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Dithiol-based Compounds Maintain Expression of Antioxidant Protein Peroxiredoxin 1 That Counteracts Toxicity of Mutant Huntingtin*

Open AccessPublished:May 10, 2012DOI:https://doi.org/10.1074/jbc.M111.334565
      Mitochondrial dysfunction and elevated reactive oxygen species are strongly implicated in both aging and various neurodegenerative disorders, including Huntington disease (HD). Because reactive oxygen species can promote the selective oxidation of protein cysteine sulfhydryl groups to disulfide bonds we examined the spectrum of disulfide-bonded proteins that were specifically altered in a HD context. Protein extracts from PC12 cells overexpressing the amino-terminal fragment of the Huntingtin (Htt) protein with either a nonpathogenic or pathogenic polyglutamine repeat (Htt-103Q) were resolved by redox two-dimensional PAGE followed by mass spectrometry analysis. Several antioxidant proteins were identified that exhibited changes in disulfide bonding unique to Htt-103Q expressing cells. In particular, the antioxidant protein peroxiredoxin 1 (Prx1) exhibited both decreased expression and hyperoxidation in response to mutant Htt expressed in either PC12 cells or immortalized striatal cells exposed to 3-nitropropionic acid. Ectopic expression of Prx1 in PC12 cells attenuated mutant Htt-induced toxicity. In contrast, short hairpin RNA-mediated knockdown of Prx1 potentiated mHtt toxicity. Furthermore, treatment with the dithiol-based compounds dimercaptopropanol and dimercaptosuccinic acid suppressed toxicity in both HD cell models, whereas monothiol compounds were relatively ineffective. Dimercaptopropanol treatment also prevented mutant Htt-induced loss of Prx1 expression in both cell models. Our studies reveal for the first time that pathogenic Htt can affect the expression and redox state of antioxidant proteins; an event countered by specific dithiol-based compounds. These findings should provide a catalyst to explore the use of dithiol-based drugs for the treatment of neurodegenerative diseases.
      Background: Decreased antioxidant activity is implicated in neurodegenerative diseases, including Huntington disease (HD).
      Results: Mutant Huntingtin (mHtt) triggered loss of peroxiredoxin 1 (Prx1) expression in two nerve cell models. Dimercaptopropanol treatment attenuated both mHtt-induced toxicity and loss of Prx1 expression.
      Conclusion: Chemical or genetic means of maintaining Prx1 expression counters mHtt toxicity.
      Significance: Dithiol compounds may offer new treatment options for neurodegenerative diseases.

      Introduction

      Huntington disease (HD)
      The abbreviations used are: HD
      Huntington disease
      β-ME
      β-mercaptoethanol
      CoQ10
      coenzyme Q10
      Cys
      cysteamine
      Cys-SH
      cysteine sulfhydryl
      DMP
      dimercaptopropanol
      DMSA
      dimercaptosuccinic acid
      DSBP
      disulfide-bonded protein
      eeGSH
      glutathione-reduced ethyl ester
      Htt
      Huntingtin protein
      Htt-103Q
      Huntingtin with a pathogenic 103 polyglutamine repeat
      mHtt
      mutant Huntingtin protein
      NAC
      N-acetylcysteine
      Prx
      peroxiredoxin
      ROS
      reactive oxygen species
      SOD
      superoxide dismutase
      MTT
      3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyltetrazolium bromide
      3-NP
      3-nitropropionic acid
      Nrf2
      NF-E2 related factor-2
      GFP
      green fluorescent protein
      ANOVA
      analysis of variance
      SCR
      scrambled.
      is an inherited adult onset neurodegenerative disorder characterized clinically by chorea, psychiatric disturbances and dementia, and pathologically by the loss of striatal and cortical cell neurons. The disease is caused by an expansion of a CAG trinucleotide repeat region in exon 1 of the HTT gene, which encodes Huntingtin (Htt), a ubiquitously expressed protein in the brain and peripheral tissues with an uncertain molecular function (
      • Imarisio S.
      • Carmichael J.
      • Korolchuk V.
      • Chen C.W.
      • Saiki S.
      • Rose C.
      • Krishna G.
      • Davies J.E.
      • Ttofi E.
      • Underwood B.R.
      • Rubinsztein D.C.
      Huntington disease. From pathology and genetics to potential therapies.
      ). Individuals with HD have a CAG expansion that results in enlargement of the polyglutamine (poly(Q)) tract within the N terminus of Htt to greater than 36 residues. Longer poly(Q) stretches are associated with earlier onset of HD and more severe disease symptoms (
      • Ross C.A.
      When more is less. Pathogenesis of glutamine repeat neurodegenerative diseases.
      ). The precise mechanism of HD pathophysiology is poorly defined but evidence exists that multiple neurodegenerative pathways are involved including mitochondrial impairment, oxidative stress, transcriptional dysregulation, elevated apoptosis, changes in intracellular transport, signaling dysfunction, and altered protein interactions and activity (
      • Imarisio S.
      • Carmichael J.
      • Korolchuk V.
      • Chen C.W.
      • Saiki S.
      • Rose C.
      • Krishna G.
      • Davies J.E.
      • Ttofi E.
      • Underwood B.R.
      • Rubinsztein D.C.
      Huntington disease. From pathology and genetics to potential therapies.
      ).
      Mutant Htt (mHtt) containing a poly(Q) repeat greater than 36 has a high predisposition to misfold and disrupt normal processes essential for cellular homeostasis (
      • Ross C.A.
      • Tabrizi S.J.
      Huntington disease. From molecular pathogenesis to clinical treatment.
      ). Among these, mitochondrial dysfunction and elevated reactive oxygen species (ROS) production are strongly involved in HD progression (
      • Reddy P.H.
      • Mao P.
      • Manczak M.
      Mitochondrial structural and functional dynamics in Huntington disease.
      ). Although mitochondria produce most of the cellular ATP, they are also a major source of ROS production via electron leakage from the respiratory chain (especially complexes I and III). Several studies have shown that mHtt is found in association with the outer mitochondrial membrane in brain tissue from HD transgenic mice and in isolated mitochondria from both lymphoblasts and postmortem brain tissue from HD patients (
      • Choo Y.S.
      • Johnson G.V.
      • MacDonald M.
      • Detloff P.J.
      • Lesort M.
      Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release.
      ,
      • Panov A.V.
      • Gutekunst C.A.
      • Leavitt B.R.
      • Hayden M.R.
      • Burke J.R.
      • Strittmatter W.J.
      • Greenamyre J.T.
      Early mitochondrial calcium defects in Huntington disease are a direct effect of polyglutamines.
      ,
      • Shirendeb U.
      • Reddy A.P.
      • Manczak M.
      • Calkins M.J.
      • Mao P.
      • Tagle D.A.
      • Reddy P.H.
      Abnormal mitochondrial dynamics, mitochondrial loss, and mutant huntingtin oligomers in Huntington disease. Implications for selective neuronal damage.
      ). In addition, isolated mitochondria from HD mice exhibit decreased membrane potential, increased propensity to depolarize at lower calcium loads, and elevated sensitivity to calcium-induced cytochrome c release compared with controls (
      • Choo Y.S.
      • Johnson G.V.
      • MacDonald M.
      • Detloff P.J.
      • Lesort M.
      Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release.
      ,
      • Panov A.V.
      • Gutekunst C.A.
      • Leavitt B.R.
      • Hayden M.R.
      • Burke J.R.
      • Strittmatter W.J.
      • Greenamyre J.T.
      Early mitochondrial calcium defects in Huntington disease are a direct effect of polyglutamines.
      ). Transcription of peroxisome proliferator-activated receptor, a coactivator 1α (PGC1α), a key transcriptional co-activator that induces expression of genes that regulate mitochondrial respiration and oxidative stress, is repressed in mHtt-expressing neurons (
      • Cui L.
      • Jeong H.
      • Borovecki F.
      • Parkhurst C.N.
      • Tanese N.
      • Krainc D.
      Transcriptional repression of PGC-1α by mutant huntingtin leads to mitochondrial dysfunction and neurodegeneration.
      ). Impaired mitochondrial respiration and ATP synthesis have been detected in postmortem brain samples from HD patients and in various HD cell and animal models (
      • Bossy-Wetzel E.
      • Petrilli A.
      • Knott A.B.
      Mutant huntingtin and mitochondrial dysfunction.
      ). Collectively these findings strongly indicate that perturbed mitochondrial function contributes to HD pathogenesis.
      Expression of mHtt in cultured non-neuronal or neuronal cells has been shown to increase both ROS production and toxicity, which can be rescued by treatment with the thiol-based antioxidants N-acetyl-l-cysteine (NAC) and glutathione (GSH) (
      • Wyttenbach A.
      • Sauvageot O.
      • Carmichael J.
      • Diaz-Latoud C.
      • Arrigo A.P.
      • Rubinsztein D.C.
      Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin.
      ,
      • Firdaus W.J.
      • Wyttenbach A.
      • Diaz-Latoud C.
      • Currie R.W.
      • Arrigo A.P.
      Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant.
      ). Antioxidant treatment has also been shown to reduce cytotoxicity associated with the expression of mHtt in transgenic nematode and mouse models of HD (
      • Parker J.A.
      • Arango M.
      • Abderrahmane S.
      • Lambert E.
      • Tourette C.
      • Catoire H.
      • Néri C.
      Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons.
      ,
      • Ferrante R.J.
      • Andreassen O.A.
      • Dedeoglu A.
      • Ferrante K.L.
      • Jenkins B.G.
      • Hersch S.M.
      • Beal M.F.
      Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington disease.
      ). However, the exact mechanism by which antioxidants counter mHtt-induced toxicity is poorly defined.
      Although ROS have traditionally been viewed as agents that cause nonspecific damage to DNA, lipids, and proteins, recent evidence has shown that ROS can act as second messengers and selectively target proteins leading to a change in their activity or function (
      • Finkel T.
      Signal transduction by reactive oxygen species.
      ). The specificity of ROS-mediated signaling has become an extremely active area of research over the last decade. Cysteine represents one of the major amino acids modified by ROS in a highly selective and reversible manner. Oxidation of protein cysteine sulfhydryl groups (Cys-SH) can lead to the formation of covalent disulfide bonds (Cys-S-S-Cys) or higher oxidized species such as sulfenic (Cys-SOH), sulfinic (Cys-SO2H), or sulfonic (Cys-SO3H) acids. Functional consequences of Cys-SH oxidation include altered protein interactions, misfolding, catalytic inactivation, and decreased antioxidant capacity (
      • Sitia R.
      • Molteni S.N.
      Stress, protein (mis)folding, and signaling. The redox connection.
      ). The specificity of ROS-mediated oxidation of cysteine residues within proteins that participate in a wide array of cellular processes has made these proteins attractive targets for therapeutic manipulation in diseases strongly linked to oxidative stress (
      • Cumming R.C.
      • Andon N.L.
      • Haynes P.A.
      • Park M.
      • Fischer W.H.
      • Schubert D.
      Protein disulfide bond formation in the cytoplasm during oxidative stress.
      ).
      One of the best examples of redox regulation is found in the peroxiredoxin family of proteins. Peroxiredoxins (Prxs) were originally characterized as abundant antioxidant proteins that contain a catalytic cysteine residue and detoxify hydrogen peroxide (H2O2), peroxynitrate, and a range of organic hydroperoxides using reducing equivalents supplied by the thioredoxin system (
      • Rhee S.G.
      • Chae H.Z.
      • Kim K.
      Peroxiredoxins. A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling.
      ). However, recent studies have shown that Prxs participate in a wide array of cellular processes including neuronal differentiation, cell signaling, molecular chaperoning, and mitochondrial function in both a catalytic dependent and independent manner (
      • Woo H.A.
      • Yim S.H.
      • Shin D.H.
      • Kang D.
      • Yu D.Y.
      • Rhee S.G.
      Inactivation of peroxiredoxin 1 by phosphorylation allows localized H2O2 accumulation for cell signaling.
      ,
      • Yan Y.
      • Sabharwal P.
      • Rao M.
      • Sockanathan S.
      The antioxidant enzyme Prdx1 controls neuronal differentiation by thiol-redox-dependent activation of GDE2.
      ,
      • Jang H.H.
      • Lee K.O.
      • Chi Y.H.
      • Jung B.G.
      • Park S.K.
      • Park J.H.
      • Lee J.R.
      • Lee S.S.
      • Moon J.C.
      • Yun J.W.
      • Choi Y.O.
      • Kim W.Y.
      • Kang J.S.
      • Cheong G.W.
      • Yun D.J.
      • Rhee S.G.
      • Cho M.J.
      • Lee S.Y.
      Two enzymes in one. Two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function.
      ). For example, Prxs are able to interact with and inactivate protein kinases such as JNK, c-Abl, and ASK1 in a redox-regulated manner (
      • Neumann C.A.
      • Cao J.
      • Manevich Y.
      Peroxiredoxin 1 and its role in cell signaling.
      ).
      In an effort to determine the spectrum of disulfide-bonded proteins (DSBP) that are altered in an HD context, and hence potential targets of antioxidants, we resolved protein extracts from PC12 cells that inducibly express exon 1 of the HTT gene with either a 25 (nonpathogenic) or 103 (pathogenic) poly(Q) repeat using a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) technique to separate DSBP. Following mass spectrometry analysis, a number of antioxidant proteins were identified that displayed alterations in disulfide bonding only in Htt-103Q expressing cells. In particular, Prx1 was shown to exhibit a progressive decrease in expression and a concomitant increase in protein sulfonylation following induction of mHtt expression. Testing of various thiol-based antioxidants revealed that dimercaptopropanol (DMP) and the structurally related compound dimercaptosuccinic acid (DMSA) were specifically able to rescue mHtt-induced toxicity in PC12 cells, whereas monothiol reducing agents were relatively ineffective. In addition, DMP was able to protect against 3-nitropropionic acid-induced toxicity in a rodent HD striatal cell line. DMP-mediated protection correlated with the maintenance of Prx expression and suppression of Prx1 sulfonylation. These novel findings suggest that dithiol-based compounds can selectively protect against mHtt-induced toxicity.

      DISCUSSION

      ROS have traditionally been viewed as deleterious molecules that nonspecifically damage cellular macromolecules and contribute to various forms of neurodegeneration and aging. Elevated oxidation of DNA, protein, and lipids has frequently been detected in postmortem brain tissue from patients afflicted with various neurodegenerative diseases. Whether the elevation in oxidative damage associated with disease states is a causal or correlative event has long been the subject of debate. However, over the last decade numerous studies have shown that ROS-mediated oxidation of cysteine residues acts as a key signaling mechanism controlling multiple cellular processes including protein phosphorylation, gene expression, chromatin remodeling, protein turnover, molecular chaperoning, metabolism, autophagy, antioxidant defense, and mitochondrial function in a specific and reversible manner (
      • Paulsen C.E.
      • Carroll K.S.
      Orchestrating redox signaling networks through regulatory cysteine switches.
      ). In fact, it is now recognized that under physiological conditions transient low level generation of ROS appears to be essential to maintain cellular homeostasis (
      • Trachootham D.
      • Lu W.
      • Ogasawara M.A.
      • Nilsa R.D.
      • Huang P.
      Redox regulation of cell survival.
      ). However, relatively few studies have attempted to detect oxidative modifications to cysteine residues in a disease context. One study revealed that glucose-6-phosphate dehydrogenase, a key enzyme that generates NADPH reducing equivalents in cells, is elevated in Alzheimer disease brain along with a concomitant increase in reactive sulfhydryls in the neuronal cytoplasm (
      • Russell R.L.
      • Siedlak S.L.
      • Raina A.K.
      • Bautista J.M.
      • Smith M.A.
      • Perry G.
      Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer disease.
      ). However, the identity of proteins with reactive sulfhydryl groups that are altered in neurodegenerative diseases is poorly defined.
      Here we show for the first time that increased expression of pathogenic Htt promotes aberrant disulfide bonding in nerve cell lines. In particular, mHtt expression promotes increased disulfide bonding of the antioxidant proteins Cu/Zn-superoxide dismutase 1 (SOD1) and Prx4 while decreasing disulfide bonding of Prx1 and Prx2. SOD1 is a cytosolic protein responsible for the enzymatic conversion of the superoxide anion to hydrogen peroxide. Prx1 and Prx2 are predominately cytosolic, whereas Prx4 is a secreted peroxiredoxin isoform that is also found in the endoplasmic reticulum. All three Prx isoforms belong to the typical 2-cysteine family of peroxiredoxins that undergo a catalytic cycle in which the N-terminal cysteine is oxidized by H2O2 to a sulfenic acid that then reacts with the C-terminal cysteine of another subunit to produce an intermolecular disulfide (
      • Rhee S.G.
      • Chae H.Z.
      • Kim K.
      Peroxiredoxins. A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling.
      ). This disulfide is then reduced by the Trx/TrxR system, completing the catalytic cycle. During oxidative stress the sulfenic intermediate is hyperoxidized to a sulfinic acid or a sulfonic acid, which is incapable of forming a disulfide bond leading to inactivation of Prx peroxidase activity (
      • Yang K.S.
      • Kang S.W.
      • Woo H.A.
      • Hwang S.C.
      • Chae H.Z.
      • Kim K.
      • Rhee S.G.
      Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid.
      ). Oxidation of the catalytic cysteine to a sulfinic acid in Prxs 1–4 is reversed by a reaction catalyzed by sulfiredoxin (
      • Jeong W.
      • Park S.J.
      • Chang T.S.
      • Lee D.Y.
      • Rhee S.G.
      Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin.
      ). However, overoxidation of all Prx isoforms to a sulfonic state is irreversible.
      In this study, we observed that mHtt expression promoted a decrease in Prx1 expression and a concurrent increase in Prx1 sulfonylation. Hyperoxidation of Prxs has been show to render these proteins susceptible to ubiquitin-mediated proteolysis (
      • Bae S.H.
      • Woo H.A.
      • Sung S.H.
      • Lee H.E.
      • Lee S.K.
      • Kil I.S.
      • Rhee S.G.
      Induction of sulfiredoxin via an Nrf2-dependent pathway and hyperoxidation of peroxiredoxin III in the lungs of mice exposed to hyperoxia.
      ,
      • Kim B.J.
      • Hood B.L.
      • Aragon R.A.
      • Hardwick J.P.
      • Conrads T.P.
      • Veenstra T.D.
      • Song B.J.
      Increased oxidation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells.
      ). Therefore, mHtt may affect Prx1 expression at both transcriptional and post-translational levels. In contrast, DMP treatment prevented both mHtt-induced loss of Prx1 expression and increased sulfonylation. Prx1 expression is controlled by the transcription factor NF-E2 related factor-2 (Nrf2) (
      • Kim Y.J.
      • Ahn J.Y.
      • Liang P.
      • Ip C.
      • Zhang Y.
      • Park Y.M.
      Human prx1 gene is a target of Nrf2 and is up-regulated by hypoxia/reoxygenation. Implication to tumor biology.
      ). Elevated ROS can promote oxidative modifications to Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, leading to the stabilization and nuclear translocalization of Nrf2 (
      • Dinkova-Kostova A.T.
      • Holtzclaw W.D.
      • Cole R.N.
      • Itoh K.
      • Wakabayashi N.
      • Katoh Y.
      • Yamamoto M.
      • Talalay P.
      Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.
      ). Once in the nucleus, Nrf2 dimerizes with a small Maf protein and the heterodimer then binds antioxidant response element sequences within the promoters of antioxidant and phase II detoxifying genes. Besides activating transcription of Prx1, Nrf2 can also activate expression of sulfiredoxin, an enzyme that can reverse oxidation of Prx sulfinic acids to sulfhydryl groups and various other antioxidant enzymes (
      • Soriano F.X.
      • Léveillé F.
      • Papadia S.
      • Higgins L.G.
      • Varley J.
      • Baxter P.
      • Hayes J.D.
      • Hardingham G.E.
      Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator [3H]1,2-dithiole-3-thione.
      ). The observation that DMP exposure can partially rescue mHtt-induced toxicity even when Prx1 expression is knocked down (Fig. 7) suggests that this compound is likely to alter expression of other neuroprotective genes. It is possible that DMP may affect Nrf2 activation via a redox-dependent mechanism leading to increased expression of Prx1, sulfiredoxin, and other antioxidant enzymes even in the presence of mHtt. Future studies will evaluate this hypothesis.
      We have previously shown that Prx1 levels are elevated and overoxidized Prx2 isoforms predominate in Alzheimer diseased brain tissue (
      • Cumming R.C.
      • Dargusch R.
      • Fischer W.H.
      • Schubert D.
      Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid β-resistant nerve cells.
      ). Overexpression of Prx1 alone can protect against amyloid-β toxicity in nerve cells. Furthermore, amyloid-β-resistant nerve cells up-regulate multiple Prx isoforms, in addition to the reductive enzymes required to maintain Prxs in an active state, thereby countering toxicity initiated by amyloid-β exposure (
      • Cumming R.C.
      • Dargusch R.
      • Fischer W.H.
      • Schubert D.
      Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid β-resistant nerve cells.
      ). A recent proteomic study showed that the Prx isoforms 1, 2, and 6 are elevated in the striatum and cortex of HD patients compared with unaffected individuals (
      • Sorolla M.A.
      • Reverter-Branchat G.
      • Tamarit J.
      • Ferrer I.
      • Ros J.
      • Cabiscol E.
      Proteomic and oxidative stress analysis in human brain samples of Huntington disease.
      ). However, this study also detected the accumulation of acidic (i.e. sulfonylated) forms of Prx1 and Prx6 in HD patients as well. Collectively these findings indicate that maintenance of Prxs at a high level and in an active state is key to ensuring optimal neuronal cell survival in the presence of pathogenic proteins associated with various neurodegenerative diseases.
      The observation that oxidative damage to macromolecules is increased in HD has spurred interest in evaluating the ability of various antioxidants to attenuate mHtt-induced toxicity. A number of studies have focused on the use of the molecule Coenzyme Q10 (CoQ10) for the treatment of HD (
      • Chaturvedi R.K.
      • Beal M.F.
      Mitochondrial approaches for neuroprotection.
      ). CoQ10 functions in the inner membrane of mitochondria as an electron carrier from enzyme complex I and complex II to complex III and can also act as a lipid soluble antioxidant, particularly in its reduced form (ubiquinol, CoQ10H2). CoQ10 has been shown to significantly increase survival and delay motor symptoms in transgenic HD mouse models (
      • Ferrante R.J.
      • Andreassen O.A.
      • Dedeoglu A.
      • Ferrante K.L.
      • Jenkins B.G.
      • Hersch S.M.
      • Beal M.F.
      Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington disease.
      ). However, clinical trials testing various formulations of CoQ10 in HD patients have, so far, failed to fully recapitulate the positive effects of this compound observed in animal models (
      Huntington Study Group
      A randomized, placebo-controlled trial of coenzyme Q10 and remacemide in Huntington disease.
      ,
      • Verbessem P.
      • Lemiere J.
      • Eijnde B.O.
      • Swinnen S.
      • Vanhees L.
      • Van Leemputte M.
      • Hespel P.
      • Dom R.
      Creatine supplementation in Huntington disease. A placebo-controlled pilot trial.
      ). Whether CoQ10 is able to completely target affected brain regions in HD patients and either increase mitochondrial function or detoxify elevated ROS is uncertain. The modest effect of this antioxidant in HD patients may be related to its relative lack of specificity in targeting oxidized intracellular targets. Although, CoQ10 can detoxify lipid peroxides, it does so to varying extents in different tissues and in a relatively nonselective manner. Furthermore, it is now recognized that under physiological conditions, the transient generation of ROS, within certain limits, appears to be essential to maintain cellular homeostasis, whereas countering low level ROS with broad-acting antioxidants can actually be detrimental (
      • Trachootham D.
      • Lu W.
      • Ogasawara M.A.
      • Nilsa R.D.
      • Huang P.
      Redox regulation of cell survival.
      ). For example, administration of antioxidants has been shown to inhibit autophagy and increase toxicity of poly(Q) proteins (
      • Underwood B.R.
      • Imarisio S.
      • Fleming A.
      • Rose C.
      • Krishna G.
      • Heard P.
      • Quick M.
      • Korolchuk V.I.
      • Renna M.
      • Sarkar S.
      • García-Arencibia M.
      • O'Kane C.J.
      • Murphy M.P.
      • Rubinsztein D.C.
      Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease.
      ). Therefore, identification of compounds that possess good bioavailability and display some degree of specificity in targeting oxidized proteins may be key factors for the treatment of neurodegenerative diseases.
      In this study we demonstrate for the first time that specific dithiol-based compounds confer selective resistance to mHtt-induced neurotoxicity. Although many commonly used monothiol antioxidants such as β-ME, NAC, and eeGSH were tested in the mHtt-inducible PC12 cell model, only the dithiols DMP, DMSA, and to a lesser extent DTT, showed neuroprotection. This observation suggests that the protective effect of DMP cannot fully be attributed to the nonspecific and widespread reduction in intracellular disulfide bonding. DMP was previously shown in an unbiased blind screen of over 1000 FDA approved compounds to be neuroprotective against mHtt toxicity in the same PC12 model used in this study (
      • Aiken C.T.
      • Tobin A.J.
      • Schweitzer E.S.
      A cell-based screen for drugs to treat Huntington disease.
      ). In addition, DMP, also known as British anti-Lewisite (BAL), was shown to reduce pathophysiological symptoms of HD and halt disease progression in a long term study of two patients conducted in 1955 (
      • Nielsen J.M.
      • Butt E.M.
      Treatment of Huntington chorea with BAL.
      ). At the time DMP was believed to work by chelating metals but subsequent studies using the clinically more tolerable metal chelator penicillamine failed to alleviate HD clinical symptoms (
      • Haslam M.T.
      Cellular magnesium levels and the use of penicillamine in the treatment of Huntington chorea.
      ). It is intriguing to note that penacillamine did not confer protection against mHtt expression in PC12 cells (Fig. 5) suggesting that the protective effect of DMP is unlikely to be mediated by metal chelation. Although DMP exhibited the highest level of protection against mHtt toxicity, it has poor bioavailability properties and requires intramuscular injections. DMSA also exhibited protection in this study, albeit to a lesser extent than DMP. DMSA is orally active and can cross the blood-brain barrier (
      • Cory-Slechta D.A.
      Mobilization of lead over the course of DMSA chelation therapy and long-term efficacy.
      ,
      • Aaseth J.
      • Jacobsen D.
      • Andersen O.
      • Wickstrøm E.
      Treatment of mercury and lead poisonings with dimercaptosuccinic acid and sodium dimercaptopropanesulfonate. A review.
      ) making it an excellent candidate drug for further testing as a therapy for HD. Moreover, the structural relationship between the dithiols DMP and DMSA and their strong neuroprotective effects in HD cell culture models suggest that these molecules may be a good starting point for further development of dithiol-based compounds to treat HD and other neurodegenerative disorders.

      REFERENCES

        • Imarisio S.
        • Carmichael J.
        • Korolchuk V.
        • Chen C.W.
        • Saiki S.
        • Rose C.
        • Krishna G.
        • Davies J.E.
        • Ttofi E.
        • Underwood B.R.
        • Rubinsztein D.C.
        Huntington disease. From pathology and genetics to potential therapies.
        Biochem. J. 2008; 412: 191-209
        • Ross C.A.
        When more is less. Pathogenesis of glutamine repeat neurodegenerative diseases.
        Neuron. 1995; 15: 493-496
        • Ross C.A.
        • Tabrizi S.J.
        Huntington disease. From molecular pathogenesis to clinical treatment.
        Lancet Neurol. 2011; 10: 83-98
        • Reddy P.H.
        • Mao P.
        • Manczak M.
        Mitochondrial structural and functional dynamics in Huntington disease.
        Brain Res. Rev. 2009; 61: 33-48
        • Choo Y.S.
        • Johnson G.V.
        • MacDonald M.
        • Detloff P.J.
        • Lesort M.
        Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release.
        Hum. Mol. Genet. 2004; 13: 1407-1420
        • Panov A.V.
        • Gutekunst C.A.
        • Leavitt B.R.
        • Hayden M.R.
        • Burke J.R.
        • Strittmatter W.J.
        • Greenamyre J.T.
        Early mitochondrial calcium defects in Huntington disease are a direct effect of polyglutamines.
        Nat. Neurosci. 2002; 5: 731-736
        • Shirendeb U.
        • Reddy A.P.
        • Manczak M.
        • Calkins M.J.
        • Mao P.
        • Tagle D.A.
        • Reddy P.H.
        Abnormal mitochondrial dynamics, mitochondrial loss, and mutant huntingtin oligomers in Huntington disease. Implications for selective neuronal damage.
        Hum. Mol. Genet. 2011; 20: 1438-1455
        • Cui L.
        • Jeong H.
        • Borovecki F.
        • Parkhurst C.N.
        • Tanese N.
        • Krainc D.
        Transcriptional repression of PGC-1α by mutant huntingtin leads to mitochondrial dysfunction and neurodegeneration.
        Cell. 2006; 127: 59-69
        • Bossy-Wetzel E.
        • Petrilli A.
        • Knott A.B.
        Mutant huntingtin and mitochondrial dysfunction.
        Trends Neurosci. 2008; 31: 609-616
        • Wyttenbach A.
        • Sauvageot O.
        • Carmichael J.
        • Diaz-Latoud C.
        • Arrigo A.P.
        • Rubinsztein D.C.
        Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin.
        Hum. Mol. Genet. 2002; 11: 1137-1151
        • Firdaus W.J.
        • Wyttenbach A.
        • Diaz-Latoud C.
        • Currie R.W.
        • Arrigo A.P.
        Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant.
        FEBS J. 2006; 273: 3076-3093
        • Parker J.A.
        • Arango M.
        • Abderrahmane S.
        • Lambert E.
        • Tourette C.
        • Catoire H.
        • Néri C.
        Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons.
        Nat. Genet. 2005; 37: 349-350
        • Ferrante R.J.
        • Andreassen O.A.
        • Dedeoglu A.
        • Ferrante K.L.
        • Jenkins B.G.
        • Hersch S.M.
        • Beal M.F.
        Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington disease.
        J. Neurosci. 2002; 22: 1592-1599
        • Finkel T.
        Signal transduction by reactive oxygen species.
        J. Cell Biol. 2011; 194: 7-15
        • Sitia R.
        • Molteni S.N.
        Stress, protein (mis)folding, and signaling. The redox connection.
        Sci. STKE. 2004; 2004: pe27
        • Cumming R.C.
        • Andon N.L.
        • Haynes P.A.
        • Park M.
        • Fischer W.H.
        • Schubert D.
        Protein disulfide bond formation in the cytoplasm during oxidative stress.
        J. Biol. Chem. 2004; 279: 21749-21758
        • Rhee S.G.
        • Chae H.Z.
        • Kim K.
        Peroxiredoxins. A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling.
        Free Radic. Biol. Med. 2005; 38: 1543-1552
        • Woo H.A.
        • Yim S.H.
        • Shin D.H.
        • Kang D.
        • Yu D.Y.
        • Rhee S.G.
        Inactivation of peroxiredoxin 1 by phosphorylation allows localized H2O2 accumulation for cell signaling.
        Cell. 2010; 140: 517-528
        • Yan Y.
        • Sabharwal P.
        • Rao M.
        • Sockanathan S.
        The antioxidant enzyme Prdx1 controls neuronal differentiation by thiol-redox-dependent activation of GDE2.
        Cell. 2009; 138: 1209-1221
        • Jang H.H.
        • Lee K.O.
        • Chi Y.H.
        • Jung B.G.
        • Park S.K.
        • Park J.H.
        • Lee J.R.
        • Lee S.S.
        • Moon J.C.
        • Yun J.W.
        • Choi Y.O.
        • Kim W.Y.
        • Kang J.S.
        • Cheong G.W.
        • Yun D.J.
        • Rhee S.G.
        • Cho M.J.
        • Lee S.Y.
        Two enzymes in one. Two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function.
        Cell. 2004; 117: 625-635
        • Neumann C.A.
        • Cao J.
        • Manevich Y.
        Peroxiredoxin 1 and its role in cell signaling.
        Cell Cycle. 2009; 8: 4072-4078
        • Aiken C.T.
        • Tobin A.J.
        • Schweitzer E.S.
        A cell-based screen for drugs to treat Huntington disease.
        Neurobiol. Dis. 2004; 16: 546-555
        • Trettel F.
        • Rigamonti D.
        • Hilditch-Maguire P.
        • Wheeler V.C.
        • Sharp A.H.
        • Persichetti F.
        • Cattaneo E.
        • MacDonald M.E.
        Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells.
        Hum. Mol. Genet. 2000; 9: 2799-2809
        • Cumming R.C.
        Analysis of global and specific changes in the disulfide proteome using redox two-dimensional polyacrylamide gel electrophoresis.
        Methods Mol. Biol. 2008; 476: 165-179
        • Li X.
        • Valencia A.
        • Sapp E.
        • Masso N.
        • Alexander J.
        • Reeves P.
        • Kegel K.B.
        • Aronin N.
        • Difiglia M.
        Aberrant Rab11-dependent trafficking of the neuronal glutamate transporter EAAC1 causes oxidative stress and cell death in Huntington disease.
        J. Neurosci. 2010; 30: 4552-4561
        • Degli Esposti M.
        Measuring mitochondrial reactive oxygen species.
        Methods. 2002; 26: 335-340
        • Yang K.S.
        • Kang S.W.
        • Woo H.A.
        • Hwang S.C.
        • Chae H.Z.
        • Kim K.
        • Rhee S.G.
        Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid.
        J. Biol. Chem. 2002; 277: 38029-38036
        • Bae S.H.
        • Woo H.A.
        • Sung S.H.
        • Lee H.E.
        • Lee S.K.
        • Kil I.S.
        • Rhee S.G.
        Induction of sulfiredoxin via an Nrf2-dependent pathway and hyperoxidation of peroxiredoxin III in the lungs of mice exposed to hyperoxia.
        Antioxid. Redox Signal. 2009; 11: 937-948
        • Kim B.J.
        • Hood B.L.
        • Aragon R.A.
        • Hardwick J.P.
        • Conrads T.P.
        • Veenstra T.D.
        • Song B.J.
        Increased oxidation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells.
        Proteomics. 2006; 6: 1250-1260
        • Ruan Q.
        • Lesort M.
        • MacDonald M.E.
        • Johnson G.V.
        Striatal cells from mutant huntingtin knock-in mice are selectively vulnerable to mitochondrial complex II inhibitor-induced cell death through a nonapoptotic pathway.
        Hum. Mol. Genet. 2004; 13: 669-681
        • Paulsen C.E.
        • Carroll K.S.
        Orchestrating redox signaling networks through regulatory cysteine switches.
        ACS Chem. Biol. 2010; 5: 47-62
        • Trachootham D.
        • Lu W.
        • Ogasawara M.A.
        • Nilsa R.D.
        • Huang P.
        Redox regulation of cell survival.
        Antioxid. Redox Signal. 2008; 10: 1343-1374
        • Russell R.L.
        • Siedlak S.L.
        • Raina A.K.
        • Bautista J.M.
        • Smith M.A.
        • Perry G.
        Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer disease.
        Arch. Biochem. Biophys. 1999; 370: 236-239
        • Jeong W.
        • Park S.J.
        • Chang T.S.
        • Lee D.Y.
        • Rhee S.G.
        Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin.
        J. Biol. Chem. 2006; 281: 14400-14407
        • Kim Y.J.
        • Ahn J.Y.
        • Liang P.
        • Ip C.
        • Zhang Y.
        • Park Y.M.
        Human prx1 gene is a target of Nrf2 and is up-regulated by hypoxia/reoxygenation. Implication to tumor biology.
        Cancer Res. 2007; 67: 546-554
        • Dinkova-Kostova A.T.
        • Holtzclaw W.D.
        • Cole R.N.
        • Itoh K.
        • Wakabayashi N.
        • Katoh Y.
        • Yamamoto M.
        • Talalay P.
        Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11908-11913
        • Soriano F.X.
        • Léveillé F.
        • Papadia S.
        • Higgins L.G.
        • Varley J.
        • Baxter P.
        • Hayes J.D.
        • Hardingham G.E.
        Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator [3H]1,2-dithiole-3-thione.
        J. Neurochem. 2008; 107: 533-543
        • Cumming R.C.
        • Dargusch R.
        • Fischer W.H.
        • Schubert D.
        Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid β-resistant nerve cells.
        J. Biol. Chem. 2007; 282: 30523-30534
        • Sorolla M.A.
        • Reverter-Branchat G.
        • Tamarit J.
        • Ferrer I.
        • Ros J.
        • Cabiscol E.
        Proteomic and oxidative stress analysis in human brain samples of Huntington disease.
        Free Radic. Biol. Med. 2008; 45: 667-678
        • Chaturvedi R.K.
        • Beal M.F.
        Mitochondrial approaches for neuroprotection.
        Ann. N.Y. Acad. Sci. 2008; 1147: 395-412
        • Huntington Study Group
        A randomized, placebo-controlled trial of coenzyme Q10 and remacemide in Huntington disease.
        Neurology. 2001; 57: 397-404
        • Verbessem P.
        • Lemiere J.
        • Eijnde B.O.
        • Swinnen S.
        • Vanhees L.
        • Van Leemputte M.
        • Hespel P.
        • Dom R.
        Creatine supplementation in Huntington disease. A placebo-controlled pilot trial.
        Neurology. 2003; 61: 925-930
        • Underwood B.R.
        • Imarisio S.
        • Fleming A.
        • Rose C.
        • Krishna G.
        • Heard P.
        • Quick M.
        • Korolchuk V.I.
        • Renna M.
        • Sarkar S.
        • García-Arencibia M.
        • O'Kane C.J.
        • Murphy M.P.
        • Rubinsztein D.C.
        Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease.
        Hum. Mol. Genet. 2010; 19: 3413-3429
        • Nielsen J.M.
        • Butt E.M.
        Treatment of Huntington chorea with BAL.
        Bull. Los Angel Neuro. Soc. 1955; 20: 38-39
        • Haslam M.T.
        Cellular magnesium levels and the use of penicillamine in the treatment of Huntington chorea.
        J. Neurol. Neurosurg. Psychiatry. 1967; 30: 185-188
        • Cory-Slechta D.A.
        Mobilization of lead over the course of DMSA chelation therapy and long-term efficacy.
        J. Pharmacol. Exp. Ther. 1988; 246: 84-91
        • Aaseth J.
        • Jacobsen D.
        • Andersen O.
        • Wickstrøm E.
        Treatment of mercury and lead poisonings with dimercaptosuccinic acid and sodium dimercaptopropanesulfonate. A review.
        Analyst. 1995; 120: 853-854