Methamphetamine Exposure During Development Causes Lasting Changes to Mesolimbic Dopamine Signaling in Mice.

Methamphetamine (MA) abuse remains a public health issue. Prenatal MA exposure (PME) poses a significant health problem, as we know very little about the drug's long-term physiological impact on the developing human brain. We investigated the long-term consequences of early MA exposure using a mouse model that targets the brain growth spurt, which occurs during human third-trimester. Adult mice previously subjected to acute MA during post-natal days 4-9 exhibited hyperactivity during the Open-Field Test, while exhibiting no motor coordination changes during the Rotarod Test. Neonatal MA exposure reduced basal dopamine (DA) uptake rates in adult nucleus accumbens slices compared with saline-injected controls. Although slices from neonatal MA-exposed mice showed no change in evoked DA signals in the presence of MA, they exhibited potentiated non-evoked DA release through DA efflux in response to MA. These data suggest that developmental MA exposure alters brain development to produce long-lasting physiological changes to the adult mesolimbic DA system, as well as altering responses to acute MA exposure in adulthood. This study provides new insights into an important, under-investigated area in drugs of abuse research.

Selenoprotein P Regulates Synaptic Zinc and Reduces Tau Phosphorylation.

Selenoprotein P (SELENOP1) is a selenium-rich antioxidant protein involved in extracellular transport of selenium (Se). SELENOP1 also has metal binding properties. The trace element Zinc (Zn2+) is a neuromodulator that can be released from synaptic terminals in the brain, primarily from a subset of glutamatergic terminals. Both Zn2+ and Se are necessary for normal brain function. Although these ions can bind together with high affinity, the biological significance of an interaction of SELENOP1 with Zn2+ has not been investigated. We examined changes in brain Zn2+ in SELENOP1 knockout (KO) animals. Timm-Danscher and N-(6-methoxy-8-quinolyl)-p-toluenesulphonamide (TSQ) staining revealed increased levels of intracellular Zn2+ in the SELENOP1-/- hippocampus compared to wildtype (WT) mice. Mass spectrometry analysis of frozen whole brain samples demonstrated that total Zn2+ was not increased in the SELENOP1-/- mice, suggesting only local changes in Zn2+ distribution. Unexpectedly, live Zn2+ imaging of hippocampal slices with a selective extracellular fluorescent Zn2+ indicator (FluoZin-3) showed that SELENOP1-/- mice have impaired Zn2+ release in response to KCl-induced neuron depolarization. The zinc/metal storage protein metallothionein 3 (MT-3) was increased in SELENOP1-/- hippocampus relative to wildtype, possibly in response to an elevated Zn2+ content. We found that depriving cultured cells of selenium resulted in increased intracellular Zn2+, as did inhibition of selenoprotein GPX4 but not GPX1, suggesting the increased Zn2+ in SELENOP1-/- mice is due to a downregulation of antioxidant selenoproteins and subsequent release of Zn2+ from intracellular stores. Surprisingly, we found increased tau phosphorylation in the hippocampus of SELENOP1-/- mice, possibly resulting from intracellular zinc changes. Our findings reveal important roles for SELENOP1 in the maintenance of synaptic Zn2+ physiology and preventing tau hyperphosphorylation.

Selenoprotein P Modulates Methamphetamine Enhancement of Vesicular Dopamine Release in Mouse Nucleus Accumbens Via Dopamine D2 Receptors.

Dopamine (DA) transmission plays a critical role in processing rewarding and pleasurable stimuli. Increased synaptic DA release in the nucleus accumbens (NAc) is a central component of the physiological effects of drugs of abuse. The essential trace element selenium mitigates methamphetamine-induced neurotoxicity. Selenium can also alter DA production and turnover. However, studies have not directly addressed the role of selenium in DA neurotransmission. Selenoprotein P (SELENOP1) requires selenium for synthesis and transports selenium to the brain, in addition to performing other functions. We investigated whether SELENOP1 directly impacts (1) DA signaling and (2) the dopaminergic response to methamphetamine. We used fast-scan cyclic voltammetry to investigate DA transmission and the response to methamphetamine in NAc slices from C57/BL6J SELENOP1 KO mice. Recordings from SELENOP1 KO mouse slices revealed reduced levels of evoked DA release and slower DA uptake rates. Methamphetamine caused a dramatic increase in vesicular DA release in SELENOP1 KO mice not observed in wild-type controls. This elevated response was attenuated by SELENOP1 application through a selenium-independent mechanism involving SELENOP1-apolipoprotein E receptor 2 (ApoER2) interaction to promote dopamine D2 receptor (D2R) function. In wild-type mice, increased vesicular DA release in response to methamphetamine was revealed by blocking D2R activation, indicating that the receptor suppresses the methamphetamine-induced vesicular increase. Our data provide evidence of a direct physiological role for SELENOP1 in the dopaminergic response to methamphetamine and suggest a signaling role for the protein in DA transmission.

Methamphetamine Induces Dopamine Release in the Nucleus Accumbens Through a Sigma Receptor-Mediated Pathway.

Methamphetamine (METH) is a drug with a high addictive potential that is widely abused across the world. Although it is known that METH dysregulates both dopamine transmission and dopamine reuptake, the specific mechanism of action remains obscure. One promising target of METH is the sigma receptor, a chaperone protein located on the membrane of the endoplasmic reticulum. Using fast-scan cyclic voltammetry, we show that METH-enhancement of evoked dopamine release and basal efflux is dependent on sigma receptor activation. METH-induced activation of sigma receptors results in oxidation of a cysteine residue on VMAT2, which decreases transporter function. Unilateral injections of the sigma receptor antagonist BD-1063 prior to METH administration increased dopamine-related ipsilateral circling behavior, indicating the involvement of sigma receptors. These findings suggest that interactions between METH and the sigma receptor lead to oxidative species (most likely superoxide) that in turn oxidize VMAT2. Altogether, these findings show that the sigma receptor has a key role in METH dysregulation of dopamine release and dopamine-related behaviors.

Selenoprotein S Reduces Endoplasmic Reticulum Stress-Induced Phosphorylation of Tau: Potential Role in Selenate Mitigation of Tau Pathology.

Previous studies demonstrated that selenium in the form of sodium selenate reduces neurofibrillary tangle formation in Alzheimer's disease models. Hyperphosphorylation of tau, which leads to formation of neurofibrillary tangles in Alzheimer's disease, is increased by endoplasmic reticulum (ER) stress. Selenoprotein S (SelS) is part of an ER membrane complex that removes misfolded proteins from the ER as a means to reduce ER stress. Selenate, as with other forms of selenium, will increase selenoprotein expression. We therefore proposed that increased SelS expression by selenate would contribute to the beneficial actions of selenate in Alzheimer's disease. SelS expression increased with ER stress and decreased under conditions of elevated glucose concentrations in the SH-SY5Y neuronal cell line. Reducing expression of SelS with siRNA promoted cell death in response to ER stress. Selenate increased SelS expression, which significantly correlated with decreased tau phosphorylation. Restricting SelS expression during ER stress conditions increased tau phosphorylation, and also promoted aggregation of phosphorylated tau in neurites and soma. In human postmortem brain, SelS expression coincided with neurofibrillary tangles, but not with amyloid-ß plaques. These results indicate that selenate can alter phosphorylation of tau by increasing expression of SelS in Alzheimer's disease and potentially other neurodegenerative disorders.

Methamphetamine acutely inhibits voltage-gated calcium channels but chronically up-regulates L-type channels.

In neurons, calcium (Ca(2+) ) channels regulate a wide variety of functions ranging from synaptic transmission to gene expression. They also induce neuroplastic changes that alter gene expression following psychostimulant administration. Ca(2+) channel blockers have been considered as potential therapeutic agents for the treatment of methamphetamine (METH) dependence because of their ability to reduce drug craving among METH users. Here, we studied the effects of METH exposure on voltage-gated Ca(2+) channels using SH-SY5Y cells as a model of dopaminergic neurons. We found that METH has different short- and long-term effects. A short-term effect involves immediate (< 5 min) direct inhibition of Ca(2+) ion movements through Ca(2+) channels. Longer exposure to METH (20 min or 48 h) selectively up-regulates the expression of only the CACNA1C gene, thus increasing the number of L-type Ca(2+) channels. This up-regulation of CACNA1C is associated with the expression of the cAMP-responsive element-binding protein (CREB), a known regulator of CACNA1C gene expression, and the MYC gene, which encodes a transcription factor that putatively binds to a site proximal to the CACNA1C gene transcription initiation site. The short-term inhibition of Ca(2+) ion movement and later, the up-regulation of Ca(2+) channel gene expression together suggest the operation of cAMP-responsive element-binding protein- and C-MYC-mediated mechanisms to compensate for Ca(2+) channel inhibition by METH. Increased Ca(2+) current density and subsequent increased intracellular Ca(2+) may contribute to the neurodegeneration accompanying chronic METH abuse. Methamphetamine (METH) exposure has both short- and long-term effects. Acutely, methamphetamine directly inhibits voltage-gated calcium channels. Chronically, neurons compensate by up-regulating the L-type Ca(2+) channel gene, CACNA1C. This compensatory mechanism is mediated by transcription factors C-MYC and CREB, in which CREB is linked to the dopamine D1 receptor signaling pathway. These findings suggest Ca(2+) -mediated neurotoxicity owing to over-expression of calcium channels.

Increased selenoprotein P in choroid plexus and cerebrospinal fluid in Alzheimer's disease brain.

Subjects with Alzheimer's disease (AD) have elevated brain levels of the selenium transporter selenoprotein P (Sepp1). We investigated if this elevation results from increased release of Sepp1 from the choroid plexus (CP). Sepp1 is significantly increased in CP from AD brains in comparison to non-AD brains. Sepp1 localizes to the trans-Golgi network within CP epithelia, where it is processed for secretion. The cerebrospinal fluid from AD subjects also contains increased levels Sepp1 in comparison to non-AD subjects. These findings suggest that AD pathology induces increased levels of Sepp1 within CP epithelia for release into the cerebrospinal fluid to ultimately increase brain selenium.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal ß-amyloid fragment.

Soluble ß-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar ß-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within ß-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and ß-secretases, and resident carboxypeptidase. The N-terminal ß-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length ß-amyloid, the N-terminal ß-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal ß-amyloid fragment proved to be highly potent and more effective than full-length ß-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal ß-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length ß-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal ß-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal ß-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal ß-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

Selenium and selenoprotein function in brain disorders.

Selenoproteins are important for normal brain function, and decreased function of selenoproteins can lead to impaired cognitive function and neurological disorders. This review examines the possible roles of selenoproteins in Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and epilepsy. Selenium deficiency is associated with cognitive decline, and selenoproteins may be helpful in preventing neurodegeneration in AD. PD is associated with impaired function of glutathione peroxidase selenoenzymes. In HD, selenium deters lipid peroxidation by increasing specific glutathione peroxidases. Selenium deficiency increases risk of seizures in epilepsy, whereas supplementation may help to alleviate seizures. Further studies on the mechanisms of selenoprotein function will increase our understanding of how selenium and selenoproteins can be used in treatment and prevention of brain disorders.

Isoform-specific binding of selenoprotein P to the ß-propeller domain of apolipoprotein E receptor 2 mediates selenium supply.

Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD ß-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery.

Early postnatal repeated maternal deprivation causes a transient increase in OMpg and BDNF in rat cerebellum suggesting precocious myelination.

Repetitive maternal deprivation (MD) of neonatal rats during early life is known as one of the strongest stressors to pre-weaned animals. There is increasing evidence that the cerebellum is involved in cognition and emotion. In the present study, we examined how neurotrophic factors and myelin-associated molecules and their receptors (NGF, BDNF, OMgp, TrkA, TrkB, p75 NTR, and NgR) in the cerebellum are affected by early postnatal maternal separation. Rat pups were separated from their mothers for 3h/day during postnatal days (PND) 10-15. At PND 16 and 30, the levels of mRNA and protein in the cerebellum were determined using real-time PCR and Western blot analysis. Cerebellar mRNA and protein levels of BDNF, TrkB, and OMgp were significantly increased in MD rats at PND 16. However, by PND 30 these variables normalized to control levels. In contrast, the levels of mRNA and protein for NGF, TrkA, p75 NTR, and NgR were unchanged at both ages examined. Transient enhancement of neurotrophic system and myelin-associated molecule expression may cause interference of normal development of the cerebellum such as precocious myelination, which may lead to functional and cognitive deficits later in life.

Methamphetamine decreases levels of glutathione peroxidases 1 and 4 in SH-SY5Y neuronal cells: protective effects of selenium.

Methamphetamine interferes with dopamine reuptake, and the resulting increased dopamine oxidation that creates oxidative stress can lead to degeneration of dopaminergic terminals. Previous studies have shown that the trace element selenium protects against methamphetamine toxicity. However, the specific selenoproteins responsible for protection have not been elucidated. Glutathione peroxidases 1 and 4 (GPx1 and GPx4) incorporate selenium into the amino acid selenocysteine, and their known antioxidant functions make them good candidates for protection from methamphetamine-induced oxidative damage. We differentiated SH-SY5Y neuronal cells in serum-free media with defined supplement containing 0, 10 and 100 nM selenium, and then challenged the cells with a 24-h exposure to methamphetamine. We found that 100 µM methamphetamine decreased GPx1 and GPx4 protein levels. However, both proteins were upregulated with increasing media selenium concentration. GPx enzymatic activity was also increased by selenium and decreased by methamphetamine and correlated with GPx protein levels. Total glutathione levels were reduced by methamphetamine at lower selenium conditions, while the oxidized fraction of GSH was increased at higher selenium levels. Additionally, we observed an increased generation of reactive oxygen species with methamphetamine exposure in media with 0 nM selenium, which was ameliorated by selenium supplementation. These results show that methamphetamine increases oxidative stress by reducing GPx levels, and this can be reversed with addition of selenium. These findings have important implications for treating patients with acute methamphetamine toxicity.

Evidence of oxidative stress in young and aged DJ-1-deficient mice.

Loss of DJ-1 function contributes to pathogenesis in Parkinson's disease. Here, we investigate the impact of aging and DJ-1 deficiency in transgenic mice. Ventral midbrain from young DJ-1-deficient mice revealed no change in 4-hydroxy-2-nonenal (4-HNE), but HSP60, HSP40 and striatal dopamine turnover were significantly elevated compared to wildtype. In aged mice, the chaperone response observed in wildtype animals was absent from DJ-1-deficient transgenics, and nigral 4-HNE immunoreactivity was enhanced. These changes were concomitant with increased striatal dopamine levels and uptake. Thus, increased oxidants and diminished protein quality control may contribute to nigral oxidative damage with aging in the model.

Selenoprotein W expression and regulation in mouse brain and neurons.

Background Selenoprotein W (Sepw1) is a selenium-containing protein that is abundant in brain and muscle of vertebrate animals. Muscular expression of Sepw1 is reduced by dietary selenium (Se) deficiency in mammals, whereas brain expression is maintained. However, expression of Sepw1 depends on the Se transporter selenoprotein P (Sepp1). Methods We assessed the regional and cellular expression of Sepw1 in the mouse brain and neuronal cultures. Results We found that Sepw1 is widespread in neurons and neuropil of mouse brain and appears in both the soma and processes of neurons in culture. Pyramidal neurons of cortex and hippocampus express high levels of Sepw1. It is also abundant in Purkinje neurons and their dendritic arbors in the cerebellum. Analysis of synaptosome fractions prepared from mice brains indicated that Sepw1 is present at synapses, as were several proteins involved in selenoprotein synthesis. Synaptic expression of Sepw1 expression is reduced in mice lacking Sepp1 compared with control mice, although selenoprotein synthesis factors were similarly expressed in both genotypes. Lastly, Sepw1 mRNA coimmunoprecipitates with Staufen 2 protein in a human neuronal cell line. Conclusions Our results suggest that Sepw1 may be locally synthesized in distal compartments of neurons including synapses.

Changes in selenoprotein P in substantia nigra and putamen in Parkinson's disease.

Oxidative stress and oxidized dopamine contribute to the degeneration of the nigrostriatal pathway in Parkinson's disease (PD). Selenoproteins are a family of proteins containing the element selenium in the form of the amino acid selenocysteine, and many of these proteins have antioxidant functions. We recently reported changes in expression of the selenoprotein, phospholipid hydroperoxide glutathione peroxidase GPX4 and its co-localization with neuromelanin in PD brain. To further understand the changes in GPX4 in PD, we examine here the expression of the selenium transport protein selenoprotein P (Sepp1) in postmortem Parkinson's brain tissue. Sepp1 in midbrain was expressed in neurons of the substantia nigra (SN), and expression was concentrated within the centers of Lewy bodies, the pathological hallmark of PD. As with GPX4, Sepp1 expression was significantly reduced in SN from PD subjects compared with controls, but increased relative to cell density. In putamen, Sepp1 was found in cell bodies and in dopaminergic axons and terminals, although levels of Sepp1 were not altered in PD subjects compared to controls. Expression levels of Sepp1 and GPX4 correlated strongly in the putamen of control subjects but not in the putamen of PD subjects. These findings indicate a role for Sepp1 in the nigrostriatal pathway, and suggest that local release of Sepp1 in striatum may be important for signaling and/or synthesis of other selenoproteins such as GPX4.

Expression and regulation of mouse selenoprotein P transcript variants differing in non-coding RNA.

Selenoprotein P (Sepp1), a glycoprotein rich in selenium, is thought to function in selenium transport throughout the body. The sepp1 gene locus potentially produces three alternative transcripts that differ only in their 5' untranslated regions (5'UTRs) and not in their protein coding regions, as indicated by transcript information in genomic databases. Here we investigated the distribution, relative expression, and biological significance of these transcript variants. We confirmed the expression of Sepp1 transcript variants using PCR and sequencing. Using 5'-RACE, we identified multiple 5'-termini upstream from three different splice donor sites, and a single splice acceptor site for exon 2. We found regional and temporal changes in variant expression in select adult and neonate murine tissue and brain regions. Distribution of variants in heart and kidney varied with stage of development. Notably, the Sepp1b variant was localized specifically to the hippocampus in brain. Targeted silencing of individual variants using RNAi demonstrated the biological importance for all transcript variants in cell viability. Additionally, we determined that the Sepp1b variant is a specific target for the miR-7 microRNA by means of its unique 5'UTR structure. Our results emphasize the importance of non-coding transcript variations as a regulatory means for Sepp1 expression in different tissues and stages of development. The presence of a variant localized in the hippocampus and regulated by a microRNA may have implications for the known deficits in synaptic function caused by genetic deletion of Sepp1.

Disruption of the selenocysteine lyase-mediated selenium recycling pathway leads to metabolic syndrome in mice.

Selenium (Se) is an essential trace element used for biosynthesis of selenoproteins and is acquired either through diet or cellular recycling mechanisms. Selenocysteine lyase (Scly) is the enzyme that supplies Se for selenoprotein biosynthesis via decomposition of the amino acid selenocysteine (Sec). Knockout (KO) of Scly in a mouse affected hepatic glucose and lipid homeostasis. Mice lacking Scly and raised on an Se-adequate diet exhibit hyperinsulinemia, hyperleptinemia, glucose intolerance, and hepatic steatosis, with increased hepatic oxidative stress, but maintain selenoprotein levels and circulating Se status. Insulin challenge of Scly KO mice results in attenuated Akt phosphorylation but does not decrease phosphorylation levels of AMP kinase alpha (AMPKα). Upon dietary Se restriction, Scly KO animals develop several characteristics of metabolic syndrome, such as obesity, fatty liver, and hypercholesterolemia, with aggravated hyperleptinemia, hyperinsulinemia, and glucose intolerance. Hepatic glutathione peroxidase 1 (GPx1) and selenoprotein S (SelS) production and circulating selenoprotein P (Sepp1) levels are significantly diminished. Scly disruption increases the levels of insulin-signaling inhibitor PTP1B. Our results suggest a dependence of glucose and lipid homeostasis on Scly activity. These findings connect Se and energy metabolism and demonstrate for the first time a unique physiological role of Scly in an animal model.

Glutathione Peroxidase 4 is associated with Neuromelanin in Substantia Nigra and Dystrophic Axons in Putamen of Parkinson's brain.

BACKGROUND: Parkinson's disease is a neurodegenerative disorder characterized pathologically by the loss of nigrostriatal dopamine neurons that project from the substantia nigra in the midbrain to the putamen and caudate nuclei, leading to the clinical features of bradykinesia, rigidity, and rest tremor. Oxidative stress from oxidized dopamine and related compounds may contribute to the degeneration characteristic of this disease. RESULTS: To investigate a possible role of the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4) in protection from oxidative stress, we investigated GPX4 expression in postmortem human brain tissue from individuals with and without Parkinson's disease. In both control and Parkinson's samples, GPX4 was found in dopaminergic nigral neurons colocalized with neuromelanin. Overall GPX4 was significantly reduced in substantia nigra in Parkinson's vs. control subjects, but was increased relative to the cell density of surviving nigral cells. In putamen, GPX4 was concentrated within dystrophic dopaminergic axons in Parkinson's subjects, although overall levels of GPX4 were not significantly different compared to control putamen. CONCLUSIONS: This study demonstrates an up-regulation of GPX4 in neurons of substantia nigra and association of this protein with dystrophic axons in striatum of Parkinson's brain, indicating a possible neuroprotective role. Additionally, our findings suggest this enzyme may contribute to the production of neuromelanin.

Role of selenoprotein P in Alzheimer's disease.

INTRODUCTION: Selenoprotein P (SelP) plays a critical role in neuronal survival and is associated with Alzheimer's pathology. We sought to determine a potential neuroprotective role for SelP in Alzheimer's disease. METHODS: We utilized RNAi to reduce SelP expression in neuronal N2A cells, and determined cell viability with flow cytometry. We subsequently measured neurotoxicity from exposure of aggregated amyloid beta (Abeta) peptides to SelP-knockdown and control N2A cells. RESULTS: We found that knockdown of SelP using siRNA in N2A cells reduced viability and increased apoptotic cell death. Additionally, knockdown of SelP using siRNA in N2A cells resulted in increased AB toxicity. CONCLUSIONS: Our findings demonstrate that SelP protects neuronal cells from Abeta-induced toxicity, suggesting a neuroprotective role for SelP in preventing neurodegenerative disorders.

The neuroprotective functions of selenoprotein M and its role in cytosolic calcium regulation.

Selenoproteins contain the trace element selenium incorporated as selenocysteine, the 21st amino acid. Some members of the selenoprotein family, such as the glutathione peroxidases, have well-characterized antioxidant activity, functioning in enzymatic breakdown of hydroperoxides to protect cells against oxidative stress. However, the functions of many of the 25 human selenoproteins, including the brain-enriched selenoprotein M, are unknown. We investigated selenoprotein M function by manipulating expression in murine hippocampal HT22 cells, cerebellar astrocyte C8-D1A cells, and primary neuronal cultures. Overexpression of the protein resulted in a reduction in reactive oxygen species and apoptotic cell death in response to oxidative challenge with hydrogen peroxide. In contrast, knock-down of selenoprotein M using shRNA in primary neuronal cultures caused apoptotic cell death comparable to levels resulting from addition of hydrogen peroxide. Calcium measurements with the indicator cameleon demonstrated that overexpression of selenoprotein M decreased calcium influx in response to hydrogen peroxide. Additionally, knock-down of selenoprotein M expression in cortical cultures caused higher baseline levels of cytosolic calcium than in control cells. These results suggest that selenoprotein M may have an important role in protecting against oxidative damage in the brain and may potentially function in calcium regulation.