. 2006). Those mice exhibit learning deficits, reduced locomotor activity, and impaired fear and anxiety responses. Another RTT mouse model contains MeCP2 protein truncated at amino acid 308 (MeCP2 308 ) (Shahbazian, et al. 2002). The resulting protein retains its Methyl Binding and Transcriptional Repression Domains and only eliminates the C-terminus of the protein, similar to C-terminal deletions seen in Rett patients. These mice appear normal until 6-weeks

31 21 of age when they start to develop symptoms reminiscent of the clinical features in Rett girls. However, these mice present lower penetrance of the lethality and have a milder phenotype. In this thesis, we used mice with complete MeCP2 loss of function by deleting exons 3 and 4 (MeCP2 tm1-1 ) (Guy, et al. 2001). This model has been used in the past in our laboratory with great success and we have studied it extensively. In our previous study (Asaka, et al. 2006) we correlated changes in neurotransmitter levels with functional alterations in hippocampal synapses. We focused our attention on glutamate receptors, AMPA and NMDA receptors in particular, because these are the ones that are known to be most affected in the pathophysiology of epilepsy, movement disorders, respiratory rhythm centres of brainstem and in cognitive deficits, and they also constitute the pathology of Rett syndrome (Chahrour and Zoghbi, 2007). Our immunoblot analysis of hippocampal tissue showed no change in AMPA receptor levels. However, loss of MeCP2 function affected NMDA receptor subunit composition: there were significantly lower levels of the NR2A subunit whereas NR2B levels were significantly increased; there was no change in NR1 subunit levels (Asaka, et al. 2006; also see Kaufmann, et al. 2000). Electrophysiological analysis of field excitatory postsynaptic potential (fepsp) in CA1 hippocampal slices revealed significantly reduced long-term potentiation (LTP) and, to our surprise, lack of long-term depression (LTD). These data suggest that in the Rett brain, changes in the NMDA receptor NR2A and NR2B subunit expression may be linked to a deficit in long-lasting activity-dependent synaptic plasticity. The work presented in this thesis is an extension of this study.

32 Male vs. female animal models of Rett syndrome. Rett syndrome is an X-linked neurodevelopmental disorder, and as such, is mostly seen in female patients. Cases of males with Rett syndrome are rare and the severity of the symptoms is much greater in males than in females. For example, male Rett patients rarely live past their first year of life, whereas female patients usually present a normal life span. This difference is attributable to the absence of a normal allele in males. By contrast, females are known to have a mosaic pattern on a cellular level, with roughly half the mutant allele and half the normal allele being expressed, which accounts for a less severe phenotype. The same relation holds true for animal models of Rett syndrome, among them the Mecp2 tm1-bird model (Guy, et al. 2001), which was used in this study. The symptoms reminiscent of Rett syndrome appear much earlier in male (3-5 weeks) than in female (7-8 months) mice, and the course of disease in female mice is generally milder (Guy, et al. 2001, Asaka, et al. 2006). By contrast, male Mecp2-deficient mice exhibit a much more severe phenotype, and usually do not live past 10 weeks of age. In addition, the phenotype appears to be more variable in the females than males because of unbalanced and random patterns of X-chromosome inactivation in females. Thus, male mice, even though not genderappropriate for this syndrome, are generally accepted as the standard model and as a result, were also used in this study.

33 Glutamate Receptors Classification Glutamate is a major excitatory neurotransmitter in the central nervous system and glutamate receptors are a large family of excitatory receptors that were extensively reviewed in the literature (Dingledine, et al. 1999; Pin and Duvoisin, 1995; Anwyl, 1999). Glutamate receptors can be broadly divided into iono- and metabotropic groups (Figure 3A). Metabotropic glutamate receptors (mglurs) are coupled to G-protein and this group can be further divided into eight types (mglur1 mglur8). Ionotropic glutamate receptors (iglurs) are ligand-gated ion channels and AMPA, NMDA, and KAR receptors belong to this group. Each class of glutamate receptors consists of several subunits adding to the complexity of glutamate receptors. A further level of biological complexity is achieved by the presence of multiple genes and splice variants of the genes coding for glutamate receptors. Glutamate receptors are present mostly, although not exclusively, in the central and peripheral nervous system. There are a few important exceptions: pancreatic islet cells (Weaver, et al. 1996), osteoclasts and osteoblasts (Chenu, 2002), cardiac ganglia (Gill, et al. 2007), and nerve terminals in the skin (Carlton, et al. 1995; Du, et al. 2001) have all been shown to express glutamate receptors proving their complex and broad biological function in the periphery, in modulating insulin secretion (Weaver, et al. 1998), or slow down bone resorption (Chenu, et al. 1998). Nonetheless, the focus of this introduction is on ionotropic glutamate receptors present in the central nervous system.

34 24 Figure 3 : Glutamate receptors classification. A) Glutamate receptors (GluRs) can be broadly divided into ionotropic (iglurs) and metabotropic (mglurs) types. Ionotropic receptors can be further divided into NMDAR, AMPAR, and Kainate Receptors. Metabotropic receptors are divided into three groups (I-III), each having their own subtypes. B) New nomenclature for ligand-gated ion channels has been recently proposed by The IUPHAR and the new and old nomenclature are outlined here (Collingridge et al. 2009). However, in this thesis we use the nomenclature that has been in use during my studies.

35 25 A) Glutamate Receptors (GluRs) Ionotropic (iglurs) Metabotropic (mglurs) AMPAR NMDAR KAR Group I - III Subunits: GluR1 GluR4 Subunits: NR1 NR2A - D NR3A - B Subunits: KA1-2 GluR5 GluR7 Subunits: mglur1 mglur8 B)

36 Ionotropic glutamate receptors taxonomy and general structure Ionotropic glutamate receptors (iglurs) are ligand-gated ion channels that mediate excitatory neuronal transmission through tightly controlled cation release into the cell (Nowak et al. 1984). This cation entry into the cell through glutamate receptors is particularly important during development and in certain forms of synaptic plasticity that underlie higher order processes, such as learning and memory (for review see: Maren and Baudry, 1995; Asztély and Gustafsson 1996). Ionotropic glutamate channels are permeable to Na + and K +, and in some cases to divalent cations, such as Ca 2+ and Zn 2+ (Mayer and Armstrong, 2004; Dingledine, et al. 1999). There are three types of iglurs: N-methyl-D-Aspartate (NMDA), alpha-amino-3- hydroxy-5methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors (KAR); the names of these receptors come from their pharmacological agonists. Similarity in sequence of genes coding for different iglurs, and in some instances similarities in intron-exon structure, suggest a common evolutionary origin for all of the ionotropic glutamate receptor genes (Suchanek, et al. 1995; Dingledine, et al. 1999). Ionotropic glutamate receptors are composed of several subunits that combine within their family to form functional receptor complexes. A total of 16 different iglurs subunits have been identified. There are seven NMDA subunits (NR1, NR2A NR2D, NR3A NR3B), four AMPA subunits (GluR1 GluR4), and five kainate subunits (KA1, KA2 and GluR5 GluR7) (Figure 3A). Recently a new nomenclature has been proposed for ligand-gated ion channels by The International Union of Basic and Clinical Pharmacology (IUPHAR) (Figure 3B). In their recommendation they proposed a more uniform nomenclature for ionotropic glutamate

37 27 receptors (Collingridge, et al. 2009). Subunits for AMPA receptors are proposed to be labelled GluA1 GluA5; NMDAR receptors subunits are named GluN1, GluN2A-D, and GluN3A-B; and Kainate receptor subunits are named GluK1 GluK5. However, in this thesis we adhere to a nomenclature that has been in use during my studies. The structure of ionotropic glutamate receptors is well known and has been extensively reviewed (Oswald, et al. 2007; Kew and Kemp, 2005; Pin and Duvoisin, 1995). iglurs have three transmembrane domains (M1, M3, M4) that form a functional ion channel, one intramembrane loop that does not traverse the whole lipid bilayer (M2), as well as an extracellular N-terminal, and cytosolic C-terminal (Figure 4). Near the extracellular N- terminal lies the ligand binding domain (S1, S2), whereas the intracellular C-terminal plays a critical role in protein-protein interactions and contains a variety of phosphorylation sites that regulate receptor function and structure. Additionally, C-terminal contains a PDZ interacting domain to which several membrane-associated proteins bind via their PDZ domains. Furthermore, post-transcriptional modifications, such as alternative splicing of mrna and RNA editing, warrants structural and functional diversity of the ionotropic glutamate receptors. For example, the NR1 subunit of the NMDA receptor complex has three alternatively spliced exons, giving rise to eight distinct splice variants that can assemble to form a functional NMDAR complex (Dingledine, et al. 1999). In addition, splice variants in the intracellular C-terminus may bind to different proteins thus influencing receptor targeting, anchoring at the surface, and signal transmission (Dingledine, et al. 1999; Cui, et al. 2007).

38 28 Figure 4 : NMDA Receptor complex subunit composition and structure. A) In the adult brain a functional unit of the NMDA Receptor complex is composed of two NR1 subunits and two NR2 (A-D) subunits. NMDARs are unique among receptors in that they require binding of two agonists for their activation: NR1 subunit binds glycine, whereas NR2 subunit binds glutamate. On the cytosolic side, several proteins bind to the receptor complex through their PDZ interacting motif on the C-terminus. B) Each NMDAR subunit is composed of three transmembrane domains (M1, M3, M4) and one intramembrane loop (M2), which together form a functional pore allowing for Ca 2+ influx. On the N-terminus, located on the extracellular side, lies the ligand binding domain (S1, S2), whereas the cytosolic C-terminus has the PDZ interacting motif. Also presented on the figure are the binding sites of antibodies used in this study. Figures adapted from: