UTILIZATION OF SELENIUM IN THE MOUSE BRAIN: IMPLICATIONS FOR NEUROLOGICAL DISEASE

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1 UTILIZATION OF SELENIUM IN THE MOUSE BRAIN: IMPLICATIONS FOR NEUROLOGICAL DISEASE A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL AND MOLECULAR BIOLOGY (NEUROSCIENCES) AUGUST 2012 By Arjun Venkat Raman Dissertation Committee: Marla Berry, Chairperson Frederick Bellinger Scott Lozanoff Robert Nichols Bruce Shiramizu

2 ABSTRACT Selenium is a chemical element that is an essential micronutrient associated with various aspects of human health. The biochemical activity of selenium is mediated by proteins, which incorporate it into the amino acid selenocysteine (Sec). There are 25 genes in humans encoding Sec-containing selenoproteins. The functionally characterized selenoproteins are oxidoreductase enzymes involved in cellular oxidation-reduction reactions. Most selenoproteins are expressed in the mammalian brain, and dietary selenium deficiency causes preferential retention in brain relative to other body organs. Further, dietary selenium deficiency and specific selenoproteins are associated with various brain diseases. Mouse models have been extensively used to study the function and handling of selenium in mammals. Genetic deletion of a Sec-rich protein in mice causes brain selenium deficiency, neurodegeneration and neurological impairment, and disruption of a phospholipid hydroperoxidase selenoenzyme causes rapid neurodegeneration. Therefore, selenoprotein expression and function promotes a healthy nervous system. However, selenium metabolism and the function of several selenoproteins in brain are not clearly defined. The overall purpose of this work is to clarify the function and utilization of selenium in the mammalian brain, to reveal implications for developmental and neurological diseases. The goal of these studies is to investigate changes in brain function and selenoprotein expression under conditions of altered selenium metabolism in mice. The research presented in this dissertation covers three major topics that are separated into chapters. To investigate selenium distribution in the brain, the neurological consequences of disrupting selenium transport and recycling in mice are assessed and compared. Disruption of selenium transport caused more profound neurological consequences than disruption of selenium recycling. To investigate potential functions of selenium in the nervous system, select selenoproteins were examined for cellular and subcellular expression in cells and brain tissue from transgenic and control mice. Select selenoproteins and synthesis factors were observed at synapses, suggesting localized expression and physiological relevance. To investigate a potential interaction between selenium and methamphetamine, ii

3 expression profiling of selenoproteins in mouse brain after exposure to methamphetamine is described. Selenoprotein synthesis was adversely affected by methamphetamine administration in mice. These results confirm the importance of selenium in the mammalian brain. iii

4 PAPERS ARISING FROM THIS DISSERTATION Published, in press: Raman, A. V., Pitts, M. W., Seyedali, A., Hashimoto, A. C., Seale, L. A., Bellinger, F. P. and Berry, M. J. (2012), Absence of selenoprotein P but not selenocysteine lyase results in severe neurological dysfunction. Genes, Brain and Behavior. doi: /j X x In preparation for publication: Raman, A. V., Pitts, M. W., Hashimoto, A. C., Nichols, R.A., Bellinger, F. P. and Berry, M. J. (2012), Expression of selenoprotein W in neurons extends into processes and is highly dependent on selenoprotein P. In preparation. iv

5 DEDICATION I would like to dedicate this dissertation directly to my parents, Neerja and Vasan Raman, and indirectly to all of my ancestors, family, and friends. Needless to say, nothing about me would have been possible without generations of love, dedication, and perseverance. v

6 TABLE OF CONTENTS ABSTRACT... ii PAPERS ARISING FROM THIS DISSERTATION... iv DEDICATION... v LIST OF FIGURES... viii CHAPTER 1: INTRODUCTION 1.1 Selenium in Chemistry and Biology Synthesis of Selenoproteins Selenoproteins as Oxidoreductase Enzymes Selenoproteins and Metabolism The Selenoprotein Family Selenoproteins in the Nervous System References CHAPTER 2: ABSENCE OF SELENOPROTEIN P BUT NOT SELENOCYSTEINE LYASE RESULTS IN SEVERE NEURLOGICAL DYSFUNCTION 2.1 Abstract Introduction Methods Results Discussion References CHAPTER 3: EXPRESSION OF SELENOPROTEIN W IN NEURONS EXTENDS INTO PROCESSES AND IS HIGHLY DEPENDENT ON SELENOPROTEIN P 3.1 Abstract Introduction Methods Results Discussion References vi

7 CHAPTER 4: METHAMPHETAMINE-INDUCED ALTERATIONS IN SELENOPROTEIN EXPRESSION IN MICE 4.1 Abstract Introduction Methods Results Discussion References CHAPTER 5: CONCLUSION 5.1 Summary and Discussion Oxidative Stress and Selenoproteins Redox Systems and Selenoproteins in the Brain References vii

8 LIST OF FIGURES 1.1 Assimilation of Se into selenoproteins Selenoprotein localization to subcellular domains Spontaneous activity and motor coordination is more reduced in male 65 than female Sepp1-/- mice compared to control mice fed a standard diet 2.2 High Se diet improves spontaneous activity and motor coordination 66 more in male than female Sepp1-/- mice compared to control mice 2.3 Generation of Scly-knockout mice Spontaneous activity and motor coordination is normal in male and 68 female Scly-/- mice fed a low Se diet 2.5 Spatial learning and memory is disrupted in Sepp1-/- mice fed a 69 standard diet 2.6 Spatial learning and memory is not disrupted in Scly-/- mice fed a 70 standard diet 2.7 Spatial learning is mildly impaired in Scly-/- mice fed a low Se diet Expression of selenoprotein transcripts is increased in Se-deficient 72 Scly-/- mice brains 2.9 Expression of selenoproteins is decreased in Se-deficient Scly-/- mice 73 brains 2.10 Glutathione peroxidase activity is decreased in Se-deficient Scly-/- mice 74 brains 3.1 Sepw1 is expressed in cell bodies and processes of cultured neurons Sepw1 is expressed in cell bodies and processes of pyramidal neurons in 96 cortex and hippocampus 3.3 Regional expression of Sepw1 in neurons of mouse brain Sepw1 is present in isolated nerve terminals Sepw1 expression in isolated nerve terminals is greatly reduced in mice 99 lacking Sepp1 3.6 Several selenoprotein synthesis factors are present in isolated nerve terminals 100 viii

9 4.1 Expression of selenoprotein mrnas is altered by methamphetamine Selenoprotein W and selenophosphate synthetase 2 mrna are 117 upregulated in midbrain after two weeks of methamphetamine administration 4.3 Selenoprotein expression following two weeks of methamphetamine 118 administration is not changed in mice brains 4.4 Long term methamphetamine administration dramatically reduces 119 selenoprotein mrnas in striatum 4.5 Sepp1 impacts brain expression of selenoproteins more than 120 methamphetamine 4.6 Methamphetamine reduces tyrosine hydroxylase expression in 121 substantia nigra independently of Sepp1 4.7 Apoptotic cell death is not increased in methamphetamine-treated selenoprotein P knockout mice 122 ix

10 CHAPTER 1 INTRODUCTION SELENIUM IN CHEMISTRY AND BIOLOGY In 1817 the Swedish chemist Jöns Jacob Berzelius discovered the element selenium (Se) after analyzing an impurity that was contaminating the sulfuric acid being produced at a nearby factory. Since the factory workers were suffering an illness caused by Se, it was initially considered to be a poisonous chemical. Nearly 140 years later, Se was identified as an essential micronutrient in humans and livestock (Hatfield, 2006). Se is a trace mineral present in the Earth s crust and ocean water at wide ranging abundances, averaging between 0.05 and 2.0 ppm. Perhaps due to low environmental quantities, Se has a narrow concentration range from deficiency to toxicity in animals. The profound biological effects of Se suggested that it was involved in enzymatic activity. This speculation was confirmed when Se was discovered to be present as selenocysteine in the active site of two unrelated enzymes (Behne et al., 1990, Berry et al., 1991b, Forstrom et al., 1978, Rotruck et al., 1973b). Like other group 16 elements oxygen and sulfur, Se has two unpaired electrons, producing highly reactive atoms. Se is classified a nonmetal but has some metalloid properties. For instance, it is a photoconductive semiconductor, meaning the electrical conductivity is enhanced by light and somewhere in between that of a true conductor and an insulator (Liao et al., 2010). This unique reactivity proved instrumental in production of the first commercial photocopiers made by Xerox, and has been exploited for use in solid-state electronics and photoelectric cells. Se and sulfur share some similar chemical and physical properties, such as electronegativity and atomic radius, however selenium is much heavier. Thus Se and sulfur compounds exist naturally as structurally analogous molecules typically complexed with a metal. These inorganic and organic molecules exist in a variety of oxidation states ranging from -2 to +6, and include selenides (Se 2 ), 1

11 selenites (SeO 3 2 ), and selenates (SeO 4 2 ), as well as the amino acids selenocysteine and selenomethionine, and methylated forms (Johansson et al., 2005). In biological systems, however, the substantial differences between Se and sulfur are highlighted by their non-interchangeable nature. For example, sulfur represents about one quarter of one percent of human body weight and possesses almost no toxicity risk even at very high levels. In contrast, Se is required in nanomolar concentrations, and the median lethal dose of in humans is mg/kg body weight with inorganic salts being more acutely toxic than organic forms (Koller & Exon, 1986). In vertebrates, Se is obtained almost exclusively by ingestion of plants and animals. The amount of Se in dietary sources depends on the concentration of environmental Se in the soil or water. Currently, the recommended daily allowance of dietary Se for healthy adults is 55 µg/day and the upper tolerable intake level is 400 µg/day (Monsen, 2000). Most cellular biological functions of Se are attributed to selenoproteins, which incorporate Se as the amino acid selenocysteine (Sec). Sec is structurally analogous to cysteine with the exception of Se replacing sulfur in the side chain. However, the similarity ends there. Se and sulfur have similar properties, but their differences generate divergent character between Sec and cysteine. Having Sec in the active site of an enzyme generally produces more efficient catalysis (Berry et al., 1992). The cause of the higher catalytic capacity of Sec is still under investigation, and several ideas have been proposed. For example, Sec is described as having stronger nucleophilic and electrophilic character than cysteine, potentially aiding in electron and proton transfer during catalysis. The acid dissociation constant for the selenol (pka = 5.2) of Sec is lower than the thiol (pka = 8.3) of cysteine. This means that at physiological ph, Sec tends towards ionization whereas cysteine tends towards protonation, and at low ph Sec retains much higher reactivity than cysteine. The selenol is further thought to be a superior leaving group due to higher acidity (Johansson et al., 2005). The biological rationale for Sec utilization has been enigmatic. Due to the relative stabilities of each oxidation state of Sec and cysteine near physiological ph, Sec has two 2

12 main advantages over cysteine in terms of reaction with oxygen. Kinetic calculations suggest that oxidized Sec, both selenenic and seleninic acid, will cycle more rapidly to the reduced form than oxidized forms of cysteine, sulfenic and sulfinic acid. Second, further oxidation to selenonic acid is extremely unfavorable, unlike irreversible oxidation of cysteine to sulfonic acid. The actual reactivity of the residue will ultimately depend on the context of the Sec within the local and global structure of the peptide and enzyme. However, these unique reactive properties may potentially explain the biological pressure to use Sec in proteins during evolution (Hondal & Ruggles, 2011). SYNTHESIS OF SELENOPROTEINS Beyond the chemistry, Sec is an extremely unusual amino acid, and can be considered an anomaly in the genetic code. Roughly defined, the genetic code is the system in which genetic information is first transcribed from deoxyribonucleic acid (DNA) to messenger ribonucleic acid (mrna), and subsequently interpreted by ribosomes and several transfer RNAs (trnas) to produce polypeptides. Three-nucleotide sequences in mrna termed codons specify binding to various trnas, each carrying single amino acids to be positioned during protein synthesis. This system of mrna translation also uses initiation and termination codons to determine the precise peptide sequence. Termination codons typically recruit proteins called release factors rather than a trna, thus dissociating the ribosomal subunits and releasing the peptide chain. In discordance with the standard rules of the system, the opal termination codon specified by UGA can be recognized by a specific trna (trnasec) (Diamond et al., 1981). However, in order for trnasec to efficiently recognize the in-frame UGA codon, the mrna must additionally contain a Sec insertion sequence (SECIS). The SECIS element is a stem-loop structure downstream of the UGA codon in selenoprotein mrnas that recruits proteins, which facilitate recoding of the UGA and incorporation of Sec (Fig. 1) (Berry et al., 1991a). Although selenoproteins are present in eukarya, archaea, and bacteria, the mechanism of Sec incorporation differs between domains in several respects. For example, the bacterial SECIS element is located immediately following the Sec UGA codon, whereas the 3

13 eukaryotic and archaeal SECIS elements are typically located much farther downstream in the 3 untranslated region (UTR) of the transcript. Additionally, the archaeal genus Methanococcus has a 5 UTR SECIS element, while eukaryotes have two distinct forms of 3 UTR SECIS elements (Kryukov et al., 2003, Kryukov & Gladyshev, 2004, Wilting et al., 1997). An elongation factor specific to trnasec has been identified as selb in bacteria. In eukaryotes, the coordinated function of a SECIS binding protein (SBP2) and a Sec-specific elongation factor (EFSec) allow the co-translational incorporation of Sec instead of termination (Papp et al., 2007). Biosynthesis of Sec in eukarya and archaea is accomplished by the sequential actions of O-phosphoseryl-tRNA kinase (PSTK) and O- phosphoserine-trna:sec-trna synthase (SEPSECS), which convert seryl-trnasec to phosphoseryl-trnasec and ultimately to selenocysteinyl-trnasec. Thus Sec formation in archaea and eukarya is a two-step process, whereas bacterial selenocysteine synthase (sela) can synthesize Sec directly from seryl-trnasec. The Se donor utilized by both SEPSECS and sela is selenophosphate, which is generated from selenide (H 2 Se) by selenophosphate synthetase (SPS) enzymes. Selenophosphate is first transferred to O- phosphoseryl-trnasec by SEPSECS while displacing the phosphoseryl moiety, and subsequent hydrolysis of the phosphate group yields Sec charged to its cognate trna (Palioura et al., 2009). Bacteria have one SPS enzyme termed seld, while two (SPS1, SPS2) have been identified in eukarya. Selenophosphate-synthetase 2 (SPS2) is a eukaryotic selenoprotein that is required for the synthesis of all selenoproteins including itself, and may provide feedback for global selenoprotein synthesis. Selenophosphate is generated by SPS2 in the presence of selenide and ATP (Guimaraes et al., 1996). A related protein called SPS1 contains a cysteine residue in place of Sec, but its involvement in Sec and selenoprotein biosynthesis is uncertain (Low et al., 1995). Intriguingly SPS2, but not SPS1, is required for selenoprotein synthesis in NIH3T3 mouse fibroblasts (Xu et al., 2007). Studies in Drosophila melanogaster, indicate that the function of SPS1 is primarily in metabolism of vitamin B6, but whether this holds true for vertebrates is unknown. It has alternatively been suggested that SPS2 assimilates selenite, whereas SPS1 recycles Sec in a Se-salvage pathway (Tamura et al., 2004). However an in vivo requirement of SPS2 and specificity of SPS1 and SPS2 with different Se substrates have not been reported. Selenocysteine lyase (SCLY) is a putative Se 4

14 recycling enzyme, which is able to catalyze the hydrolysis of Sec into selenide and alanine (Esaki et al., 1982). It facilitates Se incorporation into selenoproteins when Sec is the Se source, and may deliver selenide to SPS enzymes for phosphorylation and subsequent insertion into nascent selenoproteins (Kurokawa et al., 2011, Tobe et al., 2009). Pyridoxal phosphate is a prosthetic group derived from vitamin B6 and is required by enzymes involved in metabolism of amino acids, glucose, lipids and neurotransmitters. The enzymatic activity of both SCLY and SEPSECS are pyridoxal phosphate-dependent, implying that Sec metabolism shares regulatory elements with standard amino acid metabolic pathways (Lacourciere et al., 2000, Mihara et al., 2000, Palioura et al., 2009). Several steps in selenoprotein synthesis are regulated by Se availability (dietary or environmental depending on the organism), as well as by the local oxidation state. For example in addition to being a limiting substrate for SPS2, Se levels affect different SECIS elements differentially, potentially regulating selenoprotein synthesis efficiency at the level of translation (Papp et al., 2007). Oxidation of the cysteine-rich, redox-sensitive domain of SBP2 masks the nuclear-export signal (NES) causing importation into the nucleus. Subsequently it is either sequestered there during high oxidative burden, or else reduced by nuclear-specific isoforms of selenoproteins, unmasking the NES for binding and exportation by the nuclear export receptor CRM-1 (Papp et al., 2006). Individual selenoprotein expression also responds differentially to Se-availability and oxidative stress, providing another level of regulatory control for selenoprotein synthesis. Thus there is a complex, nonlinear interaction between Se-status and oxidative burden that coordinates the synthesis of the numerous selenoproteins. Despite a low environmental availability of Se and the various elaborate mechanisms for Sec incorporation, selenoproteins are widespread in organisms, with certain plants and fungi being the only major exceptions that lack selenoproteins and an essential biological function for Se (Lobanov et al., 2009). 5

15 SELENOPROTEINS AS OXIDOREDUCTASE ENZYMES The human genome codes for 25 selenoproteins, most of which have been identified recently by bioinformatics approaches looking for SECIS elements downstream of inframe UGA codons (Kryukov et al., 2003). The various members display wide subcellular and tissue distribution, and several are known to have multiple transcript variants and protein isoforms. Functionally, selenoproteins are closely linked with the cellular thiol-disulfide couples, particularly the glutathione (GSH) and thioredoxin (TXN) couples. GSH is a tripeptide made of glycine, cysteine, and glutamate and is the most abundant thiol in cells, present at millimolar concentrations. Oxidation of the cysteine thiol links two molecules of GSH to form glutathione disulfide (GSSG). This reaction can be spontaneous in the presence of electrophiles or alternatively can be catalyzed by a number of enzymes that utilize GSH as an electron donor including glutathione peroxidase (GPX), glutaredoxin, and glutathione S-transferase enzymes. Reduction of GSSG, producing two molecules of GSH, is performed by the homodimeric flavoenzyme glutathione reductase. All of the characterized selenoproteins that function as enzymes are oxidoreductases that catalyze thiol-disulfide oxidation-reduction (redox) reactions and contain Sec in the active site. The first Se-dependent enzyme discovered was glutathione peroxidase 1 (GPX1), which catalyzes the reduction of hydrogen peroxide (H 2 O 2 ) by oxidation of two molecules of GSH to GSSG (Rotruck et al., 1973a). Cytosolic and mitochondrial forms of the GPX1 enzyme are transcribed from the same gene containing one Sec-encoding TGA. The enzyme functions as a homotetramer utilizing four Se atoms per active enzyme (Esworthy et al., 1997). GPX1 is the most abundant glutathione peroxidase and the most abundant selenoprotein in rats, representing a significant fraction of the total circulating Se pool (Hawkes et al., 1985). Four homologous GPX selenoproteins have subsequently been identified in humans. GPX2, also known as gastrointestinal GPX, is a cytosolic enzyme that is specific to epithelial cells and is abundant in the gut (Chu et al., 1997). The extracellular GPX3 has 6

16 broad substrate specificity and is found in most extracellular compartments but is abundant in kidney and blood plasma (Olson et al., 2009). GPX6 is closely related to GPX3 but is only expressed in the olfactory system, and exists as a cysteine homolog in rodents (Kryukov et al., 2003). GPX4 is structurally and functionally different from the other GPX enzymes because it is active as a monomer rather than a tetramer, and can directly reduce membrane lipid hydroperoxides and free fatty acid hydroperoxides. Alternative splicing and transcription initiation generates three distinct isoforms of GPX4 that localize to the cytosol, mitochondria, and nucleus (Maiorino et al., 2003). Additionally, GPX4 translation is regulated by the mrna binding proteins guanine-rich sequence-binding factor 1 (Ufer et al., 2008) and PARK7/DJ-1 (Blackinton et al., 2009, Van Der Brug et al., 2008). Genetic deletion of GPX4 in mice causes embryonic lethality and knockdown of GPX4 in cells leads to rapid lipoxygenase-mediated lipid peroxidation and subsequent apoptosis, suggesting that removal of lipid hydroperoxides by GPX4 is essential for cell viability (Seiler et al., 2008, Yant et al., 2003). There are three additional GPX enzymes (GPX5, GPX7, and GPX8) that are not selenoproteins in humans. TXN is a small protein of ~12 kda and is present at concentrations several orders of magnitude below GSH. It contains an active site dithiol that is highly conserved in evolution and widely distributed among the TXN superfamily of proteins, which includes protein disulfide isomerase enzymes. Through oxidation of the dithiol to a disulfide, TXN can directly reduce cysteine sulfenic acids and control the state of dithiol-disulfide motifs in target proteins, and can also serve as an electron donating cofactor for enzymes such as ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductases (Arner & Holmgren, 2000). In turn, reducing oxidized TXN is mediated exclusively by the thioredoxin reductase (TXNRD) family of selenoproteins (Zhong et al., 2000). At least four selenoproteins (GPX3, GPX4, SEPP1, SEPX1) can utilize TXN as a cofactor for enzymatic reduction, and it is possible that others do as well (Bjornstedt et al., 1994, Takebe et al., 2002). There are numerous thioredoxin-like proteins that may depend on TXN or act in parallel to provide additional substrate specificity beyond that provided by TXN. Several selenoproteins contain a TXN-like fold, which is a well described 7

17 secondary/tertiary structure pattern with a conserved Cys-X-X-Cys or Cys-X-X-Ser/Thr active-site motif characteristic of oxidoreductases (Dikiy et al., 2007), where X is any amino acid. It is tempting to speculate that these selenoproteins operate similarly to TXN, namely by controlling the redox state of cysteine residues and dithiol motifs, but there is little evidence to support or deny this notion at present. Three mammalian thioredoxin reductases are selenoenzymes encoded by individual genes. TXNRD1, TXNRD2, and TXNRD3 encode homodimeric flavoproteins that localize to the cytosol, mitochondria, and testes respectively. They are members of the pyridine nucleotide-disulfide oxidoreductase family and contain two redox-sensitive sites in the N- and C-terminus that interact in the head to tail dimer conformation of the active enzyme. These enzymes are capable of reducing a number of substrates, but depend on NADPH for donating electrons, which are first transferred to the FAD group, then passed to the N-terminal dithiol of one subunit and subsequently to the C-terminal selenenylsulfide of the other subunit. The highly conserved Sec-containing C-terminal motif is absolutely critical for catalytic function of TXNRD enzymes (Arner & Holmgren, 2000, Hatfield, 2006). The main substrate for TXNRDs is the small redox-sensitive protein TXN, which is integral to physiological processes such as cell communication, metabolism, proliferation, and apoptosis. In general, the reactive dithiol of TXN will become oxidized to a disulfide during reduction of an oxidized target protein. Regeneration of reduced TXN proteins requires TXNRD, and thus the TXN/TXNRD system is completely dependent on Se in mammals. The importance of this system is highlighted by the fact that knockout of either TXNRD1 or TXNRD2 is embryonic lethal in mice (Conrad et al., 2004, Jakupoglu et al., 2005). It is worth noting that TXNRDs from mammals differ from the Se-independent enzymes of archaea, bacteria, yeast, and plants. Reactions between target proteins and TXN can be spontaneous, but several enzymes can catalyze the reduction of target proteins using TXN as an electron donating cofactor. The human genome codes for four Methionine Sulfoxide Reductase (MSR) enzymes that reduce oxidized methionine residues in proteins utilizing TXN as a cofactor. There is 8

18 now considerable evidence that like cysteine, reversible methionine oxidation can regulate protein function (Levine et al., 2000). For example, calcium/calmodulindependent protein kinase II and the phosphatase calcineurin, among many other proteins, are regulated by methionine redox status (Agbas & Moskovitz, 2009, Erickson et al., 2008). A single MSRA and three MSRB enzymes stereo-specifically reduce S- and R- sulfoxidated methionines respectively. MSRB1 is a selenoprotein also known as Selenoprotein R and SEPX1, while MSRA as well as MSRB2 and MSRB3 are Seindependent enzymes. SEPX1 is a zinc-containing protein present in the cytosol and nucleus and exhibits the highest methionine-r-sulfoxide reductase activity because of the presence of Sec in its active site (Kim & Gladyshev, 2004). Interestingly, redox status of the cysteine-rich metallothionein/thionein couple dictates zinc loading in that reduced thionein binds zinc and oxidation of metallothionein releases it. Moreover, reduction of non-selenoprotein MSRB3 by TXN, TXNRD, and NADPH is more efficient in the presence of thionein (Sagher et al., 2006). Therefore regulation of specific kinases, phosphatases, and other proteins by methionine-r-sulfoxide reduction is mediated by two selenoproteins (MSRB1, TXNRD1) and NADPH. SELENOPROTEINS AND METABOLISM Specific selenoproteins function at the intersection of cellular and organism metabolism by modulating insulin and thyroid hormone signaling. The iodothyronine deiodinases (DIO) function in activation and deactivation of thyroid hormone and were the second family of enzymes determined to be Sec-containing selenoenzymes (Berry et al., 1991b). Thyroid hormone metabolism both at the level of production in the thyroid and local hormone activity in the periphery is reliant on the DIO family of selenoenzymes. Most vertebrates have three DIO enzymes that can deiodinate thyroid hormones to control local availability. These integral membrane protein enzymes are thiol-requiring oxidoreductases that remove iodine atoms from the aromatic rings of thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rt3) (Bianco & Kim, 2006). DIO1 is a plasma membrane protein found mainly in cells of the liver and kidney, is capable of deiodinating both the inner and outer rings, and produces most of the circulating T3. 9

19 DIO2 is found in the endoplasmic reticulum of cells in several tissues including the thyroid, heart, skeletal muscle, fat, and the central nervous system and selectively removes the outer ring iodine, making it the primary tissue activator of thyroid hormone by converting T4 to T3. DIO3 is also a plasma membrane protein, however it is mainly found in fetal tissue and the placenta, selectively removes the inner ring iodine, and thus contributes to thyroid hormone inactivation. A specific role for a selenoprotein in redox regulation of insulin signaling was established when it was found that overexpression of GPX1 causes hyperinsulinemia and insulin resistance in mice (Mcclung et al., 2004). Moreover genetic deletion of GPX1 promotes glucose tolerance and insulin sensitivity in mice on a high-fat diet by enhancement of insulin-induced PI3K/Akt signaling (Loh et al., 2009). Dietary studies in humans have further suggested that supranutritional levels of Se are associated with type II diabetes, while animal studies confirm that both excessive dietary Se and GPX1 overexpression lead to hyperinsulinemia and insulin resistance (Labunskyy et al., 2011, Lippman et al., 2009, Stranges et al., 2007, Wang et al., 2008). Peroxide-induced oxidation of PTEN and S-glutathionylation of protein tyrosine phosphatase 1B are affected by GPX1 activity, which thereby modulates insulin receptor activation and insulin resistance (Mueller et al., 2009). Selenoprotein P (SEPP1) is a unique selenoprotein that contains multiple Sec residues and is also implicated in insulin resistance. Diabetic patients display an increase in hepatic SEPP1 mrna and serum SEPP1 protein, and purified SEPP1 administered to mice is able to induce insulin resistance and glucose intolerance. Furthermore, knockdown or knockout of SEPP1 in mice improves glucose tolerance and insulin sensitivity, and SEPP1 knockout mice are protected against glucose intolerance and insulin resistance even when on an obesity-inducing diet (Misu et al., 2010). Since SEPP1 expression can dictate expression of other selenoproteins including GPX1, its effect on insulin resistance may be direct or indirect. Primate and rodent SEPP1 contains up to ten Sec residues while as many as 17 Sec residues are present in zebrafish. SEPP1 is the most abundant selenoprotein in blood and accounts for as much as 65% of plasma 10

20 Se in rats (Burk & Hill, 1994). The abundant Sec residues of SEPP1 are divided into two regions with the bulk being located in the C-terminal domain that is required for the Se transport function. SEPP1 also contains an N-terminus Cys-X-X-Sec motif and catalyzes the reduction of lipid hydroperoxides in vitro utilizing TXN as a cofactor (Saito et al., 1999, Takebe et al., 2002). Aside from the high Se content in the form of Sec residues, SEPP1 is distinct in that it is one of two extracellular selenoproteins (the other being GPX3), and the carboxy-terminal Se transport domain appears to be a metazoan adaptation (Lobanov et al., 2008). SEPP1 is abundantly produced by the liver and secreted into blood, however local production and secretion in nearly all tissue systems has been described (Burk & Hill, 2005). Bodily transport of Se to extrahepatic tissues, particularly the brain, testes and kidneys, is facilitated by receptor-mediated uptake of SEPP1 by the low-density lipoprotein receptor family members ApoER2 (LRP8) and Megalin (LRP2) (Burk & Hill, 2009). In addition to a Se transport function and peroxidase activity, SEPP1 exhibits ph-dependent heparin binding and heavy metal binding that likely also function in redox-dependent processes. In our lab, selenocysteine lyase (SCLY) has also shown to be involved in metabolic syndrome in an in vivo mouse model. Although SCLY is not itself a selenoprotein, it is an enzyme with high specific activity for Sec, and liberates selenide (Mihara et al., 2000). Since selenoproteins are a reservoir of Sec and SEPP1 supplies Se in the form of Sec, SCLY is a potentially relevant factor in the global distribution of Se and the expression of all selenoproteins. Transgenic mice lacking SCLY were characterized and found to have altered energy metabolism in the liver. When given adequate Se, the animals exhibit hyperinsulinemia and mild hepatic steatosis, along with an increase in blood SEPP1. Upon dietary Se restriction, the animals develop obesity, fatty liver, hypercholesteremia, and insulin resistance (Seale, et al. 2012, unpublished). The results of studies on mice with disrupted GPX1, SEPP1, and SCLY do not reconcile with a simple explanation. Instead, they suggest a nonlinear, dynamic explanation. Regardless, these data demonstrate the importance of proper Se handling in vertebrate carbohydrate and lipid metabolism. Se utilization is tightly integrated with insulin and thyroid hormone signaling, thus disruption of selenoproteins and Sec-related proteins alters vertebrate 11

21 metabolic systems. A connection between Se and metabolism in multicellular organisms suggests that cell-autonomous regulation of redox systems and signaling may similarly depend on one or more selenoproteins. THE SELENOPROTEIN FAMILY Several selenoproteins with uncertain functions could have a role in regulating target protein oxidation state. The regulatory control of cellular redox signaling by Selenoprotein W (SEPW1) will be discussed next as an example, bearing in mind that selenoproteins with unknown roles can impact the cellular response to environmental changes, particularly in relation to growth and stress. Following the section on SEPW1, a brief summary of what is known of the remaining selenoproteins is presented, with the biological functions described possibly owing to the activity of the selenoproteins in undefined redox circuits. SEPW1 was purified in the early 1990s but putatively identified much earlier due to its absence in Se-deficient lambs suffering a myopathy called White Muscle Disease (Vendeland et al., 1993). Mammalian SEPW1 is a highly conserved cytosolic protein of just 87 amino acids, and SEPW1 orthologs are among the most widely distributed selenoproteins in all species including prokaryotes (Kryukov & Gladyshev, 2004, Zhang et al., 2005). The expression level of SEPW1 in vertebrates is very sensitive to dietary Se intake as well as the expression level of SEPP1 (Hoffmann et al., 2007, Vendeland et al., 1995, Yeh et al., 1995). Abundant SEPW1 expression is observed in muscle, and SEPW1 transcription during myocyte differentiation is maintained by binding of the myogenic transcription factor MyoD to the SEPW1 promoter (Noh et al., 2010). A putative metalresponse element in the promoter of the SEPW1 gene was probed in vitro using a luciferase reporter fusion construct, and luciferase specific activity was found to be stimulated by copper and zinc, but not cadmium (Amantana et al., 2002). Although a bona fide enzymatic activity has not been attributed to SEPW1, the presence of a Cys-X- X-Sec motif in a thioredoxin-like fold may indicate thioredoxin-like redox activity (Dikiy et al., 2007). 12

22 Recently, SEPW1 was shown to pull-down and co-immunoprecipitate with the beta and gamma isoforms of protein. This interaction was further confirmed by NMR spectroscopy, and extended to identify three loops of SEPW1 that interact with proteins (Aachmann et al., 2007) beta and gamma proteins are scaffolding proteins derived from the YWHAB and YWHAG genes respectively, and bind a diverse assortment of proteins including kinases, phosphatases, and receptors. In this way proteins coordinate molecular interactions and participate in cell cycle regulation, metabolism, apoptosis, protein trafficking and gene transcription (Fu et al., 2000). A computational study of SEPW1/ interaction suggests that a conserved cysteine of beta and gamma (Cys191 and Cys195 respectively) can be reversibly oxidized, with SEPW1 acting as a reducing agent (Musiani et al., 2010). The oxidized cysteine sulfenic acid of can putatively react with Sec of SEPW1, producing a mixed complex. Subsequently the formation of an intramolecular selenenyl-sulfide within SEPW1 would result in being fully reduced. Oxidized SEPW1 can then migrate away and likely be reduced to its parent state by GSH. This speculated reduction is supported by evidence that a SEPW1 cysteine residue conserved from rodents to primates can be S-glutathionylated (Beilstein et al., 1996, Gu et al., 1999). Redox regulation of proteins by SEPW1 could serve several cellular functions, but an intriguing possibility is presented by the in vitro finding that SEPW1 expression is regulated by the cell-cycle, and knockdown of SEPW1 induces p53-dependent cell-cycle arrest (Hawkes et al., 2009). Recently, it has been demonstrated that growth factorinduced receptor tyrosine kinase phosphorylation, and downstream JNK and p38 MAPK signaling, leading to cell proliferation requires SEPW1 (Hawkes, WC; personal communication). Therefore SEPW1, through redox regulation of proteins, may coordinate Se availability and oxidative burden with cellular proliferation, differentiation and death via the p53 pathway. Deficiency of SEPW1-mediated redox functionality may serve as the basis for the myopathies in livestock (Whanger, 2009). Interestingly SEPW1 is also associated with multiple myeloma in humans, where SEPW1 overactivity may promote malignancy and the overangiogenic phenotype of endothelial cells in active 13

23 disease (Ria et al., 2009). These studies further suggest a pivotal role for SEPW1 and Se in regulatory control of the cell cycle. A subset of selenoproteins is observed in mitochondrion to combat against electron leak during oxidative respiration and phosphorylation. Mitochondria-specific isoforms of GPX1, GPX4, and TXNRD2 regulate peroxide metabolism and oxidative tone within this organelle. The selenoproteins GPX4, TXNRD1, MSRB1, and SELH have been shown to exhibit varying degrees of nuclear localization (Fig. 2). Apart from SELH, the other three selenoproteins are presumably involved in reduction of lipid peroxides, oxidized TXN, and sulfoxidized methionine residues within the nuclear envelope. SELH is the only DNA-binding selenoprotein described and has a role in regulation of gene expression. Similar to SEPW1, Selenoprotein H (SELH) is a small selenoprotein that is highly expressed during development and is sensitive to dietary Se intake (Kipp et al., 2009, Novoselov et al., 2007). Like several selenoproteins it contains a Cys-X-X-Sec sequence within a thioredoxin-like fold, but unlike any other selenoprotein described to date, it is a DNA-binding protein of the AT-hook family. SELH is primarily located in the nucleus and is implicated in redox-sensitive transcription of genes whose products are involved in de novo glutathione synthesis and phase II detoxification (Panee et al., 2007). Multiple metal-response elements are present in the SELH gene (Stoytcheva et al., 2010), and one group has confirmed in vivo that SELH mrna and protein are upregulated under conditions of elevated copper in mouse liver (Burkhead et al., 2010). Although it is a nuclear protein, mitochondrial biogenesis and function are also linked with SELH. Overexpression of SELH in a transformed neuronal cell line attenuates the UVB-induced increase of p53 protein and caspase-mediated apoptosis (Mendelev et al., 2009). Additionally SELH overexpression increases mitochondrial size, cytochrome c content, and expression of mitochondrial biogenesis proteins while boosting respiration (Mendelev et al., 2011). Collectively these findings suggest that SELH is a Se- and metal-regulated selenoprotein that is able to transduce oxidant signals by modulating gene expression in conjunction with other redox-sensitive transcription factors. Further 14

24 investigation is warranted to determine if SELH modifies cysteine S-glutathionylation or disulfide formation in target proteins such as p53 to regulate gene expression. The endoplasmic reticulum (ER) regulates the synthesis, folding, and transport of proteins, and additionally constitutes the main intracellular store for calcium ions, which are integral in cell signaling. Seven selenoproteins are enriched in the ER (Fig. 2) and some are postulated to have a role in protein folding and ER calcium handling, since oxidative mechanisms within the ER are known to regulate these processes (Shchedrina et al., 2010). The ER is a relatively oxidizing environment compared to other intracellular organelles and contains oxidase enzymes to facilitate the formation of disulfide bonds in proteins destined for export. Simultaneously GSH and TXN system components are transported into the ER, providing both oxidation and reduction mechanisms for dynamic redox regulation associated with protein processing and secretion. Redox state affects calcium homeostasis by modulating ER calcium channels and chaperones, and oxidative stress and ER-stress are intimately related in signaling for apoptosis (Gorlach et al., 2006). Similar to the ER, the secretory and endosomal/lysosomal pathways are also more oxidized than other subcellular compartments (Austin et al., 2005, Hwang et al., 1992). Ligand binding to various receptors stimulates endocytosis of redox-active endosomes whose luminal redox activity directs spatiotemporally-regulated signaling and prevents nonspecific redox reactions (Li et al., 2006, Oakley et al., 2009). Therefore redoxmediated processes are vital for secretory and endocytic function, and the ability of selenoproteins to transmit oxidative signals from reactive intermediates to disulfide bonds or exposed thiols of target proteins may help to explain the enrichment of selenoproteins in the ER. Selenoprotein T (SELT) is an ER- and Golgi-localized selenoprotein that is ubiquitously expressed from development through adulthood, and shares some sequence similarity with SEPW1 and SELH including the thioredoxin-like fold containing a Cys-X-X-Sec motif (Dikiy et al., 2007). Deficiency of SELT in murine fibroblasts causes an upregulation of SEPW1, in addition to altering cell adhesion and redox regulation (Sengupta et al., 2009). A biological role for SELT in neuroendocrine secretion and 15

25 calcium mobilization in vitro has also been presented. SELT was identified as a target gene of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), and manipulation of SELT expression altered PACAP-induced intracellular Ca 2+ changes and growth hormone secretion (Grumolato et al., 2008). Furthermore, PACAP regulation of SELT is associated with ontogenesis, tissue maturation and regeneration of nervous, endocrine, and metabolic tissues (Tanguy et al., 2011). Two of the more abundant and ubiquitous ER selenoproteins, Selenoprotein M (SELM) and 15-kDa selenoprotein (SEP15), are both 15-kDa proteins that share 31% sequence homology and have Cys-X-X-Sec and Cys-X-Sec redox motifs respectively. SELM and SEP15 have thioredoxin-like tertiary structure and homology to protein disulfide isomerases that suggest oxidoreductase activity, but direct evidence in support of this notion is lacking (Ferguson et al., 2006). SEP15 associates with UDPglucose:glycoprotein glucosyltransferase (UGTR), a protein involved in protein conformation quality control, and has been suggested to facilitate proper protein folding (Labunskyy et al., 2005). SEP15 knockout mice have been generated, and the only major phenotype found was a predispostion to cataract formation that was suspected to be due to improper folding of lens proteins. A role for SELM in protein folding has been proposed, and recent evidence suggests that SELM may also be involved in regulating the flux of calcium ions. Overexpression of SELM in a neuronal cell line in vitro reduced peroxide-induced calcium influx, whereas knockdown of SELM increased the baseline intracellular calcium concentration (Reeves et al., 2010). Selenoprotein N (SEPN1) is a large (70 kda) single-spanning transmembrane protein localized to the ER membrane with two known isoforms generated by alternative splicing of exon 3 (Moghadaszadeh et al., 2001). Several congenital muscular dystrophy syndromes such as multiminicore disease, rigid spine muscular dystrophy, and desminrelated myopathy with Mallory body-like inclusions are directly associated with mutations in the SEPN1 gene and have been classified as SEPN1-related myopathies (Ferreiro et al., 2004). Interestingly, mutations in SEPN1 leading to congenital fiber-type disproportion are associated with insulin resistance (Clarke et al., 2006). To date, SEPN1 16

26 is the only selenoprotein gene in which mutations are directly and causatively linked to human disease. An in vivo study using zebrafish determined that SEPN1 associates with ER/SR ryanodine receptors, and that this interaction is necessary for the calcium-induced release of calcium from intracellular stores (Jurynec et al., 2008). Ryanodine receptor channels are homotetramers with several redox-regulated cysteine residues and SEPN1 contains a Cys-Sec-Gly-Ser motif, suggesting that SEPN1 regulates ryanodine receptormediated calcium flux in muscle by redox-dependent signaling. Finally, SEPN1 is integral to the generation and/or maintenance of skeletal muscle satellite cells, which are an adult stem cell population involved in muscle growth and regeneration (Castets et al., 2011). Selenoprotein K (SELK) and Selenoprotein S (SELS) are also predominantly ERlocalized single-spanning transmembrane proteins, however they are much smaller than SEPN1, and also show some localization to the plasma membrane (Chen et al., 2006, Kryukov et al., 2003, Ye et al., 2004). Both are widely expressed in a variety of tissues and have been implicated in the cellular response to ER-stress. Specifically, ER-stress agents regulated the expression of SELK in HepG2 hepatoma cells, and knockdown of SELK exacerbated cell death when challenged with ER-stress (Du et al., 2010). Genetic deletion of SELK in mice decreases receptor-mediated calcium flux in immune cells, impairs calcium-dependent immune cell function, and increases West Nile virus-induced lethality (Verma et al., 2011). An interesting link between metabolism and inflammation is presented in the case of SELS, which was originally identified as a glucose-regulated protein in a rodent model of diabetes (Walder et al., 2002). The relationship between SELS and type II diabetes was confirmed in humans, and there is evidence that SELS can be secreted from liver and identified in blood sera where it associates with LDL (Gao et al., 2007, Karlsson et al., 2004). SELS is now known to also be regulated by inflammatory cytokines (Gao et al., 2006) and reciprocally, reduced expression of SELS, due to polymorphisms in the gene promoter, influences the levels of IL-1, TNFα, and IL- 6 (Curran et al., 2005). SELS participates in removal of misfolded proteins from the ER lumen (Ye et al., 2005, Ye et al., 2004) and was demonstrated to prevent ER-stress and have anti-apoptotic function in macrophages and astrocytes (Fradejas et al., 2008, Kim et 17

27 al., 2007). Further work in mice indicates that in brain, SELS is mainly expressed neuronally under basal conditions, but is intensely upregulated in reactive astrocytes following brain injury (Fradejas et al., 2011). Three selenoproteins remain largely unexplored with very little published data currently available. The sequences of selenoprotein I (SELI), selenoprotein O (SELO), and selenoprotein V (SELV) were identified in the human genome several years ago, however almost no information is available on the cellular localizations or physiological functions of these selenoproteins. SELI mrna is known to be expressed in several tissues, and is a putative transmembrane protein hypothesized to function in phospholipid biosynthesis based on the presence of a CDP-alcohol phosphatidyltransferase motif that is conserved in phospholipid synthases (Horibata & Hirabayashi, 2007, Kryukov et al., 2003). SELO is predicted to be a 669 amino-acid selenoprotein containing a Cys-X-X- Sec motif, but experimental data demonstrating a redox function is unavailable (Kryukov et al., 2003). SELV appears to be a testes-restricted protein with a predicted thioredoxinlike fold housing a Cys-X-X-Sec motif, and also has some sequence homology with SEPW1, SELH, and SELT (Kryukov et al., 2003). SELENOPROTEINS IN THE NERVOUS SYSTEM The nervous system has a unique relationship with Se. This is most clearly demonstrated by the fact that dietary Se deprivation causes a much greater drop in peripheral tissue Se concentration than in brain Se concentration. In other words, the brain preferentially retains Se compared to other organs during times of deficiency, suggesting that it has some essential function in brain (Chen & Berry, 2003). Additionally, nearly all selenoproteins are expressed in the brain, and neurons appear to be the major functional sites of selenoprotein expression (Zhang et al., 2008). This organ-specific prioritization of Se to the brain is at least partially explained by receptor-mediated uptake of SEPP1 in a tissue specific manner. This unique selenoprotein is only found in metazoans, and has high Se content with as many as 28 18

28 Sec residues in the sea urchin Strongylocentrotus purpuratus (Lobanov et al., 2008). The C-terminal region of SEPP1, containing nine of the ten Sec residues in primates and rodents, is involved in maintaining stable brain Se concentration during dietary variation. SEPP1 is thought to be primarily produced in the liver and secreted to blood to transport Se, however hepatic SEPP1 deficiency does not alter brain Se levels (Schweizer et al., 2005). Rather, Se levels in brain, particularly hippocampus, are lowered by genetic deletion of full-length SEPP1 or the C-terminus (Hill et al., 2007, Nakayama et al., 2007). These mutations cause brain Se concentration to drop by a greater extent than can be achieved by dietary Se deficiency, providing further evidence that SEPP1 facilitates a steady supply of Se to the brain. Mice lacking SEPP1 develop sensory, motor, and cognitive neurological impairment including spasticity, hyperreflexia, gait disruption, and a spatial learning deficit. These animals also display widespread neurodegeneration which, along with the behavioral phenotype, is modulated by dietary Se status (Burk & Hill, 2009). However, Sesupplemented SEPP1-deficient mice still exhibit a profound deficit in synaptic long term potentiation (Peters et al., 2006). This suggests that beyond Se delivery, SEPP1 may function in cell signaling, which is established for the SEPP1 receptor ApoER2. ApoER2-deficient mice develop a phenotype similar to but less severe than SEPP1 knockout mice in terms of brain Se concentration and neurological impairment (Burk et al., 2007). Alternatively, SEPP1 could be a preferred Se source within brain that is not readily compensated for by dietary sources. Indeed Se supply to cultured Jurkat cells, assessed by stimulation of GPX activity, is times more efficient with SEPP1 than other Se-containing proteins and compounds (Saito & Takahashi, 2002). ApoER2 is a member of the low-density lipoprotein receptor-related protein family that is highly expressed in brain. It exists in multiple isoforms, and has been observed in all brain cell types (Fan et al., 2001, Korschineck et al., 2001). Expression at some excitatory synapses, where it is associated with the postsynaptic densities, can cause formation of a functional complex with N-methyl-D-aspartate (NMDA) receptors (Beffert et al., 2005). This association allows ApoER2 to modulate NMDA receptor 19

29 activity, synaptic neurotransmission, and memory in mice. Although these effects have been demonstrated using a different ApoER2 ligand, Reelin, alterations in synaptic plasticity and memory in SEPP1-deficient mice suggest that both ligands can actively modulate synaptic function. The central nervous system is protected from drugs, peptides, and other substances in the peripheral circulation by the blood-brain-barrier (BBB). It is chiefly composed of tight junctions between endothelial cells of the cerebral vasculature. Additionally, the vast majority of cerebral vessels are lined by astrocytic processes termed end-feet. This anatomical microarchitecture provides a high trans-endothelial electrical resistance, and prohibits passage of the vast majority of circulating blood components, except for small, lipophilic molecules. Since blood constituents, chiefly oxygen and glucose, are absolutely essential for nervous system function, the BBB contains an array of transmembrane proteins that transport required substrates (Zlokovic, 2008). Before the knowledge of particular receptors for SEPP1, endothelial cells of the cerebral vasculature were observed to bind high quantities of this protein (Burk et al., 1997). Recently, ApoER2 was identified as a specific component of the BBB based on its highly enriched mrna expression in mouse brain microvascular endothelial cells compared to liver and lung endothelial cells (Daneman et al., 2010). However at the BBB, ApoER2 is apparently present on the abluminal or basolateral side of endothelial cells, in between the capillary lumen and the astrocytic endfeet (Elali & Hermann, 2010). In other words ApoER2 is on the brain side of the cerebral microvascular endothelium. Thus, uptake of SEPP1 within brain by endothelial ApoER2 may retain Se in brain. However, transfer of SEPP1 across the BBB may involve additional receptors, such as Megalin. Megalin is the primary SEPP1 receptor in kidney and is responsible for reabsorption of SEPP1 from the glomerular filtrate by proximal convoluted tubule epithelial cells (Olson et al., 2008). Interestingly, Megalin facilitates transcytosis of apolipoprotein J from blood to brain at the cerebral vascular endothelium and the choroid plexus epithelium (Zlokovic et al., 1996). Furthermore apolipoprotein J, a.k.a. Clusterin, associates with SEPP1 in high molecular weight complexes in plasma, and Megalin-mutant mice have reduced Se in 20

30 brain and the periphery (Cheung et al., 2010, Chiu-Ugalde et al., 2010). Therefore, the two receptors may work in tandem to take up blood SEPP1 and/or retain central SEPP1 depending on local demand, physiological activity, and developmental stage. Since transgenic mice lacking SEPP1 or its receptors have some Se in brain, complementary mechanisms affecting brain and whole body Se distribution are additionally inferred to exist. The choroid plexus is a heavily vascularized cuboidal epithelium protruding from the ependymal walls of the cerebral ventricles. This structure produces cerebrospinal fluid (CSF), the extracellular fluid bathing the nervous system, essentially by filtering blood. SEPP1 is found in CSF, and choroid plexus highly expresses the mrna, implying that it is secreted (Scharpf et al., 2007). The ventricular regions are known to have high Se by autoradiography, positioning the choroid plexus and CSF as primary sites of brain Se homeostasis by virtue of SEPP1 expression and secretion (Kuhbacher et al., 2009). Choroid plexus also expresses abundant mrna for nearly all other selenoproteins, the synthesis factors, the SEPP1 receptors ApoER2 and Megalin, and SCLY, further suggesting a role in transfer of Se from peripheral blood to the central nervous system (Lein et al., 2007). The role of SCLY in the brain has received little attention. Although SEPP1 is established in promoting brain Se concentration and selenoprotein expression, a complete mechanism is lacking. This is because SEPP1 supplies Se in the form of Sec residues, while selenoprotein synthesis is thought to require selenide to generate selenophosphate and Sec co-translationally. The enzymatic activity of SCLY generates selenide from Sec, and the protein has been suggested to associate with SPS enzymes to recycle Se from Sec in support of selenoprotein synthesis (Tobe et al., 2009). SCLY protein and activity is found in mouse brain, albeit at much lower levels than in kidney and liver (Mihara et al., 2000). Curiously, the testes, which also has a blood-tissue barrier and relies on SEPP1 for Se, show a pattern whereby cells expressing ApoER2 do not express SCLY and vice versa (Kurokawa et al., 2011). Specifically, cells exposed to circulating SEPP1 express ApoER2, whereas germ cells expressing other selenoproteins express SCLY. This 21

31 suggests that uptake of SEPP1 and utilization of the Sec residues may depend on multiple interacting cell types, rather than cell autonomous utilization. In brain, where neurons, astrocytes, oligodendrocytes, microglia and endothelial cells interact in a complex system, Se distribution between cells and extracellular compartments may similarly occur by cell-type and -context dependent expression of SEPP1, ApoER2, and SCLY. However the exact nature of this system, both in body and brain, has not been fully elucidated. 22

32 FIGURE LEGENDS Figure 1: Assimilation of Se into selenoproteins. Dietary forms of Se in vertebrates include selenate, selenite, selenocysteine, and selenomethionine and are highlighted in green. Dietary forms are converted to the intermediate metabolite selenide for selenoprotein synthesis or Se excretion. Several factors are required to synthesize Sec on its trna, and position it at UGA codons during translation. Figure 2: Selenoprotein localization to subcellular domains. A partial list of mammalian selenoproteins is depicted schematically in a prototypical cell. GPX isoforms are distributed in the cytosolic, mitochondrial, nuclear, and extracellular compartments. Seven selenoproteins display localization to the endoplasmic reticulum, and other selenoproteins are also shown. Several steps involved in cellular signal transduction are known to be redox-sensitive, and may be functionally affected by selenoproteins. 23

33 FIGURE 1 24

34 FIGURE 2 25

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47 CHAPTER 2 ABSENCE OF SELENOPROTEIN P BUT NOT SELENOCYSTEINE LYASE RESULTS IN SEVERE NEUROLOGICAL DYSFUNCTION ABSTRACT Dietary selenium restriction in mammals causes bodily selenium to be preferentially retained in the brain relative to other organs. Almost all of the known selenoproteins are found in brain, where expression is facilitated by selenocysteine-laden selenoprotein P. The brain also expresses selenocysteine lyase, an enzyme that putatively salvages selenocysteine and recycles the selenium for selenoprotein translation. We compared mice with a genetic deletion of selenocysteine lyase to selenoprotein P knockout mice for similarity of neurological impairments, and whether dietary selenium modulates these parameters. We report that selenocysteine lyase knockout mice do not display neurological dysfunction comparable to selenoprotein P knockout mice. Feeding a lowselenium diet to selenocysteine lyase knockout mice revealed a mild spatial learning deficit without disrupting motor coordination. Additionally, we report that the neurological phenotype caused by the absence of selenoprotein P is exacerbated in male versus female mice. These findings indicate that selenocysteine recycling via selenocysteine lyase becomes limiting under selenium deficiency, and suggest the presence of a complementary mechanism for processing selenocysteine. Our studies illuminate the interaction between selenoprotein P and selenocysteine lyase in the distribution and turnover of body and brain selenium, and emphasize the consideration of sex differences when studying selenium and selenoproteins in vertebrate biology. 38

48 INTRODUCTION Selenium (Se) is an essential dietary micronutrient with antioxidant properties, and the human health consequences of Se-deficiency are under extensive study (Rayman, 2000). Sex-specific differences are observed in Se status and metabolism, which has complicated this research (Combs et al., 2011, Galan et al., 2005). The functions of Se in biochemical reactions and cellular processes of organisms are principally mediated by selenoproteins that incorporate Se into the amino acid selenocysteine (Sec). Biosynthesis of Sec occurs on its UGA-recognizing trna, and is catalyzed by Sec synthetase (SepSecS) in the presence of selenophosphate [reviewed in (Bellinger et al., 2009)]. Recoding the UGA stop codon for Sec incorporation requires a Sec-specific elongation factor (EFSec), an mrna stem-loop termed a Sec insertion sequence (SECIS), and a SECIS-binding protein (SBP2). Among 25 human selenoproteins, the glutathione peroxidase, thioredoxin reductase, and iodothyronine deiodinase families of enzymatic selenoproteins are relatively well characterized, and crucial for the health of mammals. Selenoprotein P (Sepp1) uniquely contains up to 10 Sec residues in primates and rodents. During Se-deficiency, brain Se is maintained compared to other organs by tissue-specific uptake of Sepp1 by ApoER2 and Megalin (Burk et al., 2007, Chiu-Ugalde et al., 2010). Mutant mice lacking full-length Sepp1 or the Sec-rich C-terminus show a greater depletion of brain Se than can be achieved through dietary Se deprivation (Hill et al., 2007). Sepp1 gene disruption in mice additionally causes cognitive, motor and sensory symptoms that can be exacerbated by dietary Se restriction and diminished by Se supplementation. These mice present spatial learning deficits, spasticity, and hyperreflexia that coincide with deficient synaptic plasticity and widespread neurodegeneration (Caito et al., 2011, Peters et al., 2006, Valentine et al., 2008, Valentine et al., 2005). If endocytosis of Sepp1 delivers Se to cells, the Sec residues from Sepp1 must be processed for incorporation into selenoproteins. Sec lyase (Scly) catalyzes the decomposition of Sec into alanine and hydrogen selenide (Esaki et al., 1982), and 39

49 promotes the production of selenophosphate in the presence of Sec and selenophosphate synthetase (SPS) (Tobe et al., 2009). Scly mrna and protein are expressed in mouse brain (Mihara et al., 2000), where it is posited to recycle Sec from Sepp1 for selenoprotein synthesis (Schweizer et al., 2005). We hypothesized that Scly liberates Se from Sepp1 in brain, and that deletion of Scly in mice would cause similar neurological deficits as observed in Sepp1-/- mice. To test this hypothesis, we assessed whether a novel transgenic mouse strain lacking functional Scly develops a phenotype similar to Sepp1-deficient mice. Here we report that, in contrast to Sepp1-/- mice, Scly-/- mice display few neurological abnormalities. However, spatial learning and selenoprotein expression are sensitive to Scly disruption when the mice are challenged with a low-se diet. In addition, we extensively characterized sex differences in the behavioral phenotype of Sepp1-/- mice, and report that male mice are more dependent on Sepp1 and Se than females for normal brain function. MATERIALS AND METHODS Animals: Genetically modified male and female mice on a C57BL/6 background lacking Sepp1 or Scly were bred on commercially available diets containing adequate Se (~0.25 ppm). Animals were given food and water ad libitum on a 12-hour light-cycle and group housed until behavioral experimentation. All behavioral experiments were conducted on single-housed adult mice aged 4-6 months during the light cycle. Male and female mice of all genotypes were used in approximately equal numbers to examine sex differences present in the animals. All animal procedures and experimental protocols were approved by the University of Hawaii Institutional Animal Care and Use Committee. Generation of Sepp1-/- and Scly-/- mice: Sepp1-/- mice were generated by electroporating a construct into 129S9/SvEvH-derived embryonic stem (ES) cells that were subsequently injected into C57BL/6 blastocysts. The resulting chimeric males were bred with C57BL/6J females (Hill et al., 2003). Mutant mice were backcrossed to C57BL/6J for at least 10 generations before arriving in our lab, and were bred with our 40

50 C57BL/6J colony to ensure congenic strains (Hoffmann et al., 2007). Sepp1+/- mice were bred to generate littermate pups of Sepp1-/- knockout and Sepp1+/+ control mice. Genotyping of the mice was carried out using methods previously described (Hoffmann et al., 2007). A targeting vector was generated by the NCRR-NIH supported KnockOut Mouse Project (KOMP) Repository, and included an ~11-kb region of the wild type Scly locus subcloned from a positively identified C57BL/6 BAC clone. The vector was designed with one homology arm extending 5.5 kb 5 to exon 4, and the other 5.5 kb homology arm terminating 3 to exon 7. A promoterless trapping cassette (L1L2_gt0) with flanking Flp-recombinase target (FRT) sites was inserted in an intron 5 of exon 4. Efficient splicing to the reporter cassette results in truncation of the endogenous transcript, causing a constitutive null mutation in the Scly gene. Cre-recombinase target loxp sites were inserted 5 and 3 of critical coding exon 4. The total size of the targeting construct, including vector backbone (L3L4_pZero_DTA_kan) and Neo cassette, was kb. The targeting vector was transfected into C57BL/6 embryonic stem cells by electroporation. After selection with antibiotic, surviving clones were expanded and analyzed by PCR to identify recombinant ES cell clones. ES cell clones were microinjected into C57BL/6 blastocysts to produce chimeras with one wild-type and one mutant Scly allele, which were then mated to generate Scly-/- mice on a pure C57BL/6 background. Mutant mice were backcrossed to C57BL/6J to ensure genetic comparability with wild-type control C57BL/6J mice. The latter were maintained not more than five generations after arrival from The Jackson Laboratory. Deletion of Scly was confirmed in all offspring using PCR that amplified a 1.2-kb product in the targeted region present in the wild-type allele (forward, 5 -CAC AGG TGC GGC CAT GAG GG-3 ; reverse, 5 - CTG GCT GTC CCT GAA CTA GCT TCA TA-3 ) and a 233-bp product in the mutant allele (forward, 5 -GAG ATG GCG CAA CGA AAT TAA T-3 ; reverse, 5 - CTG GCT GTC CCT GAA CTA GCT TCA TA -3 ). Diets: For dietary experiments, animals were switched from standard laboratory diets containing ~0.25 mg/kg Se to defined diets at the time of weaning (3 4 wk of age). Mice were fed Open Source Diets purchased from Research Diets containing either 0.08 mg/kg 41

51 (cat.#d19101) or 1.0 mg/kg (cat.#d ) Se. The diets were formulated with purified ingredients and contained 20.3% protein, 66% carbohydrate, and 5% fat. The protein source was casein, which was also the source of Se in the low Se diet. For high Se diet, sodium selenite was added to achieve final Se levels. Multiple lots were independently tested to confirm the Se concentration by inductively coupled plasma-ms (Bodycote) with lot-to-lot variation at or below the detection limit of inductively coupled plasma-ms testing (0.02 mg/kg). Mice were fed the defined diets from weaning until the time of sacrifice. Se-adequate standard lab diets containing ~0.25 mg/kg Se are not completely defined, and may be considered slightly supplemented compared to the rodent RDA of 0.15 mg/kg Se. The low-se diet containing 0.08 mg/kg Se is marginally deficient, and causes moderate selenium deficiency in mice (Hoffmann et al., 2010). The high-se diet containing 1.0 mg/kg Se has been shown to prevent many of the neurological symptoms in Sepp1-/- mice (Hill et al., 2003). This range of dietary Se concentration reflects the global spectrum of human Se intake, from deficiency to therapeutic supplementation. Animal Behavior: Spontaneous activity. Animals were placed in a transparent cylinder (20 cm diameter, 20 cm height) and activity was videotaped for three minutes. The cylinder was situated on clear plexiglass with a mirror placed at an angle underneath for clear view of movement along the ground as well as along the walls of the cylinder. The number of rears, forelimb and hindlimb steps, and time spent grooming were measured. Videotapes were scored in slow motion by an experimenter blind to the mouse genotype. A rear was counted when an animal made a vertical movement with both forelimbs removed from the ground. Forelimb and hindlimb steps were counted when an animal moved both forelimbs or both hindlimbs across the floor of the cylinder. Number of steps, rears, and time spent grooming were compared for wild-type and knockout mice (Fleming et al., 2004). Open field. Animals were placed in a square box (50 cm sides, 40 cm walls) and monitored by overhead camera linked to computer-assisted tracking software. During the test, the mice were allowed to move freely around the open field and to explore the environment for five minutes. The path of each mouse was automatically recorded, and 42

52 recordings were then analyzed. Total distance traveled, number of rears, time spent grooming, and center time was compared between groups. Vertical pole. The vertical pole descent test has been used to assess coordination and basal ganglia related movement disorders in transgenic mice (Fleming et al., 2004). Animals were placed head-up on top of a vertical wooden pole 50 cm long (1.2 cm in diameter). The base of the pole was fixed in plexiglass and put in the home cage. When placed on the pole, animals orient themselves downward and descend the length of the pole back into their home cage. Knockout and wild-type mice received two days of training consisting of five trials per day. On the third day, animals received five trials, and time to orient downward (turn) and total time to descend (total) were measured with a stopwatch. The best performance over the five trials was used for both wild-type and knockout mice. Inverted grid. The ability to hang upside down is a test of neuromuscular strength (Crawley, 1999). Each mouse was placed on a wire grid (mesh, 12 cm 2 with 0.5 cm 2 squares) 20 cm above a table top for 120 sec and videotaped. The lid was gently turned upside down, 60 cm above a soft surface to avoid injuries. The latency to fall was timed. Each mouse was given up to two attempts to hold on to the inverted grid for a maximum of 120 seconds and the longest period was recorded. Morris water maze. A circular pool (2 m in diameter, 1 m deep) surrounded by constant external cues was located in an observation room and filled with 24 C water. White nontoxic paint was added to make the water translucent. Tests were performed under dim light conditions. Lights below the height of the tank, necessary for video capture, also provided light for the swimming mouse. A small circular escape platform (7 cm diameter, located just below the water surface, or protruding just above the water) was placed in a constant location in the center of quadrant 1. Four equally spaced points around the wall of the pool were used as starting points. The mice were given one block of four trials each day with an inter-trial interval of 5 to 10 min. Each trial started from one of four different points, in a semi-random order. The mouse was allowed to swim around until it located the platform or 60 sec, at which point the mouse was placed on the platform by the experimenter and allowed to stay on the platform for 15 sec. The time to locate the platform was recorded as escape latency during training days. After sufficient training a 43

53 one-minute probe trial was performed, in which the platform was removed and the path of each mouse on each trial was automatically recorded and then analyzed. Time investigating the target, opposite, and adjacent quadrants, platform latency and platform crossings were measured. Average swim speed was calculated from total distance traveled per 60 sec trial. Animals that spent significant time floating, which occurred sporadically in very few animals independent of genotype, were excluded from this analysis. Quantitative RT-PCR: Animals were sacrificed by CO 2 asphyxiation, or deeply anesthetized with tribromoethanol and decapitated, and the brains rapidly excised, washed in PBS and snap-frozen in liquid nitrogen. Tissue was ground into powder, using a mortar and pestle on dry ice, and collected into pre-chilled tubes. Total RNA from tissue was prepared by Trizol extraction (Invitrogen, Carlsbad, CA, USA) followed by purification using the RNeasy kit (QIAgen, Valencia, CA, USA). Concentration and purity of extracted RNA and synthesized cdna was determined using A 260 /A 280 ratio measured on an ND1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Synthesis of cdna was carried out using High Capacity cdna Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), with 1 g RNA per 20 l reaction. For real-time PCR, 100 ng of the cdna was used in 5 l reactions with Platinum SYBR Green qpcr SuperMix-UDG (Invitrogen). Reactions were carried out in triplicate or quadruplicate in a LightCycler 480 II thermal cycler (Roche, Indianapolis, IN, USA). Cycling conditions followed the manufacturers suggestions in the SYBR Green kit instructions. All qpcr results were normalized to 18S rrna expression as a housekeeping gene and analyzed using Absolute Quantification Software (Roche). SDS-PAGE and Western blot: Total protein was extracted from powdered mouse tissues by light sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA), followed by centrifugation according to the manufacturers protocol. Protein was added to reduced Laemmli buffer, boiled for 10 minutes, and loaded into 4-20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gel contents were transferred to PVDF membranes, which were blocked with undiluted Odyssey Blocking 44

54 Buffer (Li-Cor Biosciences, Lincoln, NE, USA) for one hour. Membranes were then probed for 90 minutes with the following primary antibodies: Goat-anti-GPX1 (R&D Systems, Minneapolis, MN, USA), Rabbit-anti-GPX4 (AbFrontier, Seoul, Korea), Rabbit-anti-SEPW1 (Rockland, Gilbertsville, PA, USA), and Mouse-anti-alpha Tubulin (Novus, Littleton, CO, USA). After washing with PBS containing 0.05% Tween-20 (PBST), membranes were incubated in the dark in secondary antibodies labeled with infrared fluorophores (Li-Cor Biosciences). After further washes in PBST, blots were imaged and quantified with the Odyssey infrared imaging system (Li-Cor Biosciences). Glutathione peroxidase activity assay: Total glutathione peroxidase activity was measured using the Bioxytech GPx-340 Assay kit (Oxis International, Foster City, CA, USA). Mouse tissues for the assay were collected in the same manner as above. Powdered tissue was homogenized in lysis buffer by sonication and centrifuged at 15,000 x g. The resulting supernatant was serially diluted to determine the linear range of the assay. 5 l of diluted sample (1:10 for brain, 1:200 for liver) was added to 25 l of assay buffer and 25 l of NADPH reagent in a 96-well plate. 25 l of tert-butyl hydroperoxide was added to initiate the reaction, which was monitored for 10 minutes at room temperature by measuring kinetic absorbance at 340 nm on a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Statistical analysis: Data were analyzed using Microsoft Excel (Redmond, WA, USA), and plotted using GraphPad Prism software (San Diego, CA, USA). Two-way ANOVA was used to determine an interaction between genotype and sex for all experiments. If no interaction effect was observed, male and female groups were sometimes combined. Repeated measures ANOVA was used to assess training in the water maze, and genotype and quadrant were the two factors for analyzing quadrant investigation on the probe trial. Post hoc test using Bonferroni correction for multiple comparisons was used to determine significance between individual groups. When male and female groups were combined, unpaired t-tests comparing genotypes were performed for end-point measures in the water maze and for biochemical experiments. The significance criteria were set at p < 0.05 for all statistical measures. 45

55 RESULTS Neurological motor phenotype in Sepp1-/- mice is more pronounced in males Genetic deletion of Sepp1 in mice results in neuromotor impairments (Hill et al., 2004, Schomburg et al., 2003). We characterized the phenotype of Sepp1-deficient mice raised on a standard lab diet using a battery of behavioral tests to assess motor function in male and female mice. We found that motor coordination was worse in male knockout animals compared to females. Spontaneous locomotor activity, as assessed using the cylindrical chamber, was greatly decreased in the male Sepp1-deficient mice while being slightly decreased in the female knockout animals, when compared to wild-type mice [Fig. 1A]. Two-way ANOVA revealed an effect of genotype on spontaneous rearing (F(1,32)=13.06, p=0.0010) [Fig. 1A, left] and grooming (F(1,32)=16.93, p=0.0003) [Fig. 1A, center]. Post hoc analysis indicated a statistically significant decrease in rearing (t(16)=3.464, p<0.01) and grooming (t(16)=2.532, p<0.05) in male Sepp1-/- mice, whereas only grooming (t(16)=3.287, p<0.01) was decreased in female Sepp1-/- mice, when compared to wild-type. Despite these differences, we did not observe any genotype or sex interaction effects on total distance traveled in the open field [Fig. 1A, right]. To assess motor coordination and strength, animals were subjected to the pole descent and inverted hang tests. Two-way ANOVA revealed an interaction between genotype and sex on time to turn (F(1,42)=16.57, p=0.0002) [Fig. 1B, left] and descend (F(1,42)=5.222, p=0.0274) [Fig. 1B, center] a vertically oriented pole. Genotype strongly affected ability to hang upside down for 120 seconds (F(1,32)=16.10, p=0.0003), however only male Sepp1-/- mice were significantly different from wild-type control mice (t(16)=4.045, p<0.001) [Fig. 1B, right]. Dietary Se supplementation alleviates motor deficits in Sepp1-/- mice Previous studies have shown that Se supplementation can attenuate neuromotor impairments in Sepp1-/- mice (Schweizer et al., 2004). We determined if gender could influence this attenuation. Animals were supplemented with 1 mg/kg Se in the diet starting at weaning, and assayed in the same manner as those fed a standard diet. We found that Se-supplemented Sepp1-/- mice do not differ from wild-type mice in 46

56 spontaneous rearing [Fig. 2A, left], grooming [Fig. 2A, center] and distance traveled [Fig. 2A, right]. Performance on the pole descent test also improved in Se-supplemented mice, however a main effect of genotype remained for the time to invert (F(1,33)=12.68, p=0.0011) [Fig. 2B, left] and descend (F(1,33)=4.989, p=0.0324) [Fig. 2B, center] the vertical pole, and post hoc test indicated male Sepp1-/- mice were slower than wild-type littermates in turn time only (t(19)=4.004, p<0.001). Performance on the inverted hang test also improved in the Se-supplemented Sepp1-/- mice, and an effect of genotype did not reach statistical significance (F(1,31)=3.257, p=0.081) [Fig. 2B, right]. In all motor tests, Se-supplemented Sepp1-/- mice showed subtle qualitative behavioral deficits in both sexes, but the male bias was largely eliminated. Generation and characterization of Scly-/- animals Scly knockout mice were generated by a conditional knockout approach in the event that deletion of Scly caused embryonic lethality. Mice were generated with a trapping cassette inserted upstream of exon 4 of the Scly gene, resulting in a constitutive null mutation in the whole animal [Fig 3A]. Mutation of the targeted region of the Scly gene was confirmed by PCR of mouse tail DNA [Fig. 3B], and Scly mrna was undetectable by qpcr in all tissues examined from Scly-/- mice, including brain [Fig. 3C]. Scly-/- mice were born at the expected Mendelian ratio, appeared generally healthy, and developed to adulthood. Unlike Sepp1-/- mice, Scly-/- mice were fertile, producing viable offspring that did not differ in size at birth compared to wild-type control mice. Neurological motor phenotype is largely absent in Scly-/- mice Scly is a relatively uncharacterized enzyme thought to be involved in recycling Se from Sec in support of selenoprotein synthesis. Sepp1 contains up to 10 Sec residues and is proposed to be a source of Se for the brain. We hypothesized that mice lacking Scly would be unable to efficiently catalyze Sec degradation, and therefore would manifest a phenotype similar to the Sepp1-/- mice. Contrary to our prediction, we found that Scly-/- animals raised on a Se-adequate diet displayed no neurological phenotype. Spontaneous rearing and movement were unaffected by Scly genotype, and performance on the vertical pole descent was similar between Scly+/+ and Scly-/- mice [data not shown]. 47

57 As no apparent behavioral phenotype presented in the Scly-/- mice raised on a standard lab diet, we subjected the animals to a marginally low Se diet, containing 0.08 mg/kg Se, to assess whether the animals display enhanced sensitivity to Se deficiency as measured by locomotor behavior. Total distance traveled in the open field was unaffected by Scly genotype under low-se conditions, however two-way ANOVA revealed an interaction between Scly genotype and sex on rearing activity (F(1,22)=4.937, p=0.0369) [Fig. 4A]. Motor coordination assayed by turn and total time in the vertical pole descent test indicated no difference due to genotype in Se-deficient Scly-/- animals [Fig. 4B]. Spatial learning is sensitive to disruptions in Se availability The Morris water maze is a paradigm for assessing spatial learning and memory in rodents (Morris, 1984). Sepp1 knockout mice raised on a Se-supplemented diet have mild impairments in learning measured with this paradigm, despite having a large deficit in long-term potentiation, a cellular model for learning and memory (Peters et al., 2006). As Se supplementation attenuates neurological impairments in Sepp1 knockout mice, we questioned if learning deficits would be greater for Sepp1-/- mice raised on a normal Se diet. Mice were initially trained over several days to locate a hidden platform in a large tank of water. Subsequently the platform was removed for one final trial, in which the amount of time the animal investigated the area where the platform used to be was measured. We compared male and female Sepp1-/- and wild-type animals raised on standard laboratory chow with adequate Se for learning deficits. In contrast to our findings of gender differences in neuromotor function, we did not observe significant learning differences between Sepp1-/- male and female mice. Therefore male and female groups were combined after eliminating sex as an interacting variable. We found that learning was impaired in Sepp1-/- mice fed a standard diet [Fig. 5A]. Two-way ANOVA revealed an interaction between genotype and training (F(7,112)=2.361, p=0.0275). Escape latency over time was not substantially reduced in Sepp1-/- as compared to control mice, suggesting the mice did not learn the spatial location of the platform. The probe trial results are ambiguous since the training was ineffective in Sepp1-/- mice; however a genotype x quadrant interaction effect was 48

58 observed (F(3,64)=3.277, p=0.0266) during quadrant investigation [Fig. 5B]. Sepp1-/- mice spent significantly more time than controls investigating the opposite quadrant, which was not due to uncoordinated swimming, and likely indicates failure to learn the platform location. There was a strong trend towards reduced number of platform crossings (t(16)=2.064, p=0.0557) [Fig. 5C], but swim speed (t(14)=0.7262, p=0.48) [Fig. 5D] was not significantly different between genotypes by t-test. Similar to published work on Se-supplemented Sepp1-/- mice in the water maze, we found a genotype difference during training, but not on the probe trial in Sepp1-/- mice fed a high Se diet [data not shown]. Although we found little difference between Scly-/- and control mice in locomotor activity, we assayed the Scly-/- animals using the Morris water maze for comparison with Sepp1-/- mice. Scly-/- mice fed standard chow performed like wild-type mice during training [Fig. 6A], as the only effect observed by two-way ANOVA was for training day (F(7,154)=24.07, p<0.0001). Similarly, in the probe trial we found an effect of quadrant (F(3,88)=5.78, p=0.0012), but no interaction with genotype [Fig. 6B]. Platform crossings (t(22)=0.5415, p=0.59) [Fig. 6C], latency to platform location [data not shown], and swim speed (t(22)=0.4955, p=0.63) [Fig. 6D] were not significantly different between genotypes when assessed by t-test. Scly-/- mice showed a trend towards delayed learning on days three and four. The lack of spatial learning impairment in Se-adequate Scly-/- mice starkly contrasted with Sepp1-/- mice fed the same diet. The trend toward mild learning deficits in Scly-/- mice led us to question if a restricted Se diet would result in greater learning impairments in these animals. When Scly-/- animals fed a Se-deficient diet were assessed, two-way ANOVA revealed an interaction between genotype and training (F(5,120)=2.496, p=0.0345), while post hoc tests indicated longer latency times on days 2 (t(24)=2.942, p<0.05), 3 (t(24)=3.467, p<0.01), 5 (t(24)=2.895, p<0.05), and 6 (t(24)=3.037, p<0.05) in the knockout mice [Fig. 7A], which suggests reduced learning. However the mice learned the platform location and the deficit was not as severe as in non-supplemented Sepp1-/- animals. Scly-/- animals performed as well as wild-type animals in the probe trial. We found a main effect on quadrant investigation 49

59 (F(3,96)=11.19, p<0.0001) but no Scly genotype interaction [Fig. 7B]. We found no difference in platform crossings (t(24)=1.089, p=0.29) [Fig. 7C], latency [data not shown], or average swim speed (t(22)=1.60, p=0.124) [Fig. 7D] in Scly-/- mice on a low Se diet compared to control mice. Selenoprotein expression and Glutathione Peroxidase activity When dietary Se is restricted in mammals, Sec-enriched Sepp1 helps maintain selenoprotein expression in the brain (Hill et al., 2007). We therefore investigated the expression of selenoproteins in brains of Scly-/- animals by quantitative RT-PCR, western blot, and glutathione peroxidase (GPX) activity. We did not find a significant change in the mrna for Sepp1 (t(9)=1.536, p=0.1589, n=5-6), GPX1 (t(10)=1.056, p=0.3157, n=6), GPX4 (t(10)=0.6899, p=0.5060, n=6), or selenoprotein W (Sepw1) (t(8)=2.149, p=0.0639, n=5) in brains of Scly-/- mice fed normal chow [data not shown]. Thus Scly-/- mice on a Se-adequate diet show neither a behavioral phenotype, nor any major changes in selenoprotein mrna expression in brain. Therefore western blotting and GPX activity assays to assess the severity of Se-deficiency were performed in mice fed a low-se diet only. Scly-/- animals on a low-se diet displayed significantly elevated Sepp1 mrna expression in brain compared to control mice (t(14)=2.686, p=0.0177) [Fig. 8A]. GPX1 (t(12)=3.40, p=0.0053) [Fig. 8B] and GPX4 (t(14)=3.093, p=0.0079) [Fig. 8C] mrna were also significantly increased in brain, while Sepw1 was not changed (t(14)=0.1380, p=0.89) [Fig. 8D]. In the same mice, we did not detect an increase in mrna expression of Nfs1 [(t(14)=0.7154, p=0.4861, not pictured], a cysteine desulfurase enzyme with Sec lyase activity. In contrast to the selenoprotein mrna expression data, the corresponding protein levels assessed by western blot are consistently decreased in the brains of Se-deficient Scly-/- mice. Both GPX1 (t(14)=12.39, p<0.0001) [Fig. 9A] and Sepw1 (t(14)=8.294, p<0.0001)) [Fig. 9C] are expressed at ~37% of the wild-type level, while GPX4 expression (t(14)=5.365, p<0.0001) [Fig. 9B] is reduced to ~60% compared to control 50

60 brains. These results confirm that Scly contributes to selenoprotein expression in brain during Se-deficiency. GPX activity in brain was reduced to 43% of the wild-type control level (t(14)=4.495, p=0.0005) [Fig. 10A], and was reduced to 54% in liver (t(14)=9.9297, p<0.0001) [Fig. 10B] of Scly-/- mice fed a low-se diet. However, GPX activity in serum was not significantly reduced [Fig. 10C]. These data indicate that Scly helps maintain selenoprotein expression and activity when dietary Se availability is limiting. They further suggest that Se status in tissues may be more affected than plasma in the absence of Scly. The contrast of increased selenoprotein mrna expression in the Scly-/- mouse brain despite reduced selenoproteins and enzyme activity suggests that Scly is a significant contributor to the Se pool for selenoprotein translation. DISCUSSION The results reported herein describe the first characterization of a novel mouse strain deficient in Scly. These mice exhibit minimal neurological deficits, an unexpected finding given the phenotypic effects of Sepp1 knockout and the proposed role of Scly in recycling the essential trace element Se from Sec. Unlike Sepp1-/- animals, deletion of Scly does not result in neuromotor impairments or spatial learning deficits, except under low-se conditions. We also report sex differences in the motor phenotype of mice with genetic deletion of Sepp1, highlighting the importance of considering gender on studies addressing the biological functions of Se or selenoproteins in mammals. Sepp1 is a plasma protein that is considered to be the physiological transporter of Se from liver to brain. Sepp1 is additionally found in grey and white matter and cerebrospinal fluid, and may store Se within brain (Scharpf et al., 2007). Sepp1 and ApoER2 maintain a high Se concentration in the testes, and prioritize Se to brain albeit at a lower concentration (Burk & Hill, 2009). Most tissues produce Sepp1 and Scly, and the whole body turnover rate of Sepp1 is high, fluxing a significant proportion of bodily Se even when dietary availability is limiting (Burk & Hill, 2005). Brain Se level is not dependent 51

61 on hepatic Sepp1 in Se-adequate adult animals (Schweizer et al., 2005). However Sedeficiency directs Se from liver-derived Sepp1 to the brain (Nakayama et al., 2007, Renko et al., 2008). The turnover rate of Sepp1 within the nervous system and the interaction with the circulation are uncertain. Unlike most trace element transporters, Sepp1 cannot rapidly load and unload cargo because Se is covalently incorporated as the amino acid Sec, which must be degraded to supply Se. Biosynthesis and incorporation of Sec is a protracted, energy intensive process that requires organized interaction of specific proteins (SPS, SepSecS, EFSec, SBP2) and nucleic acids (trna (Sec), SECIS-containing mrna), in addition to ATP, pyridoxal phosphate, and the translation machinery. Receptor-mediated uptake by ApoER2 facilitates entry of Sepp1 into cells, but recycling of the Sec residues would depend on the lysosome or proteasome to hydrolyze peptide bonds followed by liberation of Se from Sec, presumably by Scly. To test the hypothesis that Scly recycles Sec from Sepp1 in the brain, we investigated whether Scly-/- mice manifest a phenotype similar to Sepp1-/- mice. Surprisingly, very little neurological dysfunction was present in the Scly-/- mice, even when fed a diet low in Se. The lack of behavioral changes in Scly-/- mice, compared to Sepp1-/- mice, could be due to Nfs1 catalyzing Sec to selenide conversion for selenoprotein synthesis (Lacourciere et al., 2000). Although we did not detect increased Nfs1 mrna in Scly-/- mice brains, the normal activity of the enzyme might be compensating for the absence of Scly. Alternatively, Sepp1 may have an acute function in brain not strictly related to Se delivery. Disrupted synaptic plasticity in Se-supplemented Sepp1-/- mice (Peters et al., 2006) supports the notion that Sepp1 has a role in cell signaling via its receptor, ApoER2. ApoER2 is found at synaptic sites (Beffert et al., 2005), in cultured astrocytes, oligodendrocytes, and microglia (Fan et al., 2001), and in the brain vasculature (Korschineck et al., 2001), suggesting that all brain cells can take up Sepp1. In adult brain, mrna for Sepp1 is apparent in glia (Lein et al., 2007) but protein expression is dominant in neurons (Bellinger et al., 2008, Scharpf et al., 2007). Sepp1 mrna and 52

62 protein are abundant in choroid plexus epithelium (Bellinger et al., 2008, Steinert et al., 1998, Zhang et al., 2008). Scly mrna appears enriched in grey matter and neurons of mouse brain (Lein et al., 2007). Further, a mouse proteomics study identified Scly in synaptoneurosomes (Filiou et al., 2010). We found that mrna for Scly was not significantly changed in brain of Sepp1-/- mice fed a standard diet. Since Sepp1-/- mice have depressed Se in brain, this finding suggests that Scly is not Se-regulated in brain, and the enzyme is minimally affected by dietary Se or tissue Se levels (Deagen et al., 1987). The mrna expression of Sepp1 trended up in Scly-/- mice fed normal chow, and was significantly increased in brain of Scly-/- mice fed low-se chow. Se-deficient Scly-/- mice also had increased GPX1 and GPX4 mrna, while that of Sepw1 was unchanged. However, GPX protein and activity in brain of the low-se Scly-/- mice were dramatically reduced compared to wild-type animals. Liver GPX activity was similarly reduced, while serum activity was less affected. Therefore Scly supports selenoprotein expression and function under conditions of dietary Se deficiency. Increased GPX mrna despite reduced protein and activity in the Se-deficient Scly-/- brain could be a compensatory mechanism to boost inefficient selenoprotein translation. These findings extend a recent study, which demonstrated reduced GPX1 expression and reduced incorporation of Se derived from radiolabeled Sepp1, in cells with Scly knocked down by sirna (Kurokawa et al., 2011). In addition, we observe that tissues are more reliant on Scly than blood. Our finding that Se-deficient Scly-/- mice manifest a subtle spatial learning deficit in the water maze corresponds with results on Se-supplemented Sepp1-/- mice (Peters et al., 2006). Spatial learning requires the hippocampus, which is more dependent on Sepp1 for optimal Se concentration than other brain regions (Nakayama et al., 2007). Therefore Scly, Sepp1, and probably other selenoproteins in the hippocampus support spatial learning. It is likely that the kinetics of selenoprotein degradation and synthesis are even more disrupted in Scly-/- mice than the steady-state mrna and protein expression levels. Brain regions with high metabolism or cellular turnover could be more dependent on a 53

63 putative Sepp1-Scly recycling mechanism, while other cell populations might efficiently utilize an alternate Se source. These results are the most extensive characterization of behavioral sex differences in Sepp1-/- mice to date. It has been suggested that the phenotype of Sepp1-/- mice is sexdependent (Riese et al., 2006), however studies on Sepp1-/- mice have focused on males and data regarding behavioral sex differences are limited. Despite variations in behavioral testing paradigms, our results showing impaired motor performance in male Sepp1-/- mice are in agreement with previous reports (Hill et al., 2004, Renko et al., 2008, Schweizer et al., 2004). We additionally assessed motor impairment in female Sepp1-/- mice, and found it to be minimal compared to males. We also report that the spatial learning deficit in Sepp1-/- mice on a Se-adequate diet is worse than in Se-supplemented mice, building on a previous study that used only male mice on a high-se diet (Peters et al., 2006). Selenoprotein expression is modulated by sex in mammals (Meplan et al., 2007, Riese et al., 2006, Stoedter et al., 2010), and Sepp1 is an androgen responsive gene (Takahashi et al., 2006). Additionally, testosterone secretion declines during Se-deficiency in male rats (Behne et al., 1996). Male mice displayed increased sensitivity to Sepp1 deletion, and male Sepp1-/- mice greatly improved when given supranutritional dietary Se, indicating that males have a higher demand than females for Sepp1 and Se in the nervous system. However the phenotype is not exclusive to males, suggesting that some aspect of metabolism or development that is more prominent in males is dependent on Se. Male gender is a risk factor for poor neurodevelopmental outcome after premature birth. Cerebral palsy and related developmental disorders are more common in males than females (Johnston & Hagberg, 2007). The phenotype of Sepp1-/- mice resembles cerebral palsy in that the developmental onset of spasticity and ataxia often presents with intellectual impairment and seizures. Moreover, this phenotype is not rapidly progressive and remains stable in adulthood when given adequate Se. Perinatal infection and hypoxia-ischemia are synergistic risk factors for cerebral palsy (Johnston & Hagberg, 54

64 2007, Mayoral et al., 2009), while Sepp1 is known to modulate immunity (Bosschaerts et al., 2008) and metabolism (Misu et al., 2010). Metabolically demanding brain regions and cells, with a presumably higher rate of selenoprotein synthesis, are susceptible to neurodegeneration in Sepp1-/- mice (Valentine et al., 2008). Moreover, an autosomalrecessive human disease termed progressive cerebellocerebral atrophy has been linked to mutations in SepSecS that globally disrupt selenoprotein synthesis (Agamy et al., 2010). The sequelae of these patients, including mental retardation, spasticity and seizures, are similar to those found in Sepp1-/- mice and emphasize the importance of selenoproteins in the function and health of the nervous system. In conclusion, these results indicate that a novel mouse strain lacking Scly does not develop a neurological phenotype similar to Sepp1-/- mice. A subtle learning deficit is observed when Scly-/- animals are fed a low-se diet, and these animals also have reduced expression of selenoproteins in brain. We further report a male bias in the neurological motor phenotype of Sepp1-/- mice. The disparity of neurological problems in Scly-/- and Sepp1-/- mice suggests that Sepp1 is more critical than Scly for maintenance of brain Se, but that recycling Se from Sec via Scly is physiologically important during dietary Se deficiency. Altogether these findings highlight that Se is critically important for the nervous system, and that Se metabolism through Sepp1 and Scly impacts spatial learning. 55

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70 FIGURE LEGENDS Figure 1: Spontaneous activity and motor coordination is more reduced in male than female Sepp1-/- mice compared to control mice fed a standard diet. (A) Rearing and grooming were measured in the cylinder, and total distance traveled in the open field. Sepp1-/- mice were less active as measured by rearing (left, genotype **p<0.01) and grooming (center, genotype ***p<0.001), but did not show decreased exploration of the open field (right). (B) Motor coordination was measured using the pole test and inverted hang test. Sepp1-/- mice took longer to turn (left, genotype x sex ***p<0.001) and descend the pole (center, genotype x sex *p<0.05), and had reduced ability to suspend themselves for two minutes (right, genotype ***p<0.001). Values are expressed as means ± SEM. n=8-12 per group, *p<0.05, **p<0.01, ***p<0.001, compared with control mice. Figure 2: High Se diet improves spontaneous activity and motor coordination more in male than female Sepp1-/- mice compared to control mice. (A) Rearing and grooming were measured in the cylinder, and total distance traveled in the open field. Sesupplemented Sepp1-/- mice were as active as controls when measured by rearing (left), grooming (center), and exploration of the open field (right), and no sex differences were observed. (B) Motor coordination was measured using the pole test and inverted hang test. Se-supplemented Sepp1-/- mice took longer to turn (left, genotype **p<0.01) and to descend the pole (center, genotype *p<0.05) when compared with control mice, but were capable of suspending themselves for two minutes (right). Male and female Sepp1-/- mice performed similarly except for turn time, and an interaction effect between genotype and sex was not statistically evident in any test. Values are expressed as means ± SEM. n=8-10 per group, ***p<0.001, compared with control mice. Figure 3: Generation of Scly-knockout mice. (A) Image from KOMP. A promoterless trapping cassette was inserted upstream of exon 4 of the mouse Scly locus on chromosome 1, causing splicing at the cassette and truncation of the endogenous transcript. The cassette was flanked by FRT sites for conditional excision of the cassette by breeding with FLP-recombinase transgenic mice in case of embryonic lethality. 61

71 Presence of the loxp sites flanking exon 4 allowed excision of a coding exon critical for enzymatic function by breeding with Cre-recombinase transgenic mice. (B) PCR genotyping of mice tails was performed to detect presence of wild-type allele (1.2 kb) or knockout allele (233 bp) using primers described in Materials and Methods. (C) Quantitative RT-PCR analysis of brains from Scly+/+ and Scly-/- mice indicated no detectable Scly mrna in homozygous knockout mice. Figure 4: Spontaneous activity and motor coordination is normal in male and female Scly-/- mice fed a low Se diet. (A) Rearing and total distance traveled was measured in the open field. Scly-/- mice were as active as controls when measured by exploration of the open field (right), however an interaction between genotype and sex affected rearing activity (left, genotype x sex *p<0.05). (B) Motor coordination was measured using the pole test. Scly-/- mice and control mice performed similarly to turn (left) and descend the pole (right). Male and female mice performed similarly, and no genotype effects were present. Values are expressed as means ± SEM. n=6-7 per group. Figure 5: Spatial learning and memory is disrupted in Sepp1-/- mice fed a standard diet. (A) Average escape latency per training day over time was measured in the Morris Water Maze. An interaction between Sepp1 genotype and training was present (*p<0.05) and Sepp1-/- mice had significantly longer latency on days 7 and 8 (*p<0.05). (B) In the probe trial, Sepp1-/- spent more time exploring the opposite (OP) quadrant (*p<0.05) and we found a significant interaction effect (genotype x quadrant *p<0.05). They also had non-significant trends toward fewer platform crossings (p=0.0557) (C) and a reduced swim speed (p>0.05) (D). Values are expressed as means ± SEM. n=8-10 per group. Figure 6: Spatial learning and memory is not disrupted in Scly-/- mice fed a standard diet. (A) Average escape latency per training day over time was measured in the Morris Water Maze. Scly-/- and control mice showed strongly reduced latency over time, and no interaction between genotype and training was apparent. (B) In the probe trial at the end of training, Scly-/- and control mice spent equal time exploring the target (TQ), left (LA) and right adjacent (RA), and the opposite (OP) quadrants (p>0.05). Both genotypes had 62

72 similar platform crossings (C) and swim speed (D) (p>0.05). Values are expressed as means ± SEM. n=11-13 per group. Figure 7: Spatial learning is mildly impaired in Scly-/- mice fed a low Se diet. (A) Average escape latency per training day over time was measured in the Morris Water Maze. An interaction between Scly genotype and training was present (*p<0.05), and Scly-/- mice had significantly longer latency on days 2, 3, 5 and 6 (*p<0.05). (B) In the probe trial at the end of training, Scly-/- and control mice spent equal time exploring the target (TQ), adjacent (LA, RA) and opposite (OP) quadrants (p>0.05). Both genotypes had similar platform crossings (C) and swim speed (D) (p>0.05) in the probe trial. Values are expressed as means ± SEM. n=13 per group. Figure 8: Expression of selenoprotein transcripts is increased in Se-deficient Scly-/- mice brains. (A) Sepp1 mrna level was measured in brains of Scly-/- mice (n=8). We additionally measured GPX1 (n=7) (B), GPX4 (n=8) (C), and Sepw1 (n=8) (D) mrna expression in brain of Scly-/- mice fed a low Se diet. Sepp1, GPX1, and GPX4 were increased (*p<0.05, **p<0.01), while Sepw1 was unchanged. Samples were assayed in triplicate or quadruplicate and values were normalized to 18s rrna and expressed as means ± SEM. Figure 9: Expression of selenoproteins is decreased in Se-deficient Scly-/- mice brains. (A) GPX1, (B) GPX4, and (C) Sepw1 protein was measured by western blot and quantified by integrated intensity (n=8). Below each graph is a representative sample of the blot including loading equivalence determined by tubulin. GPX1, GPX4, and Sepw1 were drastically reduced in Scly-/- mice (***p<0.0001). All selenoprotein values are normalized to relative amounts of tubulin, and expressed as means ± SEM. Figure 10: Glutathione peroxidase activity is decreased in Se-deficient Scly-/- mice brains. Total GPX activity in brain (n=8) (A), liver (n=8) (B), and serum (n=12-14) (C) was assayed with a coupled reaction measuring NADPH oxidation. Mice were fed the low Se diet from weaning until time of sacrifice, when tissues were harvested for 63

73 analysis. Although both brain and liver GPX activity were reduced by approximately half (***p<0.001), serum GPX activity was not significantly reduced. Values are standardized to total protein concentration (brain, liver) or volume of serum and expressed as means ± SEM. 64

74 FIGURE 1 Normal diet 65

75 FIGURE 2 High Se diet 66

76 FIGURE 3 67

77 FIGURE 4 Low Se diet 68

78 FIGURE 5 69

79 FIGURE 6 70

80 FIGURE 7 71

81 FIGURE 8 72

82 FIGURE 9 73

83 FIGURE 10 74

84 CHAPTER 3 EXPRESSION OF SELENOPROTEIN W IN NEURONS EXTENDS INTO PROCESSES AND IS HIGHLY DEPENDENT ON SELENOPROTEIN P ABSTRACT During dietary selenium deprivation, bodily selenium is prioritized to the brain to maintain selenoprotein expression by a process that depends on selenoprotein P. Selenoprotein W is a small thioredoxin-like protein that is abundant in brain and muscle tissues. Although peripheral expression of selenoprotein W is reduced by dietary selenium deficiency, brain expression is maintained, suggesting it has an important function in nervous tissue. We assessed the regional, cellular, and subcellular expression of selenoprotein W in brains of wild-type mice and mice lacking selenoprotein P. We found that selenoprotein W is widespread in neurons, processes, and neuropil of mouse brain. Pyramidal neurons of somatosensory, motor, piriform, and cingulate cortex, and CA1 and CA3 of hippocampus express high levels of selenoprotein W. Purkinje neurons and their heavily branched dendritic arbors in cerebellum also express abundant selenoprotein W. Analysis of synaptosome fractions indicated that selenoprotein W is present at synapses, and expression is dramatically reduced in mice lacking selenoprotein P. Several components of the selenoprotein synthesis machinery were also found in isolated nerve terminals. These results indicate that widespread neuronal expression of selenoprotein W relies on selenoprotein P for selenium. They further suggest that selenoprotein W synthesis may occur in distal comparments of neurons, far removed from the nucleus. 75

85 INTRODUCTION Selenium (Se) is a trace micronutrient that is incorporated into antioxidant enzymes. Se is unique among trace elements because it is covalently incorporated into proteins as the amino acid selenocysteine (Sec). Biosynthesis of Sec and insertion of the residues into polypeptides requires a Sec Insertion Sequence (SECIS) and several specific proteins to reinterpret in-frame UGA codons in selenoprotein mrnas as Sec incorporation sites [reviewed in (Bellinger et al., 2009)]. Of the 25 primate (24 rodent) Sec-containing selenoproteins that have been identified using bioinformatics, the glutathione peroxidase, thioredoxin reductase, and iodothyronine deiodinase enzyme families are functionally characterized. Selenoprotein W (Sepw1) is the smallest mammalian selenoprotein and is one of the most widely distributed selenoproteins across species in all domains of life (Lobanov et al., 2009, Zhang & Gladyshev, 2008). It was putatively identified in the early 1970s as being absent in muscle of myopathic lambs suffering from White Muscle disease, but was not purified, cloned, and named until some 20 years later (Vendeland et al., 1993, Vendeland et al., 1995, Whanger, 2000). White muscle disease is a Se-responsive muscular dystrophy syndrome in ruminants, and was named because of the appearance of pale and dry muscle, usually with longitudinal striations or chalky whiteness caused by abnormal calcium deposition. Leg muscles are typically disrupted first, but all muscles, including cardiac, can be affected. Like most of the selenoproteins, Sepw1 is expected to be involved in oxidation-reduction (redox) reactions. Indeed, it has been shown to act as a glutathione-dependent antioxidant that protects cells from peroxide-mediated damage (Jeong et al., 2002). However, the specific antioxidant function of Sepw1 has been disputed (Xiao-Long et al., 2010), and a prominent role in cell signaling has also been proposed (Hawkes & Alkan, 2010). Sepw1 directly interacts with proteins (Aachmann et al., 2007, Dikiy et al., 2007), and sirna knockdown of Sepw1 expression inhibits cell proliferation in a p53- and p21-dependent mechanism (Hawkes et al., 2012). 76

86 In addition to expression in muscle and proliferating myoblasts, the Sepw1 gene is also highly expressed in developing and adult mouse brain (Gu et al., 2000, Loflin et al., 2006). However, unlike muscle, dietary Se depletion does not cause a reduction in Sepw1 levels in sheep or rat brain, despite reducing brain Se concentration and GPX activity (Sun et al., 2001, Whanger, 2001). Selenoprotein P (Sepp1) supplies Se to the brain, and mice deficient in Sepp1 have greatly reduced levels of Sepw1 mrna and protein in brain (Hoffmann et al., 2007). These data suggest that preferential retention of Sepw1 in brain during dietary Se-deficiency is maintained by Sepp1. Regional analysis of Sepw1 mrna expression in mice brains suggests presence in neurons, with high expression in >90% of brain regions (Zhang et al., 2008). Intriguingly, Sepw1 mrna has also been identified in processes of cultured central and peripheral neurons [(Willis et al., 2005, Willis et al., 2007), see supplemental tables]. However, the protein expression and function of Sepw1 in brain remains largely unexplored. In this report, we analyzed expression of Sepw1 protein in mouse brain and mouse brain-derived primary cells. We report that Sepw1 protein expression is observed in neurons of several brain regions including cortex, hippocampus, and cerebellum. Sepw1 immunoreactivity extends into the processes of these cells, and isolation of nerve terminals by synaptosome preparations revealed the presence of Sepw1. We have also identified several components of the selenoprotein synthesis machinery in isolated nerve terminals. Additionally, expression of Sepw1 in synaptic and non-synaptic fractions was reduced in Sepp1-deficient mice, despite no change in selenoprotein synthesis machinery. Taken together these data suggest that Sepw1 is highly expressed in neurons and may be locally synthesized in distal processes far removed from the nucleus, including synaptic sites. The widespread neuronal expression of Sepw1 is dependent on Sepp1, although the cells producing Sepp1 for Sepw1 expression are undetermined. The enzymatic role of Sepw1 is unclear, but the combined data argue for a role in oxidant-mediated cell signaling rather than detoxification of oxidizing agents. 77

87 MATERIALS AND METHODS Primary cell culture: Glass bottom tissue culture plates (World Precision Instruments, Sarasota, FL) were coated with 0.1 mg/ml laminin in 0.1 mg/ml poly-l-lysine solution for 1 h, and then rinsed with PBS. Primary cells from cortex, hippocampus, and cerebellum were harvested from postnatal day one C57BL/6 mice, gently dispersed by trituration, and plated on coated dishes. Cultures were maintained at 5.0% CO 2 and 5.0% relative humidity in Neurobasal-A medium (Invitrogen) with 5% fetal bovine serum (FBS) with the addition of 100 μm glutamate (Invitrogen) to reduce growth of glial cells and enrich growth of neurons. B27 supplement (Invitrogen) was added to replace FBS after 24 h, and glutamate omitted from the media after 3 days. FBS lots were tested for Se content (Bodycote, Santa Fe Springs, CA), and the selenium concentration of media containing 10% FBS was 105 nm as determined using inductively coupled plasma-mass spectrometry. B27 was tested for Se content (Bodycote) and the selenium concentration of media containing 2% B27 was 93.8 nm by the same method. Animals: C57BL/6 mice and genetically modified male mice on a C57BL/6 background lacking Sepp1 were bred on commercially available diets containing adequate Se (~0.25 ppm). Animals were given food and water ad libitum on a 12-hour light-cycle and group housed until experimentation. All experiments were conducted on adult mice aged 3-4 months during the light cycle. All animal procedures and experimental protocols were approved by the University of Hawaii Institutional Animal Care and Use Committee. Tissue Preparation: Mice were anesthetized with ketamine-xylazine, and sacrificed by transcardial perfusion. Mice were initially perfused with phosphate-buffered saline (PBS) to flush out blood, followed by perfusion with 4% paraformaldehyde (PFA) to fix the tissue. The mice heads were cut off and the brains dissected out. Brains were post-fixed in 4% PFA overnight, followed by cryoprotecting the tissue in 10% and 30% sucrose for at least 4 hours each. The brains were then embedded in optimal cutting temperature (OCT) compound and frozen until time of sectioning. 40 μm sections were cut on a Leica CM1900 cryostat and saved in cryoprotectant solution, containing 0.1 M phosphate buffer, 78

88 30% sucrose (w/v), and 30% ethylene glycol (v/v), until time of further experimentation. Immunohistochemistry: Primary cortical, hippocampal, and cerebellar cultures maintained for three weeks in vitro were used for immunolabeling. Brain sections stored in cryoprotectant in the freezer were warmed to room temperature, and sections containing cortex and hippocampus were selected for analysis. After thorough washing, sections were blocked in 5% normal goat serum with 0.3% Triton X-100 in PBS. After blocking, the sections were incubated in diluted primary antibody solution overnight at 4C. The following antibodies were used: Rabbit-anti-Sepw1 (Rockland) and Mouse-anti-Tuj1 (Covance). A control section where primary antibody was omitted was also included in the procedure. After washing out primary antibody, sections were incubated in species-matched secondary antibody. The secondary antibody was directly conjugated to fluorophores (Alexa Fluor dyes, Invitrogen) for fluorescence imaging. Additonally, some sections were dual-labeled with a fluorescent Nissl stain to label neurons (Neurotrace, Invitrogen). Sections were then mounted onto slides, and coverslipped in VectaShield containing DAPI for fluorescent labeling of nuclei. Additional sections were colorimetrically developed using 3,3 -diaminobenzidene (DAB), after signal amplification using the avidin-biotin complex method (Vector), and coverslipped using Permount. Synaptosome Preparation: Synaptosomes were prepared by the method of Dunkley (Dunkley et al., 1986). Mice were anesthetized with Tribromoethanol and sacrificed by decapitation. The brain was rapidly excised, rinsed in ice-cold 0.32 M sucrose, and immersed in ice-cold 0.32 M sucrose with 1 mm EDTA. Brain tissue was homogenized in 5 ml of ice-cold sucrose/edta by 10 strokes at 900 rpm using a pre-chilled Teflon/glass homogenizer. The homogenate was centrifuged at 3,600 rpm for 10 minutes at 4C in polycarbonate tubes. The resulting supernatant was collected and diluted with sucrose/edta to a total volume of 9 ml, and the pellet was resuspended in sucrose/edta and saved for whole cell lysis. Approximately 3 ml of diluted supernatant was loaded on the top of a discontinuous three layer Percoll gradient. Three gradients per brain were made by adding 2 ml of 23% Percoll to each polycarbonate tube, and slowly layering 2 79

89 ml each of 10% and 3% Percoll sequentially using a peristaltic pump. The gradients with sample were centrifuged at 20,000 rpm for 5 minutes at 4C to isolate synaptosomes. Isolated synaptosomes were collected from the interface band between the 23% and 10% Percoll layers in each gradient, and transferred and pooled directly to a large polycarbonate centrifuge tube. To wash synaptosomes, 25 ml of HEPES-buffered saline (HBS) was added to the tube, and was centrifuged at 15,000 rpm for 10 minutes at 4C. The pellet was resuspended in HBS, and centrifuged at 7,000 rpm for 7 minutes at 4C. The final pellet was resuspended in HBS for analysis by SDS-PAGE followed by western blotting for select selenoproteins and related factors. SDS-PAGE and Western blot: Total protein was extracted from whole-cell lysates by light sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA) containing DTT, EDTA, and protease inhibitors, followed by centrifugation according to the manufacturers protocol. Synaptosomes were resuspeneded in CelLytic MT buffer without sonication or centrifugation. Protein was added to reduced Laemmli buffer, boiled for 10 minutes, and loaded into 4-20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gel contents were transferred to PVDF membranes, which were blocked with undiluted Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE, USA) for one hour. Membranes were then probed for 90 minutes with one of the following primary antibodies: Rabbit-anti-GPX4 (AbFrontier, Seoul, Korea), Rabbit-anti-SEPW1 (Rockland, Gilbertsville, PA, USA), Rabbit-anti-SEPHS2 (Rockland), Rabbit-anti-SEPHS1 (Rockland), Rabbit-anti-SecP43 (Santa Cruz Biotech, Santa Cruz, CA, USA), Goat-anti-SBP2 (Everest Biotech, Oxfordshire, UK), Rabbit-anti-EFSec (AbCam, Cambridge, MA, USA), Mouse-anti-Syntaxin1 (Santa Cruz Biotechnology), Mouse-anti-PSD95 (Thermo Scientific), Mouse-anti-TBP (AbCam), Mouse-anti-beta actin (Sigma, St. Louis, MO, USA) and Mouse-anti-alpha Tubulin (Novus, Littleton, CO, USA). After washing with PBS containing 0.05% tween-20 (PBST), membranes were incubated in the dark in secondary antibodies labeled with infrared fluorophores (Li-Cor Biosciences). After further washes in PBST, blots were imaged and quantified with the Odyssey infrared imaging system (Li-Cor Biosciences). 80

90 Imaging: Fluorescence imaging of primary cultures and stained sections were performed on a Zeiss LSM 5 Pascal laser confocal inverted microscope equipped Ar and HeNe lasers. AlexaFluor-488, -546, and -633 secondary antibodies with directly conjugated fluorophores were used to detect primary antibody signals. Images were acquired using the included LSM software, and were analyzed using ImageJ. Bright-field imaging was performed using an upright Zeiss AxioScope 2 Plus microscope equipped with an ASI motorized stage and Zeiss Axiocam MRc camera. Statistical analysis: Data were analyzed using Microsoft Excel (Redmond, WA, USA), and plotted using GraphPad Prism software (San Diego, CA, USA). Unpaired t-tests comparing genotypes were performed for protein expression in synaptosome experiments. The significance criteria were set at p < 0.05 for statistical measures. 81

91 RESULTS We sought to determine whether selenoprotein W (Sepw1) is expressed in neurons in mice. To address this question, we first cultured primary cells harvested from neonatal mouse brain, and assessed expression of Sepw1 along with a neuron-specific marker, class III beta-tubulin (Tuj1). As seen in figure 1, primary cultures consisted primarily of neurons, and Tuj1 immunoreactivity (magenta, middle) showed some overlap with Sepw1 expression (green, left) in neurites. Primary neuronal cultures derived from neonatal cortex (Fig. 1A), hippocampus (Fig. 1B) and cerebellum (Fig. 1C) all contained robust Sepw1 expression, and some colocalization with Tuj1, as indicated by white color in the merged panels (Fig. 1, right). Sepw1 immunoreactivity was also observed in some Tuj1-negative cells, possibly indicating expression in glia. To confirm that Sepw1 was expressed in adult mouse brain, we performed dual label fluorescent imaging of fixed mouse brain sections using antibodies directed against Sepw1 and Tuj1 or a fluorescent Nissl stain. In somatosensory cortex we observed robust Sepw1 expression in large pyramidal neurons and extensive colocalization with Tuj1 (Fig. 2A). In CA1 of hippocampus (Fig. 2B), the pyramidal layer showed similar immunolabeling of Sepw1. Intriguingly, Sepw1 expression extended into the apical and basolateral dendrites of most pyramidal neurons, and was apparent in some axonal compartments as well (Fig. 2, right). To further analyze regional expression of neuronal Sepw1, we performed immunohistochemistry with DAB development. Extending the previous results in somatosensory cortex and CA1, Sepw1 expression was observed in somas and dendrites of motor cortex (Fig. 3A, left) and CA3 of hippocampus (Fig. 3A, right). Additionally, piriform cortex (Fig. 3B) and cingulate cortex (Fig. 3C) displayed high Sepw1 immunoreactivity in pyramidal neurons. Purkinje neurons of cerebellum (Fig. 3D), and their highly branched dendritic arbors, also showed abundant expression of Sepw1. In fact, most neurons appeared to express Sepw1 to some degree and neuropil generally appeared immunopositive for Sepw1. Conspicuously, large neurons showed immunoreactivity in neuritic processes. The widespread expression in neurons of adult mouse brain, along with the punctate staining in varicose segments of cultured neurons, 82

92 led us to question if Sepw1 is expressed in the synaptic compartment. To assess if Sepw1 is expressed in synapses, we prepared synaptosomes from adult mice and performed western blotting of the purified samples. After confirming the absence of contaminating nuclear proteins by blotting for TATA-binding protein (TBP) (Fig. 4A), we blotted for Sepw1 and found it to be present in synaptosomes (Fig. 4B). Sepw1 mrna is known to be reduced in the brain of Sepp1-/- mice (Hoffman, 2007). To determine if synaptically expressed Sepw1 is reduced in the absence of Sepp1, we next prepared synaptosomes from littermate Sepp1-/- and wild-type mice. We observed a dramatic decrease in Sepw1 expression in synaptosomes isolated from Sepp1-/- mice compared to control mice (Fig. 5A). Additionally, western blot analysis of Gpx4 showed presence in wild-type synaptosomes, and slightly reduced expression in Sepp1-/- synaptosomes (Fig. 5B). We used beta actin to control for loading across samples (Fig. 5C). After normalizing to Actin, quantification of selenoprotein expression revealed that Sepw1 was significantly reduced to <25% of wild-type levels, while GPX4 was not significantly reduced in synaptosomes (Fig. 5D-E).We did not observe a qualitative enrichment of Sepw1 or Gpx4 in synaptosomes versus whole-cell lysate samples, nor did we observe selective depletion of Sepw1 in either fraction in Sepp1 knockout mice. These findings indicate that Sepw1 is roughly homogenously distributed throughout neurons, and all compartments similarly depend on Sepp1 to maintain expression. In addition to expression in the neuronal somata, Sepw1 mrna has been detected in axons, dendrites and neuropil, suggesting that it may be locally translated in neuronal processes (Cajigas et al., 2012, Willis et al., 2007). However, selenoprotein synthesis is unique, and requires several additional protein factors beyond the standard translation machinery. We sought to assess if translation of selenoproteins can occur in distal processes of neurons. Therefore, we did western blotting of synaptosomes for several proteins involved in selenoprotein translation after confirming absence of nuclear contamination by analyzing TBP (Fig. 6A). Both the Sec-specific elongation factor (EFSec) (Fig. 6B) and the SECIS-binding protein 2 (Sbp2) (Fig. 6D) are absolutely required for selenoprotein translation, and appeared to be present in synaptosomes in 83

93 addition to whole cell lysate samples. Selenophosphate, produced by the selenoenzyme selenophosphate synthetase 2 (SPS2), acts as the Se-donor during selenoprotein translation. We were able to detect the presence of SPS2 in synaptosomes (Fig. 6C). Interestingly, EFSec and SPS2 appeared to be enriched in synaptosome fractions compared to whole cell lysate samples, further suggesting the existence of selenoprotein translation in this compartment. The Sec-tRNA associated-protein, SecP43, and selenocysteine lyase (Scly) have been implicated in selenoprotein translation efficiency, but are not absolutely required (Kurokawa et al., 2011, Squires & Berry, 2008). We were unable to detect SecP43 in synaptosomes, despite robust expression in whole-cell lysates (Fig. 6E). Conversely, Scly did appear to be present in synaptosomes in similar abundance as whole cell lysates (Fig. 6F). Unlike Sepw1, and Gpx4 to a lesser degree, none of the proteins involved in selenoprotein synthesis were altered in Sepp1-/- mice, compared to wild-type controls. This is especially curious for SPS2, which is itself a selenoprotein. The combination of presented data argues that translation of selenoproteins Sepw1 and Gpx4 in synapses may be possible. 84

94 DISCUSSION The results reported herein describe the first characterization of regional selenoprotein W (Sepw1) localization in mouse brain, as well as selenoprotein and synthesis factor expression in isolated nerve terminals. Sepw1 is abundantly expressed in neuronal somata and neuropil, and is expressed along with several selenoprotein synthesis proteins in synaptosome fractions. In all regions of brain in mice lacking selenoprotein P (Sepp1), Sepw1 expression is drastically reduced without effect on synthesis factors, indicating that Sepp1 facilitates Sepw1 synthesis. Selenium (Se) is a trace micronutrient that is incorporated into the unique amino acid, selenocysteine (Sec). Sec-containing selenoproteins are typically oxidoreductase enzymes that play crucial roles in reducing reactive oxygen species and oxidized macromolecules. A selenoprotein that is widely distributed across all domains of life, Sepw1, is particularly abundant in brain and muscle of mammals (Gu et al., 2000). Sepw1 mrna expression is observed in cephalic neural folds and somites in developing rodents, with continued high expression as they become the adult brain and skeletal muscles (Loflin et al., 2006). Sepw1 was initially identified due to its absence in muscle of myopathic Se-deficient lambs, however brain expression of Sepw1, unlike in muscle, is not depleted by dietary Se deficiency (Whanger, 2001). However, Sepp1-deficient mice show reduced Sepw1 mrna and protein in brain (Hoffmann et al., 2007). Sepw1 is the smallest described mammalian selenoprotein at ~10 kda and contains an N-terminal thioredoxin-like Cys-X-X-Sec redox motif, where X is any amino acid (Lobanov et al., 2009). As with all selenoproteins, the Sec residue is encoded by a UGA codon in the mrna. A Sec Insertion Sequence (SECIS) in the 3'UTR of the mrna, the SECIS binding protein SBP2, and the Sec-specific elongation factor EFSec help to bypass translation termination and incorporate Sec during translation [reviewed in (Squires & Berry, 2008)]. Sepw1 also has another conserved Cys residue in the N-terminal region that is known to bind glutathione (GSH) (Beilstein et al., 1996, Gu et al., 1999). Antioxidant function attributed to Sepw1 is GSH dependent. In vitro 85

95 experimental studies, which increased or decreased Sepw1 expression, have demonstrated elevated and reduced resistance to oxidizing agents but only in the presence of reduced GSH (Jeong et al., 2002). However, other studies demonstrate that sirna knockdown of Sepw1 causes increased enzyme activities of glutathione peroxidase, superoxide dismutase, and catalase and total antioxidative capability and glutathione level in cultured muscle cells, which prevents oxidant-induced apoptosis (Xiao-Long et al., 2010). These authors suggested a role for Sepw1 in the antioxidative system that is not direct peroxide detoxification. Therefore, the in situ enzymatic role of Sepw1 has remained elusive. Sepw1 mrna rapidly declines in response to peroxide, suggesting that it has a role in oxidative metabolism. Similar to the metabolic enzyme glyceraldehyde phosphate dehydrogenase (GAPDH), oxidative inactivation of Sepw1 may be involved in rerouting carbohydrate flux from glycolysis to the pentose phosphate pathway, stimulating NADPH generation and reducing the intracellular pool of GSH (Loflin et al., 2006). A pull down experiment indicated that Sepw1 interacts with the cytoskeletal microtubule protein tubulin (Dikiy et al., 2007). Our data show robust colocalization of Sepw1 with the neuron-specific beta tubulin, Tuj1. Sepw1 was additionally shown to immunoprecipitate specifically with the beta and gamma isoforms of the family of scaffolding proteins (Aachmann et al., 2007). A computational study explored a putative reaction mechanism, whereby Sepw1 regulates the oxidation state of a conserved and solvent exposed Cys residue of beta and gamma. Sepw1 is suggested to reduce oxidized Cys-Sulfenic acid of back to its parental thiol using the Cys-X-X-Sec motif in combination with the bound GSH moiety (Musiani et al., 2010) proteins are multifunctional proteins that coordinate the interaction of kinases and phosphatases with other regulatory proteins, thereby affecting phosphorylation-dependent cellular processes (Fu et al., 2000). Like SEPW1 gene expression, the YWHAG gene for gamma, is highly expressed in brain, skeletal muscle, and heart in humans (Horie et al., 1999). Sepw1 has also been implicated in regulating growth factor-stimulated control of cell cycle-entry in epithelial cells. Knockdown of Sepw1 by sirna in breast and prostate 86

96 epithelial cells inhibits EGF-stimulated G1/S transition via nuclear accumulation of p53, leading to induction of p21 and G1 arrest (Hawkes & Alkan, 2011, Hawkes et al., 2012). Intriguingly, the stability of the EGFR as well as tyrosine phosphorylation was decreased by knockdown of Sepw1 (Hawkes, personal communication). EGFR tyrosine phosphorylation and reduction of the EGFR turnover rate is a process that depends on EGF-induced hydrogen peroxide production, which may oxidatively inactivate protein tyrosine phosphatases (Deyulia & Carcamo, 2005, Deyulia et al., 2005). Since proteins are established in regulating phosphorylation-mediated cell signaling, Sepw1 may function in oxidative signal transduction reactions from receptors to target proteins via reactive oxygen intermediates. High muscle expression of Sepw1 mrna is associated with myoblasts, and expression is decreased in differentiated myotubes (Loflin et al., 2006). Thus, the abundance of Sepw1 mrna and protein in post-mitotic neurons is mysterious. Sepw1 mrna and protein are widely expressed in neurons, including apparent expression in axonal and dendritic compartments (Cajigas et al., 2012, Willis et al., 2005, Willis et al., 2007). Whether translation of Sepw1 occurs in these distal cellular compartments is uncertain. Selenoprotein translation in mammals specifically requires the proteins SBP2 and EFSec, in addition to the standard translation machinery. Both of these proteins were identified in synaptosomes, along with SPS2 and SCLY which are important in Sec metabolism. Thus, the only major protein involved in selenoprotein translation that was not investigated in this study is the Sec-synthetase enzyme, SepSecS. SepSecS is also known as soluble liver antigen/liver pancreas antigen (SLA/LP) and is required to generate the Sec-loaded trnasec (Palioura et al., 2010). We were unable to test for the presence of SepSecS in synaptosomes. Selenoprotein mrnas are thought to be packaged into mrnp complexes, which aid in preventing nonsense codon-mediated decay (NMD) of transcripts with a Sec-specifying UGA that could be interpreted as a premature termination codon. Two mrna binding proteins important for nervous system function, DJ-1/Park7 and Staufen 2, have been experimentally demonstrated to bind Sepw1 mrna (Blackinton et al., 2009, Furic et al., 87

97 2008, Maher-Laporte & Desgroseillers, 2010, Van Der Brug et al., 2008). DJ-1 is a multifunctional redox-sensitive protein that is associated with Parkinson s disease, other neurodegenerative disorders, and cancer (Kahle et al., 2009). Two isoforms of staufen, Stau1 and Stau2, are known to direct subcellular localization of mrnas, and have recently been implicated in synaptic plasticity (Lebeau et al., 2011a, Lebeau et al., 2011b). Both DJ-1 and Stau2 proteins have shown varying degrees of localization to synapses, axons and dendrites, further suggesting the local regulation of Sepw1 expression in distal compartments of neurons (Jeong et al., 2007, Olzmann et al., 2007, Price et al., 2006, Usami et al., 2011). Lastly, Stau2 is reported to interact with the key NMD protein Upf1, which is implicated in selenoprotein translation because their mrnas contain in-frame termination codons. Intriguingly, Stau2 may upregulate protein expression of target mrnas in a Upf1-dependent mechanism, while upregulation of the target mrnas is Upf1-independent (Miki et al., 2011). These mechanisms may ultimately provide high specificity and sensitivity for the local expression of Sepw1 in neurons. In sum, we have shown that Sepw1 expression in mouse is abundant in neurons of several brain regions, including cingulate and piriform cortex, hippocampus, and Purkinje cells of cerebellum. We also showed Sepw1 expression in neuronal processes and some colocalization with tubulin. Analysis of isolated nerve terminals further revealed the presence of Sepw1 and much of the selenoprotein synthesis machinery in synaptic compartments. Sepw1 expression in synaptosomes and whole-cell lysates of brain was drastically reduced in Sepp1 knockout mice. Combined with previous reports documenting Sepw1 mrna expression in neuronal processes and association of Sepw1 transcripts with mrna-binding proteins, regulation of Sepw1 expression in pre- and/or post-synaptic compartments is suggested and warrants further investigation. 88

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102 FIGURE LEGENDS Figure 1: Sepw1 is expressed in cell bodies and processes of cultured neurons. Primary cultures derived from neonatal mouse cortex (A), hippocampus (B), and cerebellum (C) were grown on coverslips for 3 weeks and subsequently double immunolabeled for the presence of Sepw1 (in green, left) and a neuronal marker, Tuj1 (in magenta, center). Merged images show sporadic colocalization between Sepw1 and Tuj1 (in white, right) in neuronal somata and neurites in cultures from all three braion regions. Figure 2: Sepw1 is expressed in cell bodies and processes of pyramidal neurons in cortex and hippocampus. Fixed, frozen mouse brain sections were immunolabeled for Sepw1 in somatosensory cortex (A) and CA1 region of hippocampus (B). Cortex sections were additionally dual immunolabled with Tuj1, while hippocampus sections were labeled with a fluorescent Nissl stain. Large pyramidal neurons in both regions strongly displayed Sepw1 immunoreactivity, which prominently extended into the apical and basolateral dendrites. A few proximal axonal segments also showed Sepw1 immunoreactivity. Figure 3: Regional expression of Sepw1 in neurons of mouse brain. (A) Motor cortex (left) and CA3 region of hippocampus (right) displayed prominent Sepw1 staining in cell bodies and processes. (B-D) Low magnification (left) and high magnification (right) photomicrographs of piriform cortex (B), cingulate cortex (C), and cerebellum (D) show Sepw1 immunoreactivity in neurons. Large pyramidal (B-C) and Purkinje (D) neurons show high expression in dendritic arbors. Figure 4: Sepw1 is present in isolated nerve terminals. Synaptosomes were prepared and analyzed by SDS-PAGE followed by Western blot. Lanes 1,2 and 3,4 represent two independent preparations from wild-type C57 mice run in duplicate. (A) Analysis of TATA-binding protein (TBP) in synaptosome fractions indicated that the preparations were free of nuclear contamination. (B) Western blot showed the presence of Sepw1 in synaptosome fractions. 93

103 Figure 5: Sepw1 expression in isolated nerve terminals is greatly reduced in mice lacking Sepp1. Additional synaptosome fractions were prepared from Sepp1-/- mice (KO) and wild-type littermate conrols (WT). These fractions were analyzed in comparison to whole cell lysate (WCL) and mitochondrial (Mito) fractions. Western blotting for Sepw1 (A) and Gpx4 (B) revealed the presence of both selenoproteins, with Actin (C) used as a loading-control. Quantitation of synaptosomal expression of Sepw1 (D) and Gpx4 (E) revealed that Sepw1 was significantly decreased (p<0.01), while Gpx4 was not. Figure 6: Several selenoprotein synthesis factors are present in isolated nerve terminals. (A) TBP was analyzed to confirm that nuclear proteins, where selenoprotein synthesis factors are known to be present, are not contaminating the synaptosome fractions. (B) EFSec was found to be present in synaptosomes and qualitatively enriched compared to whole cell lysates, and absent from mitochondria. (C) SPS2, the enzyme that generates selenophosphate, showed a similar expression pattern, being apparently increased in isolated nerve terminals, and absent from mitochondria. (D) SBP2, which is required for selenoprotein translation, was found in similarly low abundance in synaptosomes and whole cell lysates, but not mitochondria. (E) The trnasec-associated protein SecP43 was found only in whole cell lysates, where as Sec lyase (Scly) (F) was found in both synaptosome and whole cell lysate fracions. 94

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110 CHAPTER 4 METHAMPHETAMINE-INDUCED ALTERATIONS IN SELENOPROTEIN EXPRESSION IN MICE ABSTRACT Selenium is an essential micronutrient in mammals. It is primarily incorporated into the amino acid selenocysteine, in a family of proteins termed selenoproteins. Some selenoproteins are known antioxidant enzymes, and many of them are expressed in the brain. Methamphetamine is a highly addictive drug with potentially neurotoxic side effects. Previous studies have demonstrated modulatory effects of dietary selenium levels on methamphetamine-induced neurotoxicity. Specifically, dietary selenium deficiency promotes excessive neurotoxicity after methamphetamine administration, while dietary selenium supplementation prevents it. Although the activity of cellular glutathione peroxidase, a well characterized selenoprotein, is largely attributed to the effects of dietary selenium, other selenoproteins may also influence the extent of methamphetamine-induced neurotoxicity. To determine what selenoproteins may be adversely impacted by methamphetamine, we screened for changes in brain selenoprotein transcripts after exposure to methamphetamine in mice. We found an upregulation of select selenoprotein mrnas without effect on protein levels, possibly indicating that methamphetamine causes increased transcription of selenoprotein genes, but an inability to upregulate selenoprotein translation. We also looked at methamphetamine-induced toxicity in mice lacking selenoprotein P, a selenium transport protein important for brain selenium supply. Selenoprotein P knockout mice did not display exacerbated toxicity after methamphetamine administration, compared to wild-type mice. These results may indicate that methamphetamine alters the expression profile of selenoproteins in brain, but less than selenoprotein P. 101

111 INTRODUCTION Methamphetamine (MA) is a psychostimulant drug with toxic side effects. Long term use of MA leads to striatal depletion of dopamine (DA), metabolites, and proteins involved in DA synthesis and transport (Cadet & Krasnova, 2009). It has also been associated with persistent neurotoxicity towards dopaminergic terminals and striatal neurons (Tulloch et al., 2011). Although the exact cellular and molecular mechanisms of toxicity are not fully elucidated, several reports indicate a prominent role for oxidative signaling via reactive oxygen species (ROS) including hydrogen peroxide and peroxynitrite (Cadet & Brannock, 1998). Selenium (Se) is a micronutrient that is incorporated into oxidoreductase enzymes with potent antioxidant activity and prominent roles in cellular oxidation-reduction (redox) balance (Kryukov et al., 2003). These enzymes use Se in the form of selenocysteine (Sec). Sec is a unique amino acid because it is encoded by the UGA codon in mrnas, which typically signals for termination of protein synthesis. To recode the UGA for Sec incorporation, the 3' untranslated regions of selenoprotein mrnas are endowed with a Sec Insertion Sequence (SECIS). The SECIS element is a stem-loop structure that binds to specific proteins, SECIS binding protein 2 and the Sec-specific elongation factor, which help bypass termination and promote insertion of Sec [reviewed in (Bellinger et al., 2009)]. Sec is a highly reactive residue, thereby rendering enzymatic selenoproteins with high catalytic activity. Cellular glutathione peroxidase 1 was the first discovered selenoenzyme, and rapidly reacts with hydrogen peroxide and reduced glutathione (GSH), releasing water and glutathione disulfide (GSSG) (Rotruck et al., 1973). Subsequently, four other GPX isozymes were determined to be selenoproteins in humans. GPX4 has high affinity for lipid and organic hydroperoxides, is essential for neuronal survival, and is required for embryonic development (Seiler et al., 2008, Yoo et al., 2012). GPX4 is associated with neuromelanin in dopaminergic neurons of human brain, and in dystrophic axons of Parkinson's disease patients (Bellinger et al., 2011). 102

112 Previous studies have shown that dietary Se status can profoundly alter the metabolism of dopamine in brain and MA-induced toxicity to dopaminergic neurons. Specifically, a Se deficient diet increases DA turnover in the substantia nigra, hippocampus and prefrontal cortex (Castano et al., 1995, Castano et al., 1997, Castano et al., 1993). Further, Se supplementation can prevent the MA-induced depletion of TH, DA and DA metabolites in the nigrostriatal pathway (Imam & Ali, 2000, Imam et al., 1999, Kim et al., 1999). GPX was suggested to be specifically inactivated by MA-induced production of peroxynitrite, and was generally attributed as the cause for protective effects of Se supplementation. However, individual selenoproteins were not examined. In this research, we investigated the regulation of selenoproteins in response to MA by mrna and protein expression to assess the contribution of select selenoproteins. 103

113 MATERIALS AND METHODS Animals: Male and female C57BL/6 mice, and genetically modified mice on the same background lacking Sepp1 were bred on commercially available diets containing adequate Se (~0.25 ppm). Animals were given food and water ad libitum on a 12-hour light-cycle and group housed until experimentation. All experiments were conducted on adult mice aged 3-4 months during the light cycle. Male and female mice of both genotypes were used in approximately equal numbers to examine sex differences present in the animals. All animal procedures and experimental protocols were approved by the University of Hawaii Institutional Animal Care and Use Committee. Animal Procedures: Mice were administered methamphetamine by intraperitoneal (i.p.) injection with various doses depending on the experiment. In initial studies, methamphetamine was chronically administered to C57/Bl6 mice 5 days per week for 2 or 8 weeks, with the dosage escalating from 2mg/kg to 20 mg /kg and then 3 mg/kg to 30 mg/kg. In two subsequent studies using Sepp1-/- mice and wild-type littermate controls, two different paradigms were used. The first study used two i.p. injections of 10 mg/kg administered approximately 4 hours apart, while the second used a single bolus of 40 mg/kg. For qpcr and Western blot analysis, animals were sacrificed 24 hours after the final injection. For histological analysis, animals were sacrificed 72 hours after the final injection. Tissue Preparation: For biochemistry, mice were sacrificed by CO 2 asphyxiation, and the brains rapidly excised, washed in PBS and snap-frozen in liquid nitrogen. The striatum and ventral mesencephalon were dissected from frozen tissue in a cold chamber maintained at -20C. Tissue was then cut into small pieces, using a razor blade, and collected into pre-chilled tubes. Tissue was homogenized by sonication in the appropriate buffer for the ensuing experiment. For histology, mice were anesthetized with ketaminexylazine, and sacrificed by transcardial perfusion. Mice were initially perfused with phosphate-buffered saline (PBS) to flush out blood, followed by perfusion with 4% paraformaldehyde (PFA) to fix the tissue. The mice heads were cut off and the brains dissected out. Brains were post-fixed in 4% PFA overnight, followed by cryoprotecting 104

114 the tissue in 10% and 30% sucrose for at least 4 hours each. The brains were then embedded in optimal cutting temperature (OCT) compound and frozen until time of sectioning. 40 μm sections were cut on a Leica CM1900 cryostat and saved in cryoprotectant solution, containing 0.1 M phosphate buffer, 30% sucrose (w/v), and 30% ethylene glycol (v/v), until time of further experimentation. Quantitative RT-PCR: Total RNA from tissue was prepared by Trizol extraction (Invitrogen, Carlsbad, CA, USA) followed by purification using the RNeasy kit (QIAgen, Valencia, CA, USA). Concentration and purity of extracted RNA and synthesized cdna was determined using A 260 /A 280 ratio measured on an ND1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Synthesis of cdna was carried out using High Capacity cdna Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), with 1 g RNA per 20 l reaction. For real-time PCR, 100 ng of the cdna was used in 5 l reactions with Platinum SYBR Green qpcr SuperMix-UDG (Invitrogen). Reactions were carried out in triplicate or quadruplicate in a LightCycler 480 II thermal cycler (Roche, Indianapolis, IN, USA). Cycling conditions followed the manufacturers suggestions in the SYBR Green kit instructions. All qpcr results were normalized to 18S rrna expression as a housekeeping gene and analyzed using Absolute Quantification Software (Roche). SDS-PAGE and Western blot: Total protein was extracted from powdered mouse tissues by light sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA), followed by centrifugation according to the manufacturers protocol. Protein was added to reduced Laemmli buffer, boiled for 10 minutes, and loaded into 4-20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gel contents were transferred to PVDF membranes, which were blocked with 5% milk in PBS containing 0.05% tween-20 (PBST) for one hour. Membranes were then probed for 90 minutes at room temperature or overnight at +4C with the following primary antibodies: Rabbitanti-Tyrosine Hydroxylase (TH) (Pel-Freez, Rogers, AR, USA), Rabbit-anti-GPX4 (AbNova, Taipei, Taiwan), Rabbit-anti-SEPW1 (Rockland, Gilbertsville, PA, USA), Rabbit-anti-SEPHS2 (Rockland) and Mouse-anti-beta actin (Sigma, St. Louis, MO, USA). After washing with PBST, membranes were incubated in secondary antibodies 105

115 conjugated to horseradish peroxidase. After further washes in PBST, blots were visualized by enhanced chemiluminesence reaction followed by exposure to film. Immunohistochemistry: Brain sections stored in cryoprotectant in the freezer were warmed to room temperature, and sections containing striatum and substantia nigra were selected for analysis. After thorough washing, sections were blocked in 5% normal goat serum with 0.3% Triton X-100 in PBS. After blocking, the sections were incubated in diluted primary antibody solution overnight at +4C. The following antibodies were used: Rabbit-anti-TH (Pel-Freez), Rabbit-anti-GPX4 (AbNova), and Rabbit-anti-SEPW1 (Rockland). A control section where primary antibody was omitted was also included in the procedure. After washing out primary antibody, sections were incubated in speciesmatched secondary antibody. The secondary antibody was either directly conjugated to fluorophores (Alexa Fluor dyes, Invitrogen) for fluorescence imaging, or was biotinylated for colorimetric visualization. After incubation in biotinylated secondary antibody, the sections were then incubated in Avidin-Biotin Complex solution, followed by visualization with 3,3-Diaminobenzidine (DAB) (Vector Labs, Burlingame, CA, USA). Additionally, some sections were dual-labeled with a fluorescent Nissl stain to label neurons (Neurotrace, Invitrogen), or phalloidin to label filamentous actin (Invitrogen). Sections were then mounted onto slides, and either coverslipped in VectaShield containing DAPI for fluorescence, or air-dried overnight and coverslipped using Permount after ethanol dehydration and Xylene clearing. TUNEL Histology: Apoptotic cell death in sections was assessed using an In Situ Cell Death Detection Kit (Roche), using the Terminal deoxynucletidyl transferase (TdT) dutp Nick End Labeling (TUNEL) method. Sections were thoroughly washed, and then blocked with 3% hydrogen peroxide in methanol for 10 minutes at room temperature. Sections were then permeabilized by incubation in 0.5% Triton X-100 at +65C for 30 minutes. After washing in PBS, TUNEL labeling was performed for 60 minutes at +37C in the dark. A negative control section lacking TdT, and a positive control section that was incubated in DNAse I was also included. The fluorescent label was then converted by incubating in POD conversion solution included in the kit for 30 minutes at +37C, and was visualized by DAB reaction. The sections were mounted and air-dried overnight. The 106

116 sections were then counterstained with 0.5% Cresyl Violet after ethanol dehydration, cleared in Xylene, and coverslipped using Permount. Imaging: Both fluorescence and bright-field imaging of stained sections were acquired on an upright Zeiss Axioscope 2 Plus microscope equipped with an ASI motorized stage and Zeiss Axiocam MRc camera. Fluorescence imaging was done with an ultraviolet lamp and appropriate filter sets for the fluorophores used, while bright-field imaging used standard white-light illumination. Images were acquired using the included AxioVision software, and were analyzed using ImageJ. Statistical analysis: Data were analyzed using Microsoft Excel (Redmond, WA, USA), and plotted using GraphPad Prism software (San Diego, CA, USA). Two-way ANOVA was used to determine an interaction between MA administration and selenoprotein expression. Post hoc test using Bonferroni correction for multiple comparisons was used to determine significance between individual groups. Unpaired t-tests comparing control and experimental groups were performed for some experiments. The significance criteria were set at p < 0.05 for all statistical measures. 107

117 RESULTS We sought to determine whether methamphetamine (MA) causes an alteration in selenoprotein transcript expression in the substantia nigra and striatum of mice brains. To achieve this, we administered MA to C57BL/6 mice for a period of 2 weeks, and harvested their brains for analysis by quantitative real-time polymerase chain reaction (qpcr). The majority of selenoprotein mrnas were analyzed, and two-way ANOVA revealed that MA had an effect on selenoprotein transcript expression in midbrain (F(1,92)=6.329, p=0.0136) and striatum (F(1,92)=6.854, p=0.0103). Qualitatively, selenoprotein expression appeared to increase in response to MA administration in both brain regions (Fig. 1). Individual selenoprotein transcripts of interest were further analyzed by t-test to compare groups. We found that selenoprotein W (Sepw1) (t(4)=3.083, p=0.0368) and selenophosphate synthetase 2 (Sps2) (t(4)=2.809, p=0.0484) mrnas were significantly increased in midbrain, but not in striatum of MA-treated mice. However, we did not find a significant increase in mrna expression of glutathione peroxidase 4 (Gpx4) or selenoprotein P (Sepp1) in either brain region (Fig. 2). These results led us to question if selenoprotein expression was also increased in the mice brains. In contrast to the mrna data, western blot analysis of Sepw1, Sps2, and Gpx4 did not reveal an increase in selenoprotein expression in response to MA in either brain region (Fig. 3). We next sought to determine if prolonged, higher-dose MA exposure has a more dramatic effect on selenoprotein expression. When mice were exposed to elevated doses of MA for 8 weeks, we found that selenoprotein mrnas in midbrain were not increased, unlike with short term MA administration. However, Sepw1 and Sps2 were decreased in midbrain, contrasting their previously seen upregulation (Fig. 4, top). This may indicate that expression of Sepw1 and Sps2 transcripts are regulated in midbrain by MA. In striatum, qpcr showed that selenoprotein mrna levels were dramatically reduced (Fig. 4, bottom). High-dose exposure to MA is known to cause degeneration of dopaminergic terminals and medium spiny neurons in striatum (Zhu et al., 2006), which may explain the reduction of selenoprotein transcripts in this region. 108

118 To determine if selenoprotein P augments selenoprotein expression in the context of MAinduced changes, we next administered MA to Sepp1-/- and wild-type littermate control mice. When administering two injections of 10 mg/kg MA and sacrificing 24 hours after the second injection, we did not detect any major changes in selenoprotein expression due to MA. However, the Sepp1-/- animals had reduced expression of several selenoprotein transcripts, most notably with Sepw1 (data not shown). As we observed little change in selenoprotein mrna expression with 2 injections of 10 mg/kg MA, we next administered high-dose MA at 40 mg/kg. We investigated protein expression by western blot and immunohistochemistry, and also performed TUNEL histology to investigate cell death. Western blot analysis of striatum revealed that Sepp1- /- mice have reduced levels of Sepw1 and Gpx4, but not Sephs2, and that MA did not significantly influence the expression of selenoproteins in wild-type or knockout mice (Fig. 5). Similar results were obtained in midbrain (data not shown). Tyrosine hydroxylase (TH) expression was investigated by western blotting and by histology. MA caused a slight upregulation of TH in midbrain lysates when measured by Western blot (data not shown). However, an apparent decrease in TH staining in substantia nigra was observed in brain sections (Fig. 6). TH expression did not show much change in striatum in response to MA either by western blot or immunohistochemistry (data not shown). Sepp1-/- mice did not respond differently than wild-type mice in terms of TH expression in midbrain or striatum. In congruence with these findings, TUNEL histology revealed an absence of apoptotic cell death in Sepp1-/- and wild-type mice exposed to MA (Fig. 7). 109

119 DISCUSSION The results presented here show that MA affects both the short- and long-term mrna expression of selenoproteins in mammalian brain. These data are in agreement with reports indicating that Se deficiency potentiates the neurotoxic profile of MA exposure, while Se repletion attenuates it (Imam et al., 1999, Kim et al., 1999). Neurons appear to be the key site of Se utilization, although these cells possess fairly low GPX activity (Zhang et al., 2008). This suggests that selenoproteins in addition to GPX1 are involved in protection from MA-induced neurotoxicity. GPX4 is a phospholipid hydroperoxidase that is particularly important for neuronal development and survival (Seiler et al., 2008). Conditional deletion of GPX4 in neurons of adult mice causes rapid lipoxygenase-mediated apoptosis (Yoo et al., 2012). GPX4 is found in dopaminergic neurons of human brain, where it has a conspicuous association with neuromelanin. It has also been found in dystrophic neurites of individuals with Parkinson s disease (Bellinger et al., 2011). These findings strongly implicate GPX4 as a necessary component of dopamine neurons. Selenoprotein P (Sepp1) is an extracellular selenoprotein that functions in Se distribution, and has roles in preventing neurodegeneration and deficits in synaptic plasticity (Caito et al., 2011, Peters et al., 2006, Valentine et al., 2008, Valentine et al., 2005). The finding that Sepp1-deficient mice did not display enhanced sensitivity to neurotoxicity in dopaminergic brain regions has not been reported, but is probably due to the severe pathology associated with deletion of Sepp1 alone. Although TUNEL labeling of apoptotic cells revealed no increase in cell death in Sepp1 knockout mice, the mice are unquestionably under more cellular stress than wild-type MA-treated control counterparts. Selenoprotein W (Sepw1) is a small thioredoxin-like protein that has a role in mediating cell signaling and is particularly abundant in muscle and brain of mammals (Gu et al., 2000, Whanger, 2009). Interestingly, dietary Se deficiency causes depletion of Sepw1 in muscle, but not brain (Whanger, 2001). Maintenance of Sepw1 expression during dietary Se deprivation is likely due to the Se-transport function of Sepp1, since mice deficient in 110

120 Sepp1 have drastically reduced expression of Sepw1 mrna and protein in brain (Hoffmann et al., 2007). Sepw1 mrna expression was altered by both short and long term MA administration, albeit in different directions, perhaps making it a specific target in MA-induced neurotoxicity. Lastly, selenophosphate synthetase 2 (Sps2) is a selenoprotein that generates selenophosphate for selenocysteine biosynthesis (Xu et al., 2007). Although we initially found that Sephs2 mrna was acutely upregulated by MA, the effect on protein was minimal. However, the mrna was downregulated upon long-term MA treatment, similar to Sepw1. Transcriptional regulation of Sepw1 and Sps2 suggests these two selenoproteins may be specifically targeted in midbrain during MA exposure. Further, the activity of the Sps2 enzyme was not assessed, and selenophosphate production may indeed be compromised by MA treatment. Collectively these data suggest that selenoproteins are neuroprotective in the context of MA exposure. The reduction of selenoprotein mrna expression in striatum after chronic MA exposure indicates that Se availability may be limiting under MA-induced stress, and therapeutic supplementation with dietary Se may be helpful. We did not detect dramatic changes in selenoprotein expression by western blot, but it remains possible that the Sec residue is oxidatively modified and that enzymatic catalysis is inactivated or reduced by high levels of MA-induced ROS production. 111

121 REFERENCES Bellinger, F.P., Bellinger, M.T., Seale, L.A., Takemoto, A.S., Raman, A.V., Miki, T., Manning-Bog, A.B., Berry, M.J., White, L.R. & Ross, G.W. (2011) Glutathione Peroxidase 4 is associated with Neuromelanin in Substantia Nigra and Dystrophic Axons in Putamen of Parkinson's brain. Molecular neurodegeneration, 6, 8. Bellinger, F.P., Raman, A.V., Reeves, M.A. & Berry, M.J. (2009) Regulation and function of selenoproteins in human disease. Biochem J, 422, Cadet, J.L. & Brannock, C. (1998) Free radicals and the pathobiology of brain dopamine systems. Neurochemistry international, 32, Cadet, J.L. & Krasnova, I.N. (2009) Molecular bases of methamphetamine-induced neurodegeneration. International review of neurobiology, 88, Caito, S.W., Milatovic, D., Hill, K.E., Aschner, M., Burk, R.F. & Valentine, W.M. (2011) Progression of neurodegeneration and morphologic changes in the brains of juvenile mice with selenoprotein P deleted. Brain research, 1398, Castano, A., Ayala, A., Rodriguez-Gomez, J.A., de la Cruz, C.P., Revilla, E., Cano, J. & Machado, A. (1995) Increase in dopamine turnover and tyrosine hydroxylase enzyme in hippocampus of rats fed on low selenium diet. Journal of neuroscience research, 42, Castano, A., Ayala, A., Rodriguez-Gomez, J.A., Herrera, A.J., Cano, J. & Machado, A. (1997) Low selenium diet increases the dopamine turnover in prefrontal cortex of the rat. Neurochemistry international, 30, Castano, A., Cano, J. & Machado, A. (1993) Low selenium diet affects monoamine turnover differentially in substantia nigra and striatum. Journal of neurochemistry, 61, Gu, Q.P., Sun, Y., Ream, L.W. & Whanger, P.D. (2000) Selenoprotein W accumulates primarily in primate skeletal muscle, heart, brain and tongue. Molecular and cellular biochemistry, 204, Hoffmann, P.R., Hoge, S.C., Li, P.A., Hoffmann, F.W., Hashimoto, A.C. & Berry, M.J. (2007) The selenoproteome exhibits widely varying, tissue-specific dependence on selenoprotein P for selenium supply. Nucleic Acids Res, 35, Imam, S.Z. & Ali, S.F. (2000) Selenium, an antioxidant, attenuates methamphetamineinduced dopaminergic toxicity and peroxynitrite generation. Brain research, 855, Imam, S.Z., Newport, G.D., Islam, F., Slikker, W., Jr. & Ali, S.F. (1999) Selenium, an antioxidant, protects against methamphetamine-induced dopaminergic neurotoxicity. Brain research, 818, Kim, H.C., Jhoo, W.K., Choi, D.Y., Im, D.H., Shin, E.J., Suh, J.H., Floyd, R.A. & Bing, G. (1999) Protection of methamphetamine nigrostriatal toxicity by dietary selenium. Brain research, 851, Kryukov, G.V., Castellano, S., Novoselov, S.V., Lobanov, A.V., Zehtab, O., Guigo, R. & Gladyshev, V.N. (2003) Characterization of mammalian selenoproteomes. Science, 300, Peters, M.M., Hill, K.E., Burk, R.F. & Weeber, E.J. (2006) Altered hippocampus synaptic function in selenoprotein P deficient mice. Molecular neurodegeneration, 1,

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123 FIGURE LEGENDS Figure 1: Expression of selenoprotein mrnas is altered by methamphetamine. Following two weeks of treatment with methamphetamine or vehicle, mice brains were analyzed for expression of selenoprotein transcripts by qpcr. The regions principally affected by methamphetamine were dissected from frozen brains. Methamphetamine administration caused significant variance in expression of selenoprotein mrnas both midbrain (top) and striatum (bottom). Data are presented as fold-change relative to PBS treated-mice for the sake of clarity, but raw data was analyzed by two-way ANOVA. Figure 2: Selenoprotein W and selenophosphate synthetase 2 mrna are upregulated in midbrain after two weeks of methamphetamine administration. Select selenoprotein mrnas of interest were analyzed by t-test to determine significance between treatment groups. Sepw1 and Sps2 had significantly elevated expression in midbrain (n=3, p<0.05), but not in striatum of methamphetamine-treated mice. Trends towards increased expression of Sepp1 and Gpx4 did not reach statistical significance in either brain region. Figure 3: Selenoprotein expression following two weeks of methamphetamine administration is not changed in mice brains. SDS-PAGE and Western blotting for Sepw1 and Gpx4 was performed on the same samples as assessed by qpcr (n=3). Qualitatively, expression of Sepw1 and Gpx4 appeared to be reduced, however considerable variation and low sample size resulted in no statistically significant alteration. Figure 4: Long term methamphetamine administration dramatically reduces selenoprotein mrnas in striatum. Following eight weeks of treatment with methamphetamine or vehicle, mice brains were analyzed by qpcr for expression of selenoprotein transcripts (n=3-4). Sepw1 and Sps2 showed reduced expression in midbrain of methamphetamine 114

124 treated mice, but none of the other selenoproteins differed in midbrain, compared to vehicle treated animals. In contrast, reduced expression of nearly all selenoprotein mrnas was observed in striatum. Figure 5: Sepp1 impacts brain expression of selenoproteins more than methamphetamine. Sepp1-deficient mice were administered a high-dose bolus of methamphetamine, and expression of selenoproteins in striatum was investigated by Western blot (n=3-4). Expression of Sepw1 and Gpx4 was reduced in brains of Sepp1 knockout mice, and did not differ between methamphetamine and vehicle treatment groups. Expression of Sps2 was stable across all experimental groups. Figure 6: Methamphetamine reduces tyrosine hydroxylase expression in substantia nigra independently of Sepp1. Sepp1 knockout (KO) and wild-type (WT) control mice were administered a bolus of MA or PBS as a vehicle control, and the brains were subsequently analyzed by immunohistochemistry for the presence of tyrosine hydroxylase (TH). An apparent decrease in TH-immunoreactivity in neurons of the substantia nigra was observed in MA-treated animals. Sepp1 KO mice had similar expression of TH as WT control mice, under both MA and PBS treatment conditions. Figure 7: Apoptotic cell death is not increased in methamphetamine-treated selenoprotein P knockout mice. Following a high-dose bolus administration of MA or vehicle control, TUNEL labeling was performed on brain sections from Sepp1 KO and WT littermate mice to investigate apoptosis. When compared to the negative (C) and positive (D) control sections, both WT (A) and KO (B) methamphetamine-treated mice showed virtually no TUNEL positive cells, indicating an absence of apoptotic cell death. 115

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