YNBDI-03264; No. of pages: 9; 4C: 4 Neurobiology of Disease xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

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Article history: Received 7 April 2014 Revised 10 June 2014 Accepted 28 June 2014 Available online xxxx

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Keywords: Huntington's disease Selenium Selenoprotein Diet Neurodegeneration Neuroprotection

Department of Veterinary Sciences, University of Wyoming, Laramie, WY 82070, USA Neuroscience Graduate Program, University of Wyoming, Laramie, WY 82070, USA Mental Health Research Institute, University of Melbourne, Parkville, Victoria 3010, Australia d MassGeneral Institute for Neurodegenerative Disease, Charlestown, MA 02129, USA c

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Disruption of redox homeostasis is a prominent feature in the pathogenesis of Huntington's disease (HD). Selenium an essential element nutrient that modulates redox pathways and has been reported to provide protection against both acute neurotoxicity (e.g. methamphetamine) and chronic neurodegeneration (e.g. tauopathy) in mice. The objective of our study was to investigate the effect of sodium selenite, an inorganic form of selenium, on behavioral, brain degeneration and biochemical outcomes in the N171-82Q Huntington's disease mouse model. HD mice, which were supplemented with sodium selenite from 6 to 14 weeks of age, demonstrated increased motor endurance, decreased loss of brain weight, decreased mutant huntingtin aggregate burden and decreased brain oxidized glutathione levels. Biochemical studies revealed that selenite treatment reverted HDassociated changes in liver selenium and plasma glutathione in N171-82Q mice and had effects on brain selenoprotein transcript expression. Further, we found decreased brain selenium content in human autopsy brain. Taken together, we demonstrate a decreased selenium phenotype in human and mouse HD and additionally show some protective effects of selenite in N171-82Q HD mice. Modification of selenium metabolism results in beneficial effects in mouse HD and thus may represent a therapeutic strategy. © 2014 Published by Elsevier Inc.

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Introduction

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Selenium is an essential nutrient that has critical roles in maintenance of redox homeostasis. Effects are mediated largely by expression of selenoproteins, all of which are expressed in brain (Zhang et al., 2008), and also through inorganic selenium species that directly modulate redox status (Ramoutar and Brumaghim, 2007). There are 25 human and 24 mouse selenoproteins and many of these regulate redox pathways (Mandal et al., 2010; Panee et al., 2007). These selenoproteins translationally incorporate selenocysteine into critical functional sites using a unique encoding mechanism (Donovan and Copeland, 2010). Mutations disrupting selenoprotein synthesis cause progressive cerebello-cerebral atrophy in children (Agamy et al., 2010). Further, selenium deficiency in genetically modified mice results

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Zhen Lu a,b, Eileen Marks a,b, Jianfang Chen a,b, Jenna Moline a, Lorraine Barrows a, Merl Raisbeck a, Irene Volitakis c, Robert A. Cherny c, Vanita Chopra d, Ashley I. Bush c, Steven Hersch d, Jonathan H. Fox a,b,⁎

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Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model

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Abbreviations: HD, Huntington's disease; htt, huntingtin protein; mhtt, mutant huntingtin protein; FRET, Förster resonance energy transfer; DARPP32, Dopamine and cyclic AMP-regulated phosphoprotein; GSH, glutathione; GSSG, oxidized glutathione; AD, Alzheimer's disease; ICP-MS, inductively-coupled plasma mass spectroscopy. ⁎ Corresponding author at: 1174 Snowy Range Road, Laramie, WY 82070, USA. Fax: +1 307 721 2051. E-mail address: [email protected] (J.H. Fox). Available online on ScienceDirect (www.sciencedirect.com).

in neurodegeneration involving somatosensory cortex and striatum (Caito et al., 2011), regions affected in HD. Selenium is therefore critical for brain function and disruption of selenium homeostasis is sufficient to cause neurodegeneration. Huntington's disease (HD) is a chronic progressive neurodegenerative condition that is caused by an autosomal dominant glutamine-encoding CAG expansion within exon-1 of the huntingtin gene (1993). Mutant huntingtin (mhtt) is expressed in brain from in-utero to old-age (Bhide et al., 1996); however, the disease typically manifests clinically in early to mid-adult life (Sturrock and Leavitt, 2010). Mutant htt misfolds and forms soluble and insoluble species that drive the disease process. Disease signs include a hyperkinetic movement disorder dominated by chorea, as well as psychiatric and cognitive disturbances that progress to dementia (Margolis et al., 1999; Sturrock and Leavitt, 2010). There is progressive brain atrophy, primarily of striatum and cerebral cortex, first detectable before clinical onset (Tabrizi et al., 2011). Mouse and human HD brains have pervasive macromolecular oxidative injury that includes damage to DNA, protein and lipids (Bogdanov et al., 2001; Browne et al., 1997; Lee et al., 2011; Sorolla et al., 2010). Oxidative damage in HD brain results from a convergence of pathways involving elevations of the pro-oxidants copper, iron and 3-hydroxykyenurine (Chen et al., 2013; Fox et al., 2007; Guidetti et al., 2000) as well as defective antioxidant defenses, including

http://dx.doi.org/10.1016/j.nbd.2014.06.022 0969-9961/© 2014 Published by Elsevier Inc.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

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Materials and methods

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Chemicals

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All chemicals were from Sigma (St. Louis, MO) unless stated otherwise. Fluorescent Nissl and secondary antibodies were from Invitrogen (Carlsbad, CA).

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Mouse breeding and maintenance

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N171-82Q HD mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained under 12 hour light-dark periods. The colony was maintained by crossing HD male mice with C57BL/6 x C3 F1 females. Tails were cut for genotyping at 2.5 weeks and mice were weaned into study cages at 4 weeks. Study mice were housed at 4–5/ cage and fed ad-libitum mouse chow (LabDiet® 5 K67). Each cage had a single mouse igloo® (Bio-Serv) and Sani Chip bedding (Harlan) Genotyping was by PCR as described (Schilling et al., 1999). CAGrepeat sizes were in the range 82–83 as determined by Laragen, Inc (Culver City, CA). All animal procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee and followed NIH guidelines.

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Experimental design and treatments

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Female N171-82Q HD mice were used in experiments. Rota-rod endurance (Fox et al., 2010) and body weights were determined at 5 weeks. Mice were fed a standard mouse chow containing 0.3 ppm selenium (AIN76A). Selenium was delivered in acidified drinking water (pH 3–4) as sodium selenite because this is the same form of selenium present in the mouse chow. Mice were supplemented from 6 weeks at 0.25 and 1.00 ppm selenium in drinking water. Dedicated water bottles were used for selenium delivery and these were given four daily changes of 1 ppm selenite before first use to ensure saturation of selenite-binding sites. Estimated combined food and water selenium intakes were 0.9, 1.65 and 3.9 μg/day in the control, low and high groups per 20 g body weight. This calculation was based on a consumption of 3 mls water and 3 g food/day for a 20 g mouse. Mice were sacrificed at 14 weeks corresponding to early-advanced disease.

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Neuropathology Studies

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Mice were anesthetized then perfused for 3 minutes with heparinized 0.9% (w/v) saline. This was followed immediately by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 minutes. Brains were post-fixed for 24 hours at 4°C then cryopreserved in 10% glycerol, 2% DMSO and 0.1 M phosphate buffer for 5 days. Brains were sectioned frontally at 40 μm and stored in phosphate buffer containing 0.05% azide. For cavalieri estimation of striatal volume every 12th section was mounted on a glass slide and stained using the thionin method. For nucleator estimation of striatal neuronal cell body volume a section at the level of the anterior commissure

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DARPP32 and mutant huntingtin analysis

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Sections at the level of the anterior commissure were stained while ‘floating’. Primary antibody incubations were for 2 days at 4° C. DARPP32 (Cell Signaling) and mutant huntingtin antibodies (5374, Millipore) were used at a 1:200 and 1:1000 dilution, respectively, followed by 24 hour incubations with AF488-labeled secondary antibody. We used fluorescent Nissl Neurotrace® 530/615 (Invitrogen) to detect neurons. Two sets of image stack were collected from each striatum (DARPP32) or motor cortex (mhtt aggregates) using a Zeiss 710 confocal microscope and 40x oil objective. DARPP32 staining in neuronal cell bodies was quantified using Image J software (NIH). Mutant huntingtin aggregates were quantified in layer VI primary motor cortex. Confocal images stack files were imported into Stereoinvestigator software (Microbrightfield). The number of mhtt aggregates in layer VI neuron cell bodies was estimated using the optical fractionator tool. The counting frame size was 25 x 25 μm and there were 10–15 frames per image. In each counting frame neuronal cell bodies and associated aggregates were counted. Aggregates per neuronal cell body were determined.

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Time-resolved FRET assay for soluble mutant huntingtin

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Mice were anesthetized then perfused for 3 minutes with cold heparinized 0.9% (w/v) saline. Brain regions were frozen on dry ice then stored at − 70° C. We measured soluble mutant huntingtin using a time-resolved Förster resonance energy transfer (FRET) method described by us previously (Weiss et al., 2009). The mean value of three wild-type cortices was used to determine background and this was subtracted from transgenic values.

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Quantification of Brain Glutathione

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Glutathione (GSH) and oxidized glutathione (GSSG) were measured as we described previously (Fox et al., 2004). In brief, mice were perfused with cold heparinized saline for 2 minutes to flush out blood; brain regions were then removed, dissected and frozen immediately in dry ice. Dissected brain regions were homogenized in cold 5% (w/v) sulfosalicyclic acid and supernatant fractions stored at − 70° C. The method detects GSH by reaction with the chromagen 5, 5′-dithiobis(2-nitrobenzoic acid) with detection at 405 nm. ‘Total GSH’ is measured by reduction of all GSSG to GSH using NADPH and glutathione reductase. GSSG is determined as for ‘total GSH’ except that GSH is first inactivated by reaction with 2-vinylpyridine. ‘True GSH’ is calculated by subtraction. GSSG standards were used.

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Quantitative PCR of brain selenoprotein-encoding transcripts

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Total RNA was extracted from cortex and striatum using the RNeasy Lipid Tissue Mini Kit (Qiagen) with on-column DNase 1 digestion according to the manufacture's instruction. RNA samples were quantified by absorption at 260 nm then reverse transcribed using Superscript III (Life Technologies). Taqman® gene expression assays (Applied Biosystems) were used with an ABI 7500 fast thermal cycler to quantify mRNA. Duplex reactions were performed for the target gene and actin normalizer. The primer/probe combinations used are listed in Supplementary Information. Primer/probes were validated for linearity of amplification and non-interference of the duplex pair before analysis. Each reaction contained 10 ng RNA equivalence of cDNA. All duplex reactions were performed in duplicate.

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only was analyzed. The stereologic methods have been described by us previously (Chopra et al., 2007). We used Stereoinvestigator software (MicroBrightField, Williston, VT) and an Olympus BX51 microscope with a motorized stage.

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decreased expression of the transcriptional co-activator PGC1α (Cui et al., 2006). Selenium has protective effects in toxicant rodent models of neurodegeneration (Ishrat et al., 2009; Khan, 2010). In addition, two independent studies have demonstrated protective effects of selenium in mouse models of tauopathy and implicated increased protein phosphatase 2A mediated de-phosphorylation of tau as the protective mechanism (Song et al., 2014; van Eersel et al., 2010). Selenium therapy has not previously been reported in HD mice. In this study, we investigated the effects of selenium supplementation in the N171-82Q mouse model of HD. We report our findings supporting neuroprotective activities in HD mice and showing changes in selenium status in mouse and human HD.

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Z. Lu et al. / Neurobiology of Disease xxx (2014) xxx–xxx

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

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Human brains

Results

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Selenium analyses

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Tissue selenium was measured by inductively-coupled plasma mass spectroscopy (ICP-MS) as described (Maynard et al., 2006). In brief, brain regions were weighed wet, lyophilized then digested in concentrated nitric acid and allowed to digest overnight at 25° C, then further digested by heating for 20 minutes at 90 °C. Samples were digested in hydrogen peroxide then heated to 70 °C for 15 minutes. They were then diluted in triplicate with 1% nitric acid. As controls, blank tubes (no tissue added) and bovine liver SRM 1577b (National Institute of Science and Technology) were used. Measurements were made using an UltraMass 700 (Varian Inc.) ICP-MS instrument under operating conditions suitable for multi-element analysis. The instrument was calibrated using 0, 10, 50 and 100 ppb of a certified multi-element ICP-MS standard solution (ICP-MS- CAl2-1, AccuStandard). The internal standard solution contained 100 ppb of Yttrium (89Y) (ICP-MS- IS-MIX1-1, AccuStandard). Selenium isotopes of mass 77 and 78 were quantified in triplicate. Plasma selenium was dissolved in water and analyzed directly as above.

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Statistical analyses

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All data was analyzed using SAS software version 9.2 (Cary, NC). The GLM procedure was used for one-way ANOVA (brain selenium, DARPP32, glutathione, oxidized glutathione, brain weights, huntingtin aggregates). For the human brain data, sex and age were used as covariates. The MIXED procedure was used for repeated-measures analysis (body weights, rota-rod). Significant differences were determined by pair-wise comparisons (t-test). Data are given as mean ± SEM. Quantitative PCR data was analyzed using the MIXED procedure (split plot analysis) taking into account region. Data is presented as means ± 95% confidence intervals.

N171-82Q HD mice receiving both 0.25 and 1.00 ppm in drinking water demonstrated significantly greater Rota-rod endurance as compared to litter-mate control HD mice at 14 weeks of age (p = 0.021) (Fig. 1A). To rule out a non-specific effect of selenite on motor endurance we tested the effect of 1.00 ppm selenite in wild-type mice. We used wild-type mice derived from a subsequent breeding cycle of our N171-82Q HD colony. As shown in Fig. 1B, while baseline latency was greater than the previous experiment, there was no effect on Rota-rod performance. We undertook a detailed analysis of HD brain to determine the effect of selenite on brain morphology. HD control mice at 14 weeks had a 9.9% less mean brain weight than wild-type littermates (p b 0.0001). HD mice receiving 1.00 ppm selenite had significantly higher mean brain weights than HD controls mice at 14 weeks (p = 0.0279) (Fig. 2A). This increase corresponded to a 53% recovery towards wild-type mean brain weight. Interestingly, HD mice at 6 weeks already had significantly lower mean brain weights than wild-type littermates (p = 0.0022) (Fig. 2B) showing that selenite protected against decreased brain weight even though the treatment was initiated after decreases in brain weight had occurred. Further, we calculated that selenite protected against 92% of the loss of mean brain weight that developed between 6 and 14 weeks of age in HD mice. We investigated the anatomic origin of this effect. Using stereology there was a trend towards elevated neostriatal volume in selenite supplemented mice (Fig. 2C); however, there was no effect on striatal neuronal cell body volume as measured by the nucleator method (not shown). We analyzed three brain biochemical markers in 14-week-old mice to investigate potential domains of selenium's protective mechanism. We quantified mhtt aggregates in layer VI primary motor cortex by confocal microscopy and found significantly fewer aggregates in HD mice receiving 1.0 ppm selenite in drinking water compared to HD controls (p = 0.0007) (Figs. 3A-B). However, soluble mutant huntingtin, measured by FRET, in frontal-parietal cortex did not demonstrate any significant change (Fig. 3C). Glutathione (GSH) is an essential electron donor required for several enzyme systems including glutathione peroxidases and glutaredoxins. In this process, GSH is oxidized to GSSG (oxidized glutathione), an indicator of oxidative stress. GSSG levels were increased in control HD mouse striatum (p = 0.0229) and cortex (p = 0.0058) as compared to wild-type littermates (Figs. 4A-B). In cortex, but not striatum, both 0.25 and 1.00 ppm drinking water selenium

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HD and age-matched brains were both obtained from each of the following brain banks: New York at Columbia University, Harvard Brain Tissue Resource Center, and Alzheimer's disease Research Center at Massachusetts General Hospital (MGH). Demographic information is shown in Table 3. Approval was obtained from the MGH Institutional Review Board.

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Fig. 1. Selenite treatment increases motor endurance in N171-82Q HD but not wild-type mice. A. Selenium provides protection against decline in Rota-rod endurance. Both levels of drinking water selenium (0.25 and 1.00 ppm) provide protection at 10 and 14 weeks. n = 14–19. B. No stimulatory effect of selenium on Rota-rod endurance in wild-type mice. n = 15, p-values: * b 0.05, ** b 0.01, *** b 0.001: TG = N171-82Q HD transgenic; WT = wild-type. Se water level: 0.25 = 0.25 ppm; 1.00 = 1.00 ppm.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

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Given the findings showing protective effects of selenite supplementation in HD mice we investigated potential mechanisms. As most of the effects of selenium are mediated through the expression of selenoproteins (Zhang et al., 2008) we first tested if our treatment changed selenoprotein-encoding transcript levels in brains. We found multiple effects of mhtt expression (HD) and selenium treatment in striatum and cortex. Numerous transcripts had significantly decreased levels in HD controls compared to control wild-type mice: in frontal cortex these were GPX3 (p = 0.0115), TXNRD1 (p = 0.0045), SelI (p = 0.0032), SelK (p = 0.0451), SelW (p b 0.0001), DIOD2 (p b 0.0001) and DIOD3 (p b 0.0001); in striatum this was DIOD2 (p = 0.0006). Several

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concentrations resulted in a significant decrease in GSSG (p = 0.0428 and 0.0362, respectively) (Figs. 4A-B). In a separate experiment, there was no effect of selenite supplementation on GSSG in wild-type cortex (Fig. 4C) and striatum (not shown) demonstrating that selenite treatment corrects a HD specific event. There were no significant effects of genotype or selenite on brain GSH or GSH: GSSG (not shown). Further, we found that 1.00 ppm selenite increased striatal DARPP32 expression (p = 0.0184) (Figs. S1 A-B), a marker of transcriptional dysregulation in HD (Jiang et al., 2011). Despite this evidence of neuroprotection the selenite treated HD mice did not have significantly increased survival (Fig. S2).

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Fig. 2. Selenite treatment partially rescues decreased brain weight in N171-82Q HD mice. A. Selenium has a protective effect on brain weight at 14-weeks of age. HD non-treated mice have significantly lower brain weights than wild-type litter-mate mice. One ppm selenium in drinking water provides protection against low brain weight. n = 10–15. B. Brain weights at 6 weeks. There is significantly lower brain weight in N171-82Q HD mice at 6 weeks; corresponding to time of treatment initiation. n = 9–13. C. Left striatal volume at 14 weeks. n = 12– 13, p-value: * b 0.05, ** b 0.01, *** b 0.001.

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Selenite in water (ppm) Fig. 3. Selenite treatment decreases mutant huntingtin aggregate burden in cortex. A-B. N171-82Q HD mice were studied at 14-weeks of age. A. Representative images of primary motor cortex. Perfused brains were sectioned at 40 μm. Sections at the level of the anterior commissure were stained for mutant huntingtin protein followed by an AF488 labeled secondary antibody. Confocal z-stack images of layer VI primary motor cortex were captured using a Zeiss 710 microscope. Mutant huntingtin aggregates are green. Fluorescent Nissl neuronal cell body stain is red. Bar = 20 μm. B. Aggregate quantification. Neuronal cell body aggregate load was quantified using the image-stack module in Microbright field software®. Aggregate burden is significantly decreased in the 1.00 ppm selenite group. n = 9–10, C. Soluble mutant huntingtin levels in frontal cortex of HD mice are not altered by selenite treatment. n = 11–12, p-values: ** = b0.01, *** = b0.001.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

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GPX1 GPX2 GPX3 GPX4 Txnrd1 Txnrd2 Txnrd3 SelR SelH SelS SelI SelK SelM SepN1 SelO SelP SelT SelV SelW 15-Sep SelD DIO2 DIO3

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1258 ± (56,54) 79 ± (11,9) 229 ± (56,45) 5660 ± (243,233) 21080 ± (928,889) 246 ± (20,18) 58 ± (11,9) 53 ± (5,5) 4581 ± (412,378) 325 ± (17,17) 499 ± (31,29) 21 ± (3,2) 2959 ± (161,153) 121 ± (32,25) 5075 ± (395,366) 14722 ± (1026,959) 36 ± (5,4) 7 ± (1,1) 57538 ± (2462,2361) 19450 ± (640,620) 2646 ± (137,130) 613 ± (57,52) 1329 ± (364,286)

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Table 1 Relative actin-normalized qPCR mRNA transcript expression in frontal cortex.

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each sex: females and males had ages of 47 ± 2.9 and 42 ± 2.1, respectively. The HD patients had a mean total functional capacity score of 10 (range = 2–11) (Marder et al., 2000). Plasma selenium analyses did not demonstrate differences in HD compared to controls (Fig. 5B). As glutathione metabolism is linked closely with selenium status via activity of the selenoprotein glutathione peroxidase 1–4 we determined plasma glutathione status in study mice. As shown in Fig. 6 we found that at 14-weeks of age HD mice had significantly elevated plasma total glutathione compared to wild-type mice (p = 0.0048). Further, selenite treatment resulted in a significant decrease in plasma glutathione (p = 0.0475); decreasing GSH in HD and wild-type mice. Therefore, in these HD mice plasma GSH is significantly elevated and selenite treatment reverses this effect. Liver is an important organ of body selenium metabolism because it synthesizes selenoprotein P, an important transport form of selenium in plasma. We measured liver selenium as a marker of whole body selenium status. Control N171-82Q mice had significantly decreased liver selenium as compared to wild-type controls at 14weeks of age (p b 0.0001) (Fig. 7A). Further, this effect was reversed

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transcripts were increased in HD; these were TXNRD3 (p = 0.0063), SelO (p = 0.0376) and SelV (p = 0.0061) (cortex) and TXNRD3 (p b 0.0001), SelO (p = 0.0365) and SelD (p = 0.0015) (striatum). Selenite treatment affected numerous transcript levels in striatum and cortex. In HD mice selenite induced increased levels of TXNRD1 (p = 0.0498) and SelP (p = 0.0273) in cortex and GPX3 (p = 0.0172) and SelT (p = 0.0233) in striatum. Selenite also resulted in decreased levels of other transcripts in HD; these were GPX4 (p = 0.0091) and SelK (p = 0.0049) (cortex) and SelK (p = 0.0333) (striatum). Results are summarized in Table 1 (frontal cortex) and Table 2 (striatum). We additionally tested if changes in peripheral selenium and redox status correlate with protection in HD mice. Firstly, we measured plasma selenium in mouse and human HD. As shown in Fig. 5A while N171-82Q HD mice had mildly elevated plasma selenium at 14-weeks of age (p = 0.0278) there was no effect of selenite supplementation. We obtained human plasma for parallel analyses. There were 22 control samples: 12 females and 10 males had mean ± SEM ages of 48 ± 3.2 and 46 ± 3.7 years, respectively. There were 20 HD samples, 10 of

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Fig. 4. Oxidative stress marker oxidized glutathione is decreased in cortex by selenite treatment. A-B. Oxidized glutathione (GSSG) is increased in control HD compared to wild-type mice in cortex and striatum at 14-weeks of age. A. Selenite supplementation decreases GSSG in HD cortex. B. No significant effect of selenite supplementation on GSSG in striatum. n = 17– 21. C. No effect of selenite supplementation in wild-type cortex. n = 15, p-values: * b 0.05, ** = b0.01, NS= N 0.05.

1262 ± (53,51) 87 ± (11,10) 154 ± (35,29) 5391 ± (219,211) 19276 ± (804,772) 221 ± (17,16) 81 ± (14,12) 54 ± (5,4) 4465 ± (380,350) 301 ± (15,15) 438 ± (26,24) 17 ± (2,2) 3154 ± (163,155) 145 ± (36,29) 5681 ± (418,389) 13456 ± (888,833) 37 ± (4,4) 9 ± (1,1) 50297 ± (2040,1960) 18900 ± (589,572) 2834 ± (139,132) 432 ± (38,35) 547 ± (141,112)

Selenite water

P-values

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Main effects and interaction

T-tests

1175 ± (50,48) 70 ± (9,8) 191 ± (44,36) 5311 ± (216,208) 19442 ± (811,779) 212 ± (16,15) 71 ± (12,10) 49 ± (4,4) 4022 ± (342,315) 303 ± (15,15) 439 ± (26,24) 13 ± (2,1) 2904 ± (150,143) 139 ± (34,27) 4981 ± (367,342) 12846 ± (848,795) 27 ± (3,3) 5 ± (1,1) 56943 ± (2309,2219) 17956 ± (560,543) 2461 ± (121,115) 450 ± (40,36) 742 ± (192,152)

1266 ± (54,51) 82 ± (10,9) 180 ± (42,34) 4989 ± (203,195) 20449 ± (853,819) 232 ± (18,16) 68 ± (12,10) 54 ± (5,4) 4074 ± (347,319) 291 ± (15,14) 452 ± (27,25) 13 ± (2,2) 3170 ± (164,156) 148 ± (36,29) 5450 ± (401,374) 14936 ± (985,924) 33 ± (4,4) 7 ± (1,1) 52089 ± (2112,2030) 18426 ± (575,557) 2802 ± (137,131) 431 ± (38,35) 638 ± (165,131)

Se = * G = *** I = ** Se = * I = **

1–3 = *, 3–4 = *

G = *, I = ***

1–2 1–3 1–2 1–3 1–2

= = = = =

* *, 2–4 = **, 3–4 = * **, 1–3 = **, 2–4 = * ** **

Se = ** Se = * Se = *, I = * Se = *** G = **

1–3 1–2 1–2 1–2 3–4

= = = = =

* * **, 1–3 = ** *, 1–3 = ***, 2–4 = ** *

G = ** I = ** G = *, Se = ** G = ***, Se = ** G = *** Se = ***, I = * G = *** G = ***, Se = **, I = ** G = *, Se = *, I = *

1–2 1–3 1–3 1–2 1–2 1–3 1–3 1–2 1–2

= = = = = = = = =

* **, 2–4 = *, 3–4 = ** ***, 3–4 = * **, 1–3 = *, 3–4 = ** ***, 3–4 = ** *** *, 3–4 = *** ***, 1–3 = *** ***, 1–3 = **

For each target the mean and upper/lower 95% confidence intervals of β-actin normalized selenoprotein transcript expression is shown. Data was analyzed using a GLM method with SAS software. Only results with p-values b 0.05 are shown: p b 0.05 = *; p b 0.01 = **; and, p b 0.001 = ***. Tests: G = main effect of genotype; Se = main effect of Se treatment; I = genotype x Se interaction. Group pair wise comparison are indicated using group numbers as shown in the headings for columns 2–5. NA = not applicable. n = 9–10.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321

6

GPX1 GPX2 GPX3 GPX4 Txnrd1 Txnrd2 Txnrd3 SelR SelH SelS SelI SelK SelM SepN1 SelO SelP SelT SelV SelW 15-Sep SelD DIO2 DIO3

Selenite water

Statistics

HD2

Wild-type3

HD4

Main effects and interaction

T-tests

1734 ± (77,74) 35 ± (5,4) 118 ± (29,23) 5064 ± (217,209) 20060 ± (883,846) 74 ± (6,6) 18 ± (3,3) 46 ± (4,4) 2725 ± (245,225) 294 ± (16,15) 563 ± (35,33) 14 ± (2,2) 2998 ± (164,155) 76 ± (20,16) 3667 ± (285,265) 13559 ± (945,883) 27 ± (3,3) 19 ± (3,3) 63001 ± (2696,2585) 17394 ± (572,554) 2141 ± (111,105) 438 ± (41,37) 901 ± (247,194)

1696 ± (72,69) 42 ± (55) 91 ± (21,17) 4836 ± (197,189) 18987 ± (792,761) 85 ± (6,6) 30 ± (5,4) 50 ± (5,4) 2855 ± (243,224) 280 ± (14,14) 537 ± (32,30) 15 ± (2,2) 3072 ± (159,151) 102 ± (25,20) 4203 ± (309,288) 14206 ± (982,918) 28 ± (3,3) 21 ± (3,3) 60470 ± (2452,2356) 17327 ± (540,524) 2434 ± (119,114) 348 ± (32,29) 803 ± (219,172)

1661 ± (70,67) 33 ± (4,4) 94 ± (22,18) 4774 ± (194,187) 19303 ± (806,773) 82 ± (6,6) 31 ± (5,5) 50 ± (5,4) 2298 ± (195,180) 269 ± (14,13) 527 ± (31,29) 13 ± (2,1) 2930 ± (151,144) 93 ± (23,18) 3745 ± (276,257) 13042 ± (861,807) 23 ± (3,2) 15 ± (3,2) 62511 ± (2535,2436) 16137 ± (503,488) 2229 ± (109,104) 352 ± (31,28) 654 ± (169,134)

1652 ± (70,67) 42 ± (5,5) 132 ± (30,25) 4961 ± (202,194) 19293 ± (805,773) 88 ± (7,6) 27 ± (5,4) 50 ± (5,4) 2821 ± (240,221) 288 ± (15,14) 527 ± (31,29) 12 ± (2,1) 2999 ± (155,147) 103 ± (25,20) 4141 ± (305,284) 14578 ± (962,902) 28 ± (3,3) 17 ± (3,2) 60048 ± (2435,2340) 16679 ± (520,504) 2338 ± (115,109) 349 ± (31,28) 734 ± (189,151)

Se = * G = *** I = ** Se = * I = **

1–2 = *, 3–4 = ** 2–4 = *, 3–4 = * 1–3 = * 1–2 = * 1–2 = *** 1–3 = ***

G = *, I = *** Se = ** Se = * Se = *, I = * Se = *** G = **

1–3 = ** 3–4 = *** 1–3 = *

G = ** I = ** G = *, Se = ** G = *** Se = ** G = *** Se = ***, I = * G = *** G = *** Se = **, I = ** G = *, Se = *, I = *

2–4 = *

1–2 3–4 3–4 1–3

= = = =

* * * *

1–3 = ** 1–2 = *** 1–2 = *** 1–3 = ***

P

t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27

Control water Wild-type1

F

Gene

t2:4

O

t2:3

Table 2 Relative actin-normalized qPCR mRNA transcript expression in striatum.

R O

t2:1 t2:2

Z. Lu et al. / Neurobiology of Disease xxx (2014) xxx–xxx

For each target the mean and upper/lower 95% confidence intervals of β-actin normalized selenoprotein transcript expression is shown. Data was analyzed using a GLM method with SAS software. Only results with p-values b 0.05 are shown: p b 0.05 = *; p b 0.01 = **; and, p b 0.001 = ***. Tests: G = main effect of genotype; Se = main effect of Se treatment; I = genotype x Se interaction. Group pair wise comparison are indicated using group numbers as shown in the headings for columns 2–5. NA = not applicable. n = 9–10.

322 323

by selenite treatment (p = 0.0004) (Fig. 7A). Selenite treatment had no effect in wild-type mice. To determine if decreased liver selenium could be the result of weight loss and nutrition we undertook an experiment to correlate body weight with liver selenium in HD mice. We found that at 7.5 weeks of age, there is no weight loss in our N171-82Q HD mice (Fig. 7B); however, these mice still have a small and statistically significant decrease in liver selenium (p = 0.0387) (Fig. 7C). We studied liver selenium in the R6/2 mouse HD model to determine if they have a similar phenotype. R6/2 HD mice express exon-1 of mutant Huntingtin and exhibit an aggressive disease phenotype (Mangiarini et al., 1996). We found that they have elevated liver selenium in pre-clinical disease at 5 weeks of age (p = 0.0252) and significantly decreased selenium in advanced disease at 13 weeks of age (p = 0.0245) (Fig. 7D). Finally, we sought evidence that a selenium phenotype might exist in human HD. We measured selenium levels in multiple brain regions from autopsy samples (see Table 3 for demographic information). As demonstrated in Fig. 8A we found that brain regional selenium content was

339 340

Discussion

349

N171-82Q HD mice express an N-terminal fragment of mutant huntingtin under the control of the prion promoter. This provides expression of mhtt that is largely restricted to the CNS and results in a phenotype similar to human HD that includes progressive motor decline,

350

O

R

R

E

C

T

E

significantly decreased in putamen (p = 0.0021), dorso-lateral prefrontal cortex (p = 0.0044), primary visual cortex (p = 0.0005), cingulate gyrus (p = 0.0095) and cerebellum (p = 0.0215). There were no changes in the substantia nigra pars compacta and the globus pallidus. As late-stage HD patients have significant weight loss we considered the possibility that low brain selenium in HD cases could have a nutritional origin. We measured brain regional selenium in end-stage Alzheimer's disease where there is also advanced physical debilitation. While group sizes were lower than the HD brain study, we found no significant differences between AD and controls (Fig. 8B).

B)

A)

*

7

Human plasma selenium (µM)

10 8 6 4 2

6 5 4 3 2 1

Females

D H

C

on t

ro l

D H

on t C

00 H D

-1 .

00 -0 . H D

00 1.

ro l

0

0 00

338

W T-

336 337

0.

334 335

C

332 333

W T-

330 331

N

328 329

Mouse plasma selenium (µM)

326 327

U

324 325

D

t2:28 t2:29 t2:30

Males

Fig. 5. Effect of Huntington's disease and selenite treatment on plasma total selenium. A. Mouse plasma selenium levels in control and selenium supplemented wild-type and N17182Q HD mice. HD mice have significantly elevated plasma selenium at 14-weeks of age (p = 0.0278). There is no effect of selenite treatment on plasma selenium (p = 0.2594), n = 12–15. B. Human plasma selenium levels in HD compared to age and sex matched controls reveals no change. n = 20–22. p-value: * b 0.05.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

341 342 343 344 345 346 347 348

351 352 353

Z. Lu et al. / Neurobiology of Disease xxx (2014) xxx–xxx

*

Table 3 Summary of demographic data for human subjects used for brain selenium analyses.

40

Group

Age (mean ± SE)

Total, Sex (M, F)

Vonsattel grade (average)

t3:3

BA17

HD Control HD Control HD Control HD Control HD Control HD Control HD Control HD Control

59 62 60 62 58 61 59 62 59 62 49 71 48 70 49 60

25, (15, 10) 12, (5, 7) 25, (15, 10) 11, (5, 6) 25, (15, 10) 10, (4,6) 24, (15, 9) 10, (4, 6) 25, (15,10) 12, (7, 5) 12, (6, 6) 9, (4, 5) 21, (8, 13) 15, (6, 9) 13, (4, 9) 8, (4, 4)

2.9 . 2.9 . 2.9 . 2.8 . 2.9 . NA . NA . NA .

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20

30 BA4 BA9

20

Cerebellum

10 Cingulate gyrus

0

Putamen

WT-0.00

HD-0.00

HD-1.00

WT-1.00

Substantia nigra pars compacta Globus pallidus

Genotype-treatment combination Fig. 6. Plasma glutathione levels are modified by HD and selenium treatment. N17182Q HD mice have elevated plasma ‘total’ glutathione (tGSH) at 14-weeks of age (main effect p = 0.0048). Selenium treatment decreases plasma tGSH (main effect p = 0.0475). Shown on graph are significant pair-wise comparisons: * = p b 0.05, n = 9–10.

12 12 12 12 12 12 12 13 12 12 13 20 14 17 13 7

R O

t3:21

weight) and aggregated mutant huntingtin levels (Figs. 1–4). These findings indicate that selenium may have therapeutic value in human HD and that selenium intake level through natural diet may be an environmental modifier of HD.

P

B)

***

***

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

D

A)

20

15

C

T

1.5

Body weight (grams)

E

2.0

E

1.0

0 -1 D

D

H

H

WT HD

WT HD

WT HD

WT HD

4.5

5.5

6.5

7.5

Age (weeks)

D) *

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

WT

*

1.6

Liver Se level (µg / g wet weight)

Liver Se level (µg / g wet weight)

5

0

C)

0.0

10

.0

.0

00

R 1.

TW

W

T-

0.

00

0.0

0

R

0.5

-0

Liver Se level (µg / g wet weight)

357

NA = not available.

weight loss, low brain weight and premature death (Schilling et al., 1999). In these mice, we show that selenite supplementation has protective effects as determined by positive outcomes encompassing motor endurance, oxidative stress (GSSG), neurodegeneration (brain

N C O

355 356

U

354

HD

t3:1 t3:2

Brain Region

*

F

Plasma total GSH (µM)

*

O

50

7

*

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 WT

HD

5 weeks

WT

HD

13 weeks

Fig. 7. Liver selenium levels are decreased in N171-82Q mice and restored by selenium treatment. A. Low liver selenium at 14-weeks of age in N171-82Q mice is reversed by selenite treatment. n = 9–10, B–C. Relationship between body weight and liver selenium in early N171-82Q HD. B. No effect of HD on body weight at up to 7.5-weeks of age. n = 15, C. Small decrease in liver selenium content at 8-weeks of age in N171-82Q HD mice. n = 15, C. R6/2 HD mice demonstrate significantly increased liver selenium at 5-weeks (pre-clinical) and decreased liver selenium at 12-weeks (advanced disease). n = 10.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

358 359 360 361

8

Z. Lu et al. / Neurobiology of Disease xxx (2014) xxx–xxx

A)

B)

1.2

0.8

0.8 0.6

***

*

**

**

0.4 0.2

0.6

0.4

0.2

F

1.0

BA 17

us

C

Pa l

lid

SN

gy ru s Pu ta m en

m

s bu lo

R O

C

G

in

C

gu

la

er

te

eb

el

lu

BA 9

BA 4

BA 17

O

0.0

0.0

BA 9

Selenium (µg / g wet weight)

Selenium (µg / g wet weight)

***

375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403

E

D

selenium intake on multiple transcripts in frontal cortex and striatum (Tables 1 and 2). Mutant htt and selenium both positively and negatively regulated the levels of numerous transcripts. Fold changes were not large however. Glutathione peroxidase activity (GPX), including GPX3 has recently been shown to be protective in a Drosophila HD model (Mason et al., 2013). We found that GPX3 transcript levels were significantly increased in selenite-treated HD mouse striatum compared to HD controls. However, measurement of total brain regional GPX activity and GPX3 protein levels did not reveal significant changes (data not shown). Therefore, while there were mRNA transcript changes in selenite-supplemented mice more detailed studies are needed at the protein levels to identify putative protein mediators of protection. As many selenoproteins regulate redox-sensitive molecules through processes such as thiol and methionine oxidation status, altered expression of selenoproteins could normalize redox signaling pathways, consistent with the finding of decreased oxidized glutathione with selenite treatment (Fig. 4). Health benefits of selenium supplementation are primarily in those with low, but not high, food selenium intake level (Rayman, 2012). Therefore, potential protection by selenium supplementation in HD may depend on dietary intake level. Further, altered intestinal structure and function in mouse HD indicates nutrient malabsorption and suggests altered nutrient requirements in human HD (van der Burg et al., 2011). Experimental severe selenium deficiency results in a large decrease in liver selenium concentration (Nakayama et al., 2007). The modest decrease in liver selenium status we found in R6/2 and N17182Q HD mice (Fig. 7) may indicate a mild selenium deficiency that is not sufficient to decrease plasma selenium. While selenite corrected liver selenium status, there was no effect on plasma selenium indicating that protective effects of selenite are not due to increased plasma selenium availability for brain uptake. Interestingly, N171-82Q mice had elevated plasma glutathione concentrations (Fig. 6). Plasma glutathione is derived mainly from hepatic release and is taken up by brain (Kannan et al., 1990). Oxidative stress is present in human HD blood (SanchezLopez et al., 2012). Therefore, elevated plasma glutathione in HD mice may result from increased hepatic release as part of an adaptive response to peripheral oxidative stress. Normalization of plasma glutathione in HD mice by selenite suggests reversal of this effect. Selenite is an important selenium species present in foods and dietary supplements; however, acute toxicity in rats does occur at an ~ 3 fold lower dose than selenomethionine (Vinson and Bose, 1987).

T

C

E

373 374

R

372

R

370 371

O

368 369

C

366 367

N

364 365

Selenium absorbed through the diet is mainly converted by liver to selenoprotein P, then released into blood for transport to brain and other organs (Hill et al., 2012). Plasma selenium, which measures predominantly selenoprotein P (Hill et al., 1996), was measured to determine if there are deficits in selenium status in HD that may decrease availability for brain uptake. In human HD, plasma selenium was not altered while in N171-82Q HD mice there was a small increase (Fig. 5). Despite this, advanced human HD brains had significantly decreased selenium concentrations in multiple brain regions (Fig. 8A). Decreases were present in regions affected late in HD such as cerebellum (Vonsattel et al., 1985) and were not present in end-stage AD brain (Fig. 8B) where there is significant neuronal loss and gliosis (Esiri et al., 1997). These findings support the interpretation that low selenium content in HD brain is not simply the result of changes in cell populations as occurs in advanced HD (Selkoe et al., 1982). Human nutritional selenium deficiencies manifest as decreased plasma selenium (Hurst et al., 2013). Further, brain is a selenium protected site and severe nutritional deficiencies are necessary to lower brain selenium (Nakayama et al., 2007). Therefore, low brain selenium in the presence of normal plasma selenium in human HD (Figs. 5B and 8) suggests an intrinsic effect of mutant huntingtin protein on selenium status in brain. Selenium is present in foods and supplements as protein-bound selenomethionine and selenocysteine as well as the inorganic forms sodium selenate and selenite. Both organic and inorganic forms of selenium can be metabolized to hydrogen selenide, the start point for selenoprotein synthesis (Rayman et al., 2008). We chose to utilize sodium selenite in our therapeutic study because this is the form also present in the mouse chow we used (see methods). We provide evidence that elevated selenite intake has neuroprotective effects in N171-82Q HD mice (Figs. 1–4). We measured striatal and cortical selenium concentrations in control and selenium-supplemented wild-type and HD mice. Differences between groups were not found; however, levels measured were below the level of quantification of the ICP-MS instrumentation due to the small brain regions studied (not shown). Selenium's biologic activities are mediated predominantly through the activity of 25 human selenoproteins (Zhang et al., 2008). We therefore instead focused on studying brain selenoprotein gene expression to seek potential markers and understand mechanisms of selenite-mediated protection. We determined the expression levels of 23 mouse selenoprotein-encoding transcripts in brain. The findings are consistent with complex regulatory effects of mhtt and

U

362 363

P

Fig. 8. Human HD autopsy brains demonstrate a selenium phenotype. A. Decreased selenium in multiple regions in human HD revealed by ICP-MS. See Table 3 for demographic data. B. Brain regional selenium levels are not decreased in end-stage AD. n = 8–9. BA17 = primary visual cortex; BA4 = primary motor cortex; BA9 = dorso-lateral prefrontal cortex; SNC = substantia nigra pars compacta. Bars: black = control; cross-hatched = HD (1A) or AD (1B). P-values: p b 0.001 = ***, p b 0.01 = **, p b 0.05 = * P-value for BA4 (1A) = 0.0566.

Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022

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Funding was provided by University of Wyoming Neuroscience COBRE (5P20RR015640-10), CHDI Foundation and Hatch Project #WYO-438-09. We thank Megan Stiles for assistance with mouse maintenance. The authors declare that there are no conflicts of interest.

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While we found no evidence of toxicity in our studies evaluation of other selenium species with potential for greater human tolerability may be desirable. In mouse models of tauopathy neuroprotection has been demonstrated with both selenate and selenomethionine (Song et al., 2014; van Eersel et al., 2010). Recommendations for selenium intake average 60 μg per day for men and 53 μg per day for women (Rayman, 2012); however, up to 400 μg per day supplemented selenium yeast has been used in long-term cancer prevention studies (Reid et al., 2008). Based on average mouse water intake our low dose effect was with supplementation of ~ 0.75 μg/day/20 g mouse over basal diet; this equates to supplementation with 212 μg selenium/day for a 70 kg person based on a surface area dose translation method (ReaganShaw et al., 2008). Therefore, the protective level of selenite we used is translatable to humans. Despite this, the authors stress that the current findings are not sufficient to support selenium supplementation in HD patients. More work is first needed to identify potential contributory roles of nutritional deficiency versus intrinsic alterations in selenium metabolism, understand mechanisms of neuroprotection in HD mice, determine the most potent therapeutic selenium species in HD animal models and demonstrate clinical efficacy in HD patients. In conclusion, we provide evidence for altered selenium metabolism in mouse and human HD. We further demonstrate protective effects of selenite supplementation in mouse HD. The results support further investigation into a potentially new therapeutic pathway for HD and also into the role of dietary selenium intake level as a modifier of human HD. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nbd.2014.06.022.

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Please cite this article as: Lu, Z., et al., Altered selenium status in Huntington's disease: Neuroprotection by selenite in the N171-82Q mouse model, Neurobiol. Dis. (2014), http://dx.doi.org/10.1016/j.nbd.2014.06.022