University of Groningen

Overexpression of Cystathionine gamma-Lyase Suppresses Detrimental Effects ofSpinocerebellar Ataxia Type 3Snijder, Pauline M.; Baratashvili, Madina; Grzeschik, Nicola A.; Leuvenink, Henri G. D.;Kuijpers, Lucas; Huitema, Sippie; Schaap, Onno; Giepmans, Ben N. G.; Kuipers, Jeroen;Miljkovic, Jan LjPublished in:Molecular medicine

DOI:10.2119/molmed.2015.00221

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2015

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Snijder, P. M., Baratashvili, M., Grzeschik, N. A., Leuvenink, H. G. D., Kuijpers, L., Huitema, S., ... Sibon,O. C. M. (2015). Overexpression of Cystathionine gamma-Lyase Suppresses Detrimental Effects ofSpinocerebellar Ataxia Type 3. Molecular medicine, 21, 758-768.https://doi.org/10.2119/molmed.2015.00221

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 09-03-2020

7 5 8 | S N I J D E R E T A L . | M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5

INTRODUCTIONSpinocerebellar ataxia type 3 (SCA3),

also known as Machado-Joseph disease,is a rare progressive neurodegenerativedisease and the most common domi-nantly inherited ataxia worldwide. SCA3is a polyglutamine (polyQ) disordercaused by a CAG-trinucleotide repeat ex-pansion encoding glutamine within thesequence of the ataxin-3 (ATXN3) gene.The length of the repeat expansion is di-

rectly related to the aggregation propen-sity of the ataxin3 protein and is in-versely related to the age of onset of thedisease. Protein aggregates are consid-ered to be the cause for neuronal dys-function and death, which is supportedby several lines of evidence showing thataggregate prevention or increased (au-tophagic) clearance delays neuronaldeath and degeneration in multiplemodel systems (1–4).

The pathophysiological sequel of neu-rodegeneration in SCA3 is not fully un-derstood, although proteotoxic stress,transcriptional dysregulation, mitochon-drial dysfunction, oxidative stress andinflammation have been implicated(5–7). To date, there are no disease-modi-fying treatments for polyQ diseases suchas SCA3.

Cystathionine γ-lyase (CSE) is one ofthe central enzymes in cysteine and hy-drogen sulfide metabolism (H2S). Homo-cysteine is a substrate for CSE leading tothe production of H2S, α-ketobutyrate,ammonia, homolanthionine and cys-tathionine, with the latter serving as aCSE substrate to produce cysteine (8,9).Cysteine is also a substrate for CSE lead-ing to the production of H2S, cystathion-ine and pyruvate (8,9). H2S and CSE arelinked to aging and age-related patholo-gies (10–13). H2S can act as an endoge-

Overexpression of Cystathionine γ-Lyase SuppressesDetrimental Effects of Spinocerebellar Ataxia Type 3

Pauline M Snijder,1* Madina Baratashvili,2* Nicola A Grzeschik,2 Henri G D Leuvenink,3 Lucas Kuijpers,2

Sippie Huitema,1 Onno Schaap,2 Ben N G Giepmans,4 Jeroen Kuipers,4 Jan Lj Miljkovic,5

Aleksandra Mitrovic,6 Eelke M Bos,1 Csaba Szabó,7 Harm H Kampinga,2 Pascale F Dijkers,2

Wilfred F A den Dunnen,1 Milos R Filipovic,5 Harry van Goor,1* and Ody C M Sibon2*

Departments of 1Pathology and Medical Biology, 2Cell Biology, and 3Surgery, University Medical Center Groningen, University ofGroningen, Groningen, the Netherlands; 4UMCG Microscopy and Imaging Center, University Medical Center Groningen, Universityof Groningen, Groningen, the Netherlands; 5Department of Chemistry and Pharmacy, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany; 6Faculty of Chemistry, University of Belgrade, Belgrade, Serbia; and 7Department of Anesthesiology,University of Texas Medical Branch, Galveston, Texas, United States of America

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3(ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors con-tributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegen-eration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protec-tive in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates,reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophilacystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improvesSCA3- associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a mod-ifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protectionagainst SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 pa-tients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2015.00221

*PMS, MB, HvG, and OCMS contributed equally to this work.

Address correspondence to Ody CM Sibon, Antonius Deusinglaan 1, 9713 AV, Groningen, the

Netherlands. Phone: +31-50-3632559; Fax: +31-50-363-2515; E-mail: o.c.m.sibon@umcg.nl.

Submitted October 8, 2015; Accepted for publication October 9, 2015; Published Online

(www.molmed.org) October 13, 2015.

R E S E A R C H A R T I C L E

M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5 | S N I J D E R E T A L . | 7 5 9

nous modulator of oxidative stress eitherby direct scavenging of reactive oxygenspecies (ROS) and nitrogen species (14) orthrough increasing the intracellular glu-tathione (GSH) pool (15,16). H2S also con-fers cytoprotection via suppression of in-flammation (17) and by protectingmitochondrial function and integrity(17,18). Decreased levels of H2S in braintissue are associated with neurodegenera-tive age-related diseases such as Parkin-son’s (19), and administration of H2S hasbeen shown protective in experimentalmodels for this disease (20–22). Decreasedlevels of CSE have recently been observedin human Huntington disease tissues andin a mouse Huntington model (22). Afteraddition of sodium hydrogen sulfide andL-cysteine, levels of protein persulfidation(also called protein S-sulfhydration) in-creased in a CSE-dependent manner invitro (23), suggesting an influential effectof this type of posttranslational proteinmodification. Indeed, protein persulfida-tion has been demonstrated to mediatethe activity of parkin, to serve as an anti-oxidant and to protect against cellularsenescence (24–26). A possible link be-tween SCA3, CSE and protein persulfida-tion remains to be determined as well asthe potential neuroprotective effects ofoverexpressing the CSE enzyme directly ina neurodegenerative background.

Here, we investigated the possible pro-tective role of CSE in a Drosophila modelfor SCA3. Drosophila was chosen becauseCSE is highly conserved between humansand flies (http://flybase.org/blast), andan established Drosophila model forSCA3 is available (27,28). In the flymodel for SCA3, a truncated version ofthe pathogenic human ATXN3 gene con-taining a multiple CAG repeat is ex-pressed, and key features of SCA3 dis-ease are present (27–29). This model issuitable because the CAG repeat, andnot the mutated protein, is considered tobe the disease-causing entity in SCA3and in several other polyQ diseases aswell (29,30). We used the DrosophilaSCA3 model (also called SCA3 flies) toinvestigate the effects of CSE overex-pression. We identified additional phe-

notypes in the SCA3 model, such as lossof tissue integrity in degenerativepatches in the fly eye, increased activa-tion of the innate immune response anddecreased protein persulfidation. Wefound that transgene-mediated increasedexpression of CSE rescues these noveland also previously reported pheno-types of the fly SCA3 model. Rescue wasalso observed after addition of sodiumthiosulfate, a drug approved by the U.S.Food and Drug Administration and acomponent of the transsulfuration path-way in which CSE plays a central role(9,31). The CSE-mediated rescue occursindependently of protein aggregate for-mation, indicating that rescue effectsoccur downstream of the formation ofthese toxic entities. We also found en-dogenous expression of CSE in brainareas that are affected in SCA3 patients.In addition a lower expression was ob-served in patient samples comparedwith controls. We present and discuss apossible role for CSE in polyQ diseasepathology and treatment.

MATERIALS AND METHODSBelow we provide a brief overview of

the methods used for experiments pre-sented in this article. For further details,please see the Supplementary Materialsand Methods.

Drosophila StocksAs wild-type control, the y1w1118

Drosophila line was used. Eip55E(Drosophila CSE)-overexpressing lineswere generated in the laboratory. TheGMR-GAL4 UAS-SCA3trQ78 fly stockwas a gift from Nancy M Bonini (28,32).The detailed description of the Drosophilalines and fly food, backcrossing and sup-plementation of chemical compound in-formation can be found in the Supple-mentary Materials and Methods.

Eye Degeneration AssayTo evaluate relative degeneration per-

centage, we used an eye scoring methodthat was previously described (33,34). Ir-regularly structured depigmented eyeswithout dark patches were defined as

rough. The presence of one or more blackpatches along with the irregular struc-ture and depigmentation was considereda degenerated rough eye. Each eye of1-d-old flies was scored as a singular en-tity. We scored the total amount of de-generated eyes as opposed to the totalamount of eyes (rough + degenerated).Total count of eyes scored per conditionwas between 100 and 1,000, dependingon the number of progeny of a particulargenotype.

Protein Persulfidation AssayTo detect protein persulfidation, a tag-

switch assay was used as previously de-scribed (35,36). Briefly, protein extractswere prepared from either whole flies(three flies per sample, 100 μL extractionbuffer) for persulfidation analysis of CSEoverexpression effect under control ofactin driver in a nondisease backgroundor fly heads (25 heads per sample, 65 μLof extraction buffer) for persulfidationanalysis of CSE overexpression effectunder control of a glass multiple reporter(GMR) driver in the SCA3 background.Extracts were prepared using HEN buffer(50 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [HEPES],2 mmol/L ethylenediaminetetraaceticacid [EDTA], 1 % NP-40) additionallysupplemented with 2% sodium dodecylsulfate (SDS), 1% protease inhibitors and20 mmol/L 2-benzo[d]thiazol-2- ylsulfonyl)acetic acid (MSBTA), a water-soluble methylsulfonyl benzothiazole de-rivate (37). The extracts were incubatedfor 30 min on ice and 30 min at 37°C. Pro-teins were then purified by usingwater/methanol/ chloroform precipitation(4/4/1, v/v/v). Samples were redis-solved in phosphate-buffered saline con-taining 2% SDS and treated with CN- biotin (Supplementary Figure S4A, D)overnight. Protein concentrations wereadjusted to the same value before the SDSelectrophoresis was performed. Biotinyla-tion of the samples was detected by West-ern blot, using anti- biotin antibodies(Sigma-Aldrich). To improve the assay, weused a new probe, CN-Cy3, which servedas an alternative for CN-biotin and al-

7 6 0 | S N I J D E R E T A L . | M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5

O V E R E X P R E S S I O N O F C Y S T A T H I O N I N E γ - L Y A S E

lowed direct in-gel fluorescence measure-ments. CN-Cy3 is a cyanoacetate deriva-tive of a cyanine-based Cy3 fluorescencedye. (See Figure 1).

Molecular Biology TechniquesFor a detailed description of quantita-

tive reverse transcriptase–polymerasechain reaction (qRT-PCR), Western blotand protein oxidation analyses, and im-munohistochemistry used in the currentstudy, please see the Supplementary Ma-terials and Methods.

All supplementary materials are availableonline at www.molmed.org.

RESULTS

SCA3 Flies Show Increased TissueDegeneration

A Drosophila model was used to fur-ther investigate a possible protective roleof overexpression of CSE in SCA3. First,we performed an extended phenotypicanalysis of this model. Previously, it wasshown that flies bearing UAS-SCA3trQ78—an inducible truncated ver-sion of the human ATXN3 gene contain-ing 78 CAG repeats—under the controlof the GMR driver (also called GMR-GAL4-UAS-SCA3trQ78 or SCA3 flies) de-velop progressive cellular eye degenera-tion. These flies develop a fullypenetrant “rough eye” phenotype, and acertain percentage of the rough-eyed fliespossess a variable amount of patcheswith increased degeneration in theserough eyes (27,28). Rough eyes contain-ing these degenerative patches (furthercalled “degenerated rough eyes”) are

considered to be more affected comparedwith the rough eyes without thesepatches; therefore, this is a useful tool toidentify enhancers or suppressors of theSCA3-induced toxicity (32). To visualizethese phenotypic differences at a highermagnification and to evaluate the sever-ity of the rough versus degeneratedrough eye phenotypes in more detail, weperformed correlative light microscopyand scanning electron microscopy on theeyes of wild-type flies (Figure 2A–A’’)and flies overexpressing human ATXN3(Figures 2B, C). It appeared that therough eyes consist of irregular-shapedommatidia with irregularly arrangedbristles (Figures 2B–B’’). In contrast, thedegenerative patches contained an unde-fined structure, and bristles were absent(Figures 2C–C’’). These results confirmedthat degenerative patches can indeed beclassified as more severely affected tissueparts compared with the rest of therough eye structures. Therefore, an inter-vention that causes a decreased amountof rough eyes with degenerative areaswithin the SCA3 background can be clas-sified as protective.

Generation and Characterization ofVarious CSE Transgenic Lines

To further investigate a possible mod-ulating role of CSE in SCA3 pathogene-sis, we investigated the effect of CSEoverexpression in the SCA3 fly model.Six different fly lines overexpressingEip55E, a highly conserved Drosophila or-tholog of the human CSE gene(http://flybase.org/blast) (38) under aGAL4-inducible promoter, were created.Because of the absence of an antibody

against Drosophila CSE, the characteriza-tion of the lines was performed by usingqRT-PCR analysis. CSE expression levelsof each inducible line were determinedin the presence of a daughterless driver,resulting in ubiquitous expression of theconstruct. All six lines showed increasedmRNA expression of CSE compared withthe in-house control w1118 strain (Supple-mentary Figure S1A). Because the geneticbackground of Drosophila plays an im-portant role in the severity of specificphenotypes (39), all transgenic overex-pressing lines were backcrossed for atleast six generations to create isogeniccontrols. This step resulted in the genera-tion of CSE overexpressing strain 1(called CSE1) and its specific isogeniccontrol (called control 1) and to the gen-eration of CSE-expressing strain 2 and 3(called CSE2 and CSE3) and their iso-genic control (called control 2). This ap-proach allowed us to compare the effectof CSE overexpression in two geneticbackgrounds, to investigate the effect ofvariations in overexpression levels and tocompare this to isogenic controls. Byusing qRT-PCR, we demonstrated thatCSE1 showed a 2.1-fold induction of CSEcompared with its isogenic control andthat CSE2 and CSE3 showed a 2.2- and5.3-fold increased expression of CSEcompared with their isogenic control, re-spectively (Figures 3A, B). Extensive de-scription of all used Drosophila genotypesis presented in the Supplementary Mate-rials and Methods and SupplementaryFigures S1A, B).

Overexpression of CSE PartiallyRescues the Phenotype of SCA3 inDrosophila

To investigate a possible effect of CSEoverexpression on the eye phenotype, wescored the percentage of degeneratedeyes (i.e., when degenerated patches vi-sualized in Figures 2C–C’’ are present) 1 d after eclosion by using light mi-croscopy. In the SCA3 background, CSE-overexpressing flies showed a significantdecrease in the percentage of degenera-tive rough eyes (Figures 3C, D). Suppres-sion of the SCA3-degenerative rough eye

Figure 1. Synthesis of Cy3-CN from a commercial Cy3-NHS ester. TEA, triethylamine; DMF,dimethylformamide.

R E S E A R C H A R T I C L E

M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5 | S N I J D E R E T A L . | 7 6 1

phenotype was observed in all CSE- overexpressing lines compared with theirSCA3-expressing isogenic control lines.Similar results were obtained in both ge-netic backgrounds. The CSE3 line withthe highest level of CSE overexpressionreduced the number of degenerative eyesto a greater extent than the CSE2 line(Figure 3D). To further strengthen therescue potential of CSE, we pharmaco-logically inhibited CSE with propargyl-glycine (PPG) as previously described(10). Supplementation of PPG to the flyfood reversed the protective effect of CSE

overexpression in the SCA3 background,as evidenced by an increased percentageof degenerated rough eyes (Figures 3C, D).Addition of PPG neither enhanced norsuppressed the percentage of degenera-tive eyes in the SCA3 background,strongly suggesting that the observed ef-fect in the CSE-overexpressing back-ground is due to inhibition of CSE andnot due to other effects of PPG. Together,these results indicate that the rescuingpotential is mediated by overexpressionof CSE and is not influenced by the ge-netic background.

Overexpression of CSE Does NotInduce a Change in Levels ofInsoluble Proteins

Polyglutamine diseases are thought tobe driven by protein aggregation thatsubsequently triggers a myriad of down-stream consequences ultimately leadingto neurodegeneration. We thus first testedwhether the rescue of SCA3-induced tis-sue degeneration by CSE overexpressionwas associated with reduced aggregationof the truncated human ATXN3 protein.Hereto, we determined ratios of insolubleversus soluble fractions of ATXN3 pro-teins in SCA3 flies in the absence andpresence of CSE overexpression by usingWestern blot analysis as described previ-ously (29). An increased insoluble/solubleratio indicates an increase in protein ag-gregation. Overexpression of CSE didnot significantly alter the insoluble/ soluble ratio in SCA3-expressing flies(Figures 4A, B; Supplementary Figure S2),indicating that the protective effects ofCSE overexpression are not mediated bydecreased toxic protein aggregates andmost likely work protectively againstdamaging effects downstream of the for-mation of aggregates.

Overexpression of CSE ReducesLevels of Oxidative Damage ofProteins in SCA3 Flies

Oxidative stress is associated with thepathogenesis of SCA3 disease (40,41).Previously, it was demonstrated that CSEdeficiency is linked to increased levels ofoxidative stress (42). As a readout for ox-idative stress, we used OxyBlot analysisas previously described (43). SCA3 fliesshowed increased levels of oxidized pro-teins (characteristically visible as multiplebands) compared with their isogenic non-SCA3 control lines (Figures 4C, D). Thelevel of oxidized proteins was reduced inall three CSE overexpression lines in theSCA3 background compared with theisogenic controls (Figures 4C, D).

Overexpression of CSE Prevents SCA3-Associated Immune Induction

Several findings suggest that inflam-mation contributes to the multifaceted

Figure 2. Increased tissue degeneration is present in neurodegenerative patches within therough eye background of SCA3 flies. Eyes of SCA3 and control flies were visualized in detailusing light end electron microscopy. Representative light microscopy pictures (A, B, C) withcorrelative scanning electron microscopy pictures (A’, A,” B’, B,” C’, C”) of eye phenotypesare shown. (A, A’, A’’) Normal control eye phenotype (B, B’, B”) SCA3-overexpressing eyewith a phenotype classified as rough. (C, C’, C”) SCA3-overexpressing eye with a pheno-type classified as degenerated; black patches show less preservation of tissue integrity.

7 6 2 | S N I J D E R E T A L . | M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5

O V E R E X P R E S S I O N O F C Y S T A T H I O N I N E γ - L Y A S E

pathogenesis of SCA3 disease (44). InDrosophila, the Toll and immune defi-ciency (IMD) immune signaling path-ways mediate activation of nuclear factor(NF)-κB transcription factors Dif-Dorsaland Relish, respectively. Targets of thesetranscription factors include antimicro-bial peptides (AMPs). These immunepathways are equivalent to NF-κB signal-ing in mammals and are a key factor inthe induction of the innate immune re-sponse (45). Immune induction by ex-pression of SCA3trQ78 in Drosophila hasbeen previously reported as well (46). Toinvestigate whether CSE-mediated pro-tection against SCA3 is associated with areduction in the immune response, weperformed a qRT-PCR analysis for AMPs,which are targets of either the IMD or theToll pathway. As targets of the Toll path-way, immune-induced molecule 1 (IM1), im-mune-induced molecule 2 (IM2) and dro-somycin were analyzed (47,48), and forthe IMD pathway, attacin, cecropin A1 anddiptericin (45). Expression of SCA3 acti-vated the immune response of both path-ways. CSE overexpression in the SCA3background significantly attenuated allinvestigated players of the Toll pathway(Figure 5). A comparable effect was seenfor a subset of AMPs of the IMD pathway(Supplementary Figure S3). This resultshows that the suppressive effect of CSEoverexpression on eye degeneration inSCA3 flies is associated with a dampen-ing of the Toll pathway and a partialdampening of the IMD innate immuneresponse pathway. These data suggestthat CSE may exert its beneficial effectson the SCA3 phenotype by attenuatingthe immune response.

SCA3 Flies Show Reduced Levels ofProtein Persulfidation

Decreased levels of CSE are associatedwith impaired neurological function (22).Here we demonstrated that increasedlevels of CSE are protective. Because CSEoverexpression is associated with in-creased protein persulfidation (25,49), wehypothesized that, in a SCA3 back-ground, protein persulfidation may bedecreased and CSE overexpression may

Figure 3. CSE overexpression suppresses the SCA3 phenotype. (A, B) CSE overexpressionwas determined by using qRT-PCR in CSE (CSE1; CSE2; CSE3) overexpressing transgenic flylines compared with their isogenic controls (control 1 and control 2). CSE was expressedubiquitously by using an actin-GAL4 driver. In both genetic backgrounds, CSE mRNA levelswere increased in the CSE-overexpressing lines compared with their isogenic controls. *p < 0.05, error bars indicate standard error of the mean (SEM). (C, D) SCA3 flies with andwithout overexpression of CSE (three independent lines) were analyzed. In all three trans-genic lines in the SCA3 background, CSE overexpression resulted in a decrease of the de-gree of eye degeneration compared with isogenic SCA3-expressing lines. Inhibition of CSEby 2 mmol/L PPG diminished this effect. The presence of degenerative patches (blackarea) was determined by using light microscopy. For quantification, the number of roughand degenerated eyes in at least three independent experiments (n = 100–300 per ex-periment) was counted. ***p < 0.001, error bars indicate SEM. Black area represents thepercentage of rough eyes containing neurodegenerative patches. Grey area representspercentage of rough eyes without neurodegenerative patches.

R E S E A R C H A R T I C L E

M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5 | S N I J D E R E T A L . | 7 6 3

lead to restoration of this posttransla-tional modification.

To selectively detect protein persulfi-dation, a recently reported tag-switchassay was further optimized and used.The original method labels the persulfi-dation of proteins with biotin as a re-porting tag (Supplementary FiguresS4A–C). To further enhance the signalintensity, the tag-switch assay was im-proved by using a reagent, which di-rectly introduced a cyanine-based Cy3fluorescence tag, Cy3-CN (Supplemen-tary Figure S4A), to persulfidated pro-teins. An artificial color scheme wasused to better illustrate the persulfida-tion levels (the color scale is given in thefigure). A signal in the yellow-whiterange indicates high levels of persulfida-tion, and a signal in the black-blue rangeindicates a low level (see also the Sup-plemental Data). Persulfidation of pro-teins in heads of SCA3 flies was foundto be decreased compared with wild-type controls (Figure 6A). Next, we in-vestigated whether the protective effectof CSE overexpression was associatedwith restoration of protein persulfida-tion. Consistently with our hypothesis,we found that CSE- overexpressing fliesdemonstrated elevated protein persulfi-dation compared with the controls (Fig-ure 6B, Supplementary Figure S4D). Theobserved rescue of SCA3 degenerationin CSE- overexpressing fly lines was as-sociated with normalization of proteinpersulfidation levels (Figure 6C, Supple-mentary Figures S4B, C). These datashowed that degenerative defects ofSCA3 are associated with decreased lev-els of protein persulfidation, and thiscan be reversed by overexpression ofCSE, an intervention that also protectsagainst tissue loss in the DrosophilaSCA3 model.

Treatment with Sodium ThiosulfateReduces Eye Degeneration in SCA3Flies

To try to pharmacologically rescueSCA3-induced degeneration, we usedsodium thiosulfate (STS). STS is a rela-tively stable nontoxic compound used in

Figure 4. SCA3tr-Q78 protein expression and aggregation is not altered, and oxidative stressis reduced in the presence of CSE overexpression. (A) Western blot analysis of extracts ofheads of SCA3-overexpressing flies was used to determine levels of protein aggregation.The samples were analyzed for the amount of soluble SCA3tr-78 protein (present in resolvinggel) and levels of SCA3tr-78 protein aggregation (present in stacking gel) by using an anti-HA antibody. α-Tubulin was used as a loading control. In the SCA3 flies, both solublemonomer (in the resolving gel) and aggregated protein (in the stacking gel) fractions weredetected. CSE overexpression did not significantly alter the solubility of the SCA3 protein. (B)Quantification of the ratio between the relative intensity of the protein band in the stackinggel and SCA3tr-78 monomer band in the resolving gel. There was no significant change inthe protein solubility upon the overexpression of CSE in a SCA3 background (n = 5). (C, D)OxyBlot analysis of extracts derived from fly heads was used to examine levels of oxidizedproteins. SCA3 flies had higher total levels of oxidized proteins compared with wild-type flies.CSE overexpression in the SCA3 background showed a reduction of oxidized proteins. Threeindependent CSE-overexpressing lines were used and compared with their isogenic con-trols. For quantification, optical density of oxidized proteins was normalized to tubulin; levelsof oxidized proteins in CSE-overexpressing SCA3 fly samples were comparable to levels incontrol fly heads. *p < 0.05, **p < 0.01, error bars indicate SEM.

7 6 4 | S N I J D E R E T A L . | M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5

O V E R E X P R E S S I O N O F C Y S T A T H I O N I N E γ - L Y A S E

clinical settings to treat caliciphylaxis, ex-travasations during chemotherapy orcyanide poisoning (50), and is also acomponent of the transsulfuration path-way. Used as a substrate for rhodanase-like enzymes, thiosulfate could also be asource of targeted persulfidation, as re-cently proposed by Mishanina et al. (31).Therefore, we tested the effects of thio-sulfate on SCA3 flies.

Increasing concentrations of STS werefed to SCA3-expressing flies to investi-gate possible toxicity of STS. Concentra-tions of ≥120 mmol/L STS inducedlethality (data not shown). Upon supple-mentation of lower concentrations ofSTS, a dose-dependent decrease in thepercentage of degenerated rough eyeswas observed (Figure 7A). A total of 80 mmol/L STS reduced the percentageof degenerated rough eyes in SCA3- overexpressing conditions in two geneticbackgrounds (Figure 7B). These datashow that the suppression of SCA3- associated degeneration is achieved notonly using a genetic approach by overex-pression of CSE but can also be achievedpharmacologically using STS.

Endogenous CSE Is Present in AffectedBrain Tissue of SCA3 Patients

To investigate a possible role of CSE inhuman SCA3 pathogenesis, we investi-gated the expression levels and localiza-tion of CSE in healthy tissue and inSCA3 disease tissue. To determine thepresence and localization of CSE, we per-formed immunohistochemistry for CSEon postmortem pontine tissue of controlindividuals and SCA3 patients. From thesparse tissue available for this rare dis-ease, pontine tissue was chosen to ana-lyze the features of this disorder, becausein this tissue several types of toxic pro-tein aggregates are present with enoughneurons preserved to allow immunohis-tochemical analysis (7) in contrast toother brain areas that are almost com-pletely degenerated or that hardly showdegeneration or protein aggregation(51,52). As control samples, postmortemtissue of individuals without a neurode-generative or neuropsychiatric disease

were used (Supplementary Table S1; n =7). CSE protein expression was observedin vascular endothelium, neurons and as-trocytes (Figures 7C–F). This localizationpattern was not affected in pontine tissueof SCA3 patients (Figures 7G–J; Supple-mentary Figure S5). To investigate ex-pression levels of CSE, we performedqRT-PCR analysis for CSE transcripts onpontine samples of SCA3 patients andcontrol samples. qRT-PCR data revealedthe presence mRNA levels of CSE inpontine tissue of control tissue (n = 7)and SCA3 (n = 6) patients; although, inthe latter, levels were reduced (Figure 7K).Western blot analysis using an anti-CSEantibody (53) further confirmed the pres-ence and decreased levels of endogenousCSE in pontine tissue of SCA3 patientscompared with controls (Figures 7L, L’).Together, these data demonstrated that

CSE is endogenously present but de-creased expressed in affected brain areasof SCA3 patients.

DISCUSSIONWe present evidence that CSE overex-

pression works protectively in aDrosophila model for the neurodegenera-tive disease SCA3. To our knowledge,protective effects by CSE overexpressionin neurodegenerative animal models havenot been described before. However, neu-roprotective effects of H2S have been re-ported previously, not only in experimen-tal models for Parkinson’s disease, (21),vascular dementia (54) and homocysteine-induced neurotoxicity, but also in in vitromodels for oxidative stress in neurons (55)and Alzheimer’s disease (56). In an exper-imental mouse model for Parkinson’s dis-ease, inhalation of H2S prevents the devel-

Figure 5. Overexpression of CSE prevents SCA3-related immune induction. mRNA levels ofvarious immune response genes (IM1, IM2, Drosomycin) were determined by qRT-PCR in con-trol flies, in SCA3 flies (2 genetic backgrounds) and in SCA3 flies overexpressing CSE in thesame isogenetic background. In SCA3-expressing flies, all investigated immune players wereupregulated compared with control flies of the same genetic background. In CSE-expressingflies in various SCA3 backgrounds, immune response gene expression was lower comparedwith the isogenetic SCA3 backgrounds. *p < 0.05, **p < 0.01 and ***p < 0.001, error bars indi-cate SEM. IM 1, immune-induced molecule 1; IM 2, immune-induced molecule 2.

R E S E A R C H A R T I C L E

M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5 | S N I J D E R E T A L . | 7 6 5

opment of neurodegeneration and move-ments disorders (20).

The findings in Drosophila may be ofclinical relevance because we observedthat, in SCA3 patients, CSE expression isdecreased in affected brain areas com-pared with controls. Recently, decreasedlevels of CSE were also demonstrated instriatal brain samples from patients withHuntington disease (22). CSE–/– miceshowed impaired locomotor functions(22); therefore, it is possible that low lev-els of CSE negatively influence the pro-gression of neurodegenerative pheno-types in Huntington disease and SCA3.This result is consistent with our findingsshowing that, in contrast to decreasingCSE levels, boosting CSE expression in aneurodegenerative background is benefi-cial. In contrast to our results, CSE pro-tein levels were found unaltered in thecerebellum and cerebral cortex of onespinocerebellar ataxia patient (SCA sub-type unknown) (22), suggesting that al-teration of CSE levels in SCA patientsmay be confined to pontine tissue ormay depend on the SCA subtype.

CSE is an essential enzyme in thetranssulfuration pathway and plays arole in the endogenous production of cys-teine (57) and H2S (58–60). Therefore, thebeneficial effects of CSE can be mediatedvia cysteine, H2S or both. It is also possi-ble that the increased expression of CSEcatalyzes the formation of cysteine per-sulfides that can trans-persulfidate theproteins without any H2S being produced(61). The explanation of the beneficial ef-fects by protein persulfidation is in agree-ment with our observations that thisposttranslational modification is in-creased in CSE-overexpressing flies andrestored in CSE-overexpressing flies in aSCA3 background. Moreover, it also ex-plains the rescue effect of STS, assumingthat the proposed effect of STS on proteinpersulfidation is correct (31). A protectiveeffect of protein persulfidation has beendemonstrated in other studies as well.For example, the activity of neuroprotec-tive ubiquitin ligase parkin is regulatedby protein persulfidation. Parkin persulfi-dation is markedly depleted in the brains

Figure 6. Protein persulfidation is decreased in SCA3 flies and restored by overexpression ofCSE. (A) Persulfidation levels in the SCA3 fly heads are decreased compared with control flies.Protein persulfidation was determined using the tag-switch assay with direct fluorescence la-beling and in-gel fluorescence detection. The gels were artificially colorized in ImageJ for bet-ter visualization of the changes in the signal intensity. Fluorescence intensity scale is providedat the right. Silver-stained gels were shown to demonstrate equal protein loading of the sam-ples. Note that because of the sensitivity of the methods, not all bands visualized by silverstaining will be detected by the fluorescence detection of protein persulfidation and viceversa. Fluorescence intensity scale is provided; signal in the white-yellow range of colors indi-cates relatively high levels of protein persulfidation; signal in the black-blue range indicatesrelatively low levels of protein persulfidation. Extracts of control flies and SCA3 flies wereloaded. (B) CSE overexpression in wild-type flies elevates levels of persulfidation. Protein persul-fidation was determined using the tag-switch assay with direct fluorescence labeling and in-gel fluorescence detection. Extracts of flies overexpressing CSE showed an increase of proteinpersulfidation compared with the control flies. (C) Protein persulfidation levels in the SCA3 flyheads are decreased compared with control flies, and CSE overexpression in the SCA3 back-ground resulted in the partial restoration of persulfidation levels.

7 6 6 | S N I J D E R E T A L . | M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5

O V E R E X P R E S S I O N O F C Y S T A T H I O N I N E γ - L Y A S E

of patients with Parkinson’s disease (24).Another study demonstrates that the ca-pacity of H2S to protect against oxidativestress is executed via persulfidation(25,26). These findings together with ourresults suggest that boosting the transsul-furation pathway may contribute to neu-roprotection via increased persulfidationof proteins.

Our results show that overexpressionof CSE is associated with a dampening ofthe immune response and decreased lev-els of protein oxidation. This finding isconsistent with previous findings be-cause inflammation has been implicatedas a critical mechanism responsible forthe progressive nature of neurodegenera-tion (44,62), and there is an inverse linkbetween an activated transsulfurationpathway and the immune response. Inexperimental models, H2S exerts anti-neuroinflammatory effects via inhibitionof p38/Jun nuclear kinase and NF-κBsignaling pathways (63), and the inhibi-tion of CSE by PPG leads to increased in-flammation (64). Furthermore, CSE hasbeen shown to be a modulator of oxida-tive stress in mice (42). SCA3 is associ-ated with oxidative stress because mu-tant ATXN3 is associated with asignificantly reduced capability to coun-teract oxidative stress that contributes toneuronal cell death in SCA3 (65).

On the basis of the discussed results ofothers and our observations, we proposethe following hypothetical model: PolyQdiseases lead to accumulation of toxicprotein aggregates, and this somehowreduces levels of CSE and/or proteinpersulfidation. This result, in turn, con-tributes to increased oxidative stress andan augmented immune response leadingto accelerated neurodegeneration. It ispossible that overexpression of CSE in-duces protein persulfidation (via or in-dependent from induced H2S and/orcysteine biosynthesis). Increased levelsof protein persulfidation reduces the lev-els of oxidative stress and dampens theimmune response, and, by this, the dam-aging effects of toxic protein aggregatesare reduced and tissue integrity is betterpreserved.

Figure 7. Treatment with STS suppresses SCA3-associated degeneration in Drosophila, andCSE levels are decreased in brains of SCA3 patients. (A, B) Effect of the H2S donor sodiumthiosulfate was determined on the degenerative eye phenotype by using light mi-croscopy. The number of rough and degenerated eyes was counted in three indepen-dent experiments (n = 100–300 per experiment). (A) Increasing concentrations of STS re-sulted in a reduced percentage of degenerated rough eyes of SCA3-expressing flies inthe control1 background. (B) Addition of 80 mmol/L STS to the food of SCA3-expressinglines in two genetic backgrounds partly rescued the SCA3-induced eye degenerativephenotype. *p < 0.05, error bars indicate SEM. (C–J) Immunohistochemistry by using ananti-human CSE antibody revealed that in control human pontine tissue (C–F) and inpontine tissue of SCA3 patients (sample 5, Supplementary Table S1) (representative im-ages are shown in G–J), CSE is localized in neurons of the pontine nuclei (C, G), the vas-culature (D, H) and astrocytes (E, I). Black arrows indicate the mentioned structures. Nodifferences in staining pattern were observed between control and SCA3 brain tissue.Omission of the primary antibody resulted in absence of staining; representative imagesare shown (F, J). Scale bar indicates 150 μm in all images. (K) CSE mRNA levels were deter-mined by using qRT-PCR (control, n = 7; SCA3, n = 6). (L,L’) CSE protein levels were deter-mined using Western blot analysis. Control samples correspond with control: 2, 3, 4, 6, re-spectively (Supplementary Table S1); SCA3 samples correspond with SCA3: 2, 3, 4, 5,respectively (Supplementary Table S1). *p < 0.05, error bars indicate SEM.

R E S E A R C H A R T I C L E

M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5 | S N I J D E R E T A L . | 7 6 7

Our data show that CSE levels are de-creased in tissue of SCA3 patients. How-ever, in our opinion, it is more importantthat CSE is still expressed in affected tis-sue and apparently in identical celltypes, because this result allows a strat-egy to increase CSE expression or activ-ity by pharmacological inventions to pro-tect against tissue degeneration in SCA3.Little is known about the regulation ofCSE, but there are substances availablethat are able to influence CSE activity ortranscription. There is evidence thatmyeloid zinc finger 1 and specificity pro-tein 1 transcription factor affect the tran-scription of CSE (66). Furthermore, stud-ies suggest that CSE can be upregulatedby bacterial endotoxin (66,67) and by ni-tric oxide (68). S-adenosylmethionineand pyridoxal-5′-phosphate stimulateCSE activity to increase H2S production(69,70). Alternatively to increasing CSEexpression as a therapeutic option, res-cue may be provoked by STS becauseour data show a protective effect of thiscompound as well, and it is tolerated byhumans in high concentrations (71,72).

CONCLUSIONOur data indicate a modifying role of

the transsulfuration pathway in SCA3and suggest that this is mediated viaprotein persulfidation. The presence ofCSE in SCA3-relevant brain regions, to-gether with the protective effects of CSEoverexpression in Drosophila, indicatesthe relevance for future research on de-veloping clinically applicable activatorsof CSE or other members of the transsul-furation pathway. As the protective ef-fects occur downstream of the formationof protein aggregates, it may be possiblethat activation of the transsulfurationpathway is protective for other polyglut-amine expansion–induced diseases aswell.

ACKNOWLEDGMENTSThis work was supported by a VICI

grant (to OCM Sibon) and a grant fromthe Jan Kornelis de Cock Foundation (toPM Snijder). Part of this work was per-formed at the UMCG Microscopy and

Imaging Center (UMIC), sponsored byZonMW grant 91111.006.

The authors express their gratitude toMartha Elwenspoek, Marian Bulthuisand Yi Xian Li for excellent technicalsupport and to Bart Kanon and Jan Vonkfor support and valuable advice.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

REFERENCES1. Zoghbi HY, Orr HT. (2000) Glutamine repeats

and neurodegeneration. Annu. Rev. Neurosci.23:217–47.

2. Hageman J, et al. (2010) A DNAJB chaperone sub-family with HDAC-dependent activities sup-presses toxic protein aggregation. Mol. Cell.37:355–69.

3. Dueñas AM, Goold R, Giunti P. (2006) Molecularpathogenesis of spinocerebellar ataxias. Brain.129:1357–70.

4. Frake RA, Ricketts T, Menzies FM, Rubinsztein DC.(2015) Autophagy and neurodegeneration. J. Clin.Invest. 125:65–74.

5. Lee S, Lim H, Masliah E, Lee H. (2011) Proteinaggregate spreading in neurodegenerative dis-eases: problems and perspectives. Neurosci. Res.70:339–48.

6. Takahashi T, Katada S, Onodera O. (2010)Polyglutamine diseases: where does toxicitycome from? what is toxicity? where are wegoing? J. Mol. Cell Biol. 2:180–91.

7. Seidel K, et al. (2012) Cellular protein quality con-trol and the evolution of aggregates in spinocere-bellar ataxia type 3 (SCA3). Neuropathol. Appl.Neurobiol. 38:548–58.

8. Kabil O, Banerjee R. (2014) Enzymology of H2Sbiogenesis, decay and signaling. Antioxid. RedoxSignal. 20:770–82.

9. Kabil O, Motl N, Banerjee R. (2014) H2S and itsrole in redox signaling. Biochim. Biophys. Acta.1844:1355–66.

10. Kabil H, Kabil O, Banerjee R, Harshman LG,Pletcher SD. (2011) Increased transsulfurationmediates longevity and dietary restriction inDrosophila. Proc. Natl. Acad. Sci. U. S. A.108:16831–6.

11. Zhang Y, et al. (2013) Hydrogen sulfide, the nextpotent preventive and therapeutic agent in agingand age-associated diseases. Mol. Cell. Biol.33:1104–13.

12. Chen Y, et al. (2005) Endogenous hydrogen sul-fide in patients with COPD. Chest. 128:3205–11.

13. Miller DL, Roth MB. (2007) Hydrogen sulfide increases thermotolerance and lifespan in

Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 104:20618–22.

14. Carballal S, et al. (2011) Reactivity of hydrogen sul-fide with peroxynitrite and other oxidants of bio-logical interest. Free Radic. Biol. Med. 50:196–205.

15. Kimura Y, Goto Y, Kimura H. (2010) Hydrogensulfide increases glutathione production andsuppresses oxidative stress in mitochondria. An-tioxid. Redox Signal. 12:1–13.

16. Kimura Y, Kimura H. (2004) Hydrogen sulfideprotects neurons from oxidative stress. FASEB J.18:1165–7.

17. Bos EM, et al. (2009) Hydrogen sulfide-induced hy-pometabolism prevents renal ischemia/reperfusioninjury. J. Am. Soc. Nephrol. 20:1901–5.

18. Fu M, et al. (2012) Hydrogen sulfide (H2S) metab-olism in mitochondria and its regulatory role inenergy production. Proc. Natl. Acad. Sci. U. S. A.109:2943–8.

19. Hu L, et al. (2010) Neuroprotective effects of hy-drogen sulfide on Parkinson’s disease rat mod-els. Aging Cell. 9:135–46.

20. Kida K, et al. (2011) Inhaled hydrogen sulfideprevents neurodegeneration and movement dis-order in a mouse model of Parkinson’s disease.Antioxid. Redox Signal. 15:343–52.

21. Xie L, et al. (2013) Therapeutic effect of hydrogensulfide-releasing L-Dopa derivative ACS84 on 6-OHDA-induced Parkinson’s disease rat model.PLoS One. 8:e60200.

22. Paul BD, et al. (2014) Cystathionine gamma-lyasedeficiency mediates neurodegeneration in Hunt-ington’s disease. Nature. 509:96–100.

23. Mustafa AK, et al. (2009) H2S signals throughprotein S-sulfhydration. Sci. Signal. 2:ra72.

24. Vandiver MS, et al. (2013) Sulfhydration mediatesneuroprotective actions of parkin. Nat. Commun.4:1626.

25. Yang G, et al. (2013) Hydrogen sulfide protectsagainst cellular senescence via S-sulfhydration ofKeap1 and activation of Nrf2. Antioxid. RedoxSignal. 18:1906–19.

26. Xie ZZ, et al. (2014) Sulfhydration of p66Shc atcysteine59 mediates the antioxidant effect of hy-drogen sulfide. Antioxid. Redox Signal. 21:2531–42.

27. Warrick JM, et al. (1998) Expanded polyglutamineprotein forms nuclear inclusions and causes neu-ral degeneration in Drosophila. Cell. 93:939–49.

28. Warrick JM, et al. (1999) Suppression of poly -glutamine-mediated neurodegeneration inDrosophila by the molecular chaperone HSP70.Nat. Genet. 23:425–8.

29. Bilen J, Bonini NM. (2007) Genome-wide screenfor modifiers of ataxin-3 neurodegeneration inDrosophila. PLoS Genet. 3:1950–64.

30. Gusella JF, MacDonald ME. (2000) Molecular genet-ics: unmasking polyglutamine triggers in neurode-generative disease. Nat. Rev. Neurosci. 1:109–15.

31. Mishanina TV, Libiad M, Banerjee R. (2015) Bio-genesis of reactive sulfur species for signaling byhydrogen sulfide oxidation pathways. Nat. Chem.Biol. 11:457–64.

32. Bonini NM, Lessing D. (2008) Polyglutamine

7 6 8 | S N I J D E R E T A L . | M O L M E D 2 1 : 7 5 8 - 7 6 8 , 2 0 1 5

O V E R E X P R E S S I O N O F C Y S T A T H I O N I N E γ - L Y A S E

genes interact to modulate the severity and pro-gression of neurodegeneration in Drosophila.PLoS Biol. 6:e29.

33. Carra S, et al. (2010) Identification of theDrosophila ortholog of HSPB8: implication ofHSPB8 loss of function in protein folding dis-eases. J. Biol. Chem. 285:37811–22.

34. Vos MJ, et al. (2010) HSPB7 is the most potentpolyQ aggregation suppressor within the HSPBfamily of molecular chaperones. Hum. Mol. Genet.19:4677–93.

35. Zhang D, et al. (2014) Detection of protein S-sulfhydration by a tag-switch technique. Angew.Chem. Int. Ed Engl. 53:575–81.

36. Park CM, Macinkovic I, Filipovic MR, Xian M.(2015) Use of the “tag-switch” method for the de-tection of protein s-sulfhydration. Methods Enzy-mol. 555:39–56.

37. Zhang D, Devarie-Baez NO, Li Q, Lancaster JR Jr,Xian M. (2012) Methylsulfonyl benzothiazole(MSBT): a selective protein thiol blocking reagent.Org. Lett. 14:3396–9.

38. Andres AJ, Fletcher JC, Karim FD, Thummel CS.(1993) Molecular analysis of the initiation of in-sect metamorphosis: a comparative study ofDrosophila ecdysteroid-regulated transcription.Dev. Biol. 160:388–404.

39. Burnett C, et al. (2011) Absence of effects of Sir2overexpression on lifespan in C. elegans andDrosophila. Nature. 477:482–5.

40. Kazachkova N, et al. (2013) Patterns of mitochon-drial DNA damage in blood and brain tissues ofa transgenic mouse model of Machado-Josephdisease. Neurodegener. Dis. 11:206–14.

41. Yu Y, Kuo C, Cheng W, Liu C, Hsieh M. (2009)Decreased antioxidant enzyme activity and in-creased mitochondrial DNA damage in cellularmodels of Machado-Joseph disease. J. Neurosci.Res. 87:1884–91.

42. Bos EM, et al. (2013) Cystathionine gamma-lyaseprotects against renal ischemia/reperfusion bymodulating oxidative stress. J. Am. Soc. Nephrol.24:759–70.

43. Rana A, et al. (2010) Pantethine rescues aDrosophila model for pantothenate kinase- associated neurodegeneration. Proc. Natl. Acad.Sci. U. S. A. 107:6988–93.

44. Evert BO, et al. (2001) Inflammatory genes areupregulated in expanded ataxin-3-expressingcell lines and spinocerebellar ataxia type 3brains. J. Neurosci. 21:5389–96.

45. Lemaitre B, Hoffmann J. (2007) The host defenseof Drosophila melanogaster. Annu. Rev. Immunol.25:697–743.

46. Shieh SY, Bonini NM. (2011) Genes and pathwaysaffected by CAG-repeat RNA-based toxicity inDrosophila. Hum. Mol. Genet. 20:4810–21.

47. Fullaondo A, et al. (2011) Spn1 regulates theGNBP3-dependent Toll signaling pathway inDrosophila melanogaster. Mol. Cell. Biol.31:2960–72.

48. Boutros M, Agaisse H, Perrimon N. (2002) Se-quential activation of signaling pathways dur-

ing innate immune responses in Drosophila.3:711–22.

49. Krishnan N, Fu C, Pappin DJ, Tonks NK. (2011)H2S-Induced sulfhydration of the phosphatasePTP1B and its role in the endoplasmic reticulumstress response. Sci. Signal. 4:ra86.

50. Farese S, et al. (2011) Sodium thiosulfate pharma-cokinetics in hemodialysis patients and healthyvolunteers. Clin. J. Am. Soc. Nephrol. 6:1447–55.

51. Seidel K, et al. (2010) Axonal inclusions in spin-ocerebellar ataxia type 3. Acta Neuropathol.120:449–60.

52. Seidel K, et al. (2012) Brain pathology of spin-ocerebellar ataxias. Acta Neuropathol. 124:1–21.

53. Ju Y, Untereiner A, Wu L, Yang G. (2015) HS-in-duced S-sulfhydration of pyruvate carboxylasecontributes to gluconeogenesis in liver cells.Biochim. Biophys. Acta. 1850:2293–303.

54. Zhang L, Jiang C, Liu D. (2009) Hydrogen sulfideattenuates neuronal injury induced by vasculardementia via inhibiting apoptosis in rats. Neu-rochem. Res. 34:1984–92.

55. Luo Y, et al. (2013) Hydrogen sulfide preventsOGD/R-induced apoptosis via improving mito-chondrial dysfunction and suppressing an ROS-mediated caspase-3 pathway in cortical neurons.Neurochem. Int. 63:826–31.

56. Liu Y, Bian J. (2010) Hydrogen sulfide protectsamyloid-beta induced cell toxicity in microglia.J. Alzheimers Dis. 22:1189–200.

57. Aitken SM, Kirsch JF. (2005) The enzymology ofcystathionine biosynthesis: strategies for the con-trol of substrate and reaction specificity. Arch.Biochem. Biophys. 433:166–75.

58. King AL, et al. (2014) Hydrogen sulfide cytopro-tective signaling is endothelial nitric oxide syn-thase-nitric oxide dependent. Proc. Natl. Acad.Sci. U. S. A. 111:3182–7.

59. Singh S, Padovani D, Leslie RA, Chiku T, BanerjeeR. (2009) Relative contributions of cystathioninebeta-synthase and gamma-cystathionase to H2Sbiogenesis via alternative trans-sulfuration reac-tions. 284:22457–66.

60. Paul BD, Snyder SH. (2012) H(2)S signallingthrough protein sulfhydration and beyond. Nat.Rev. Mol. Cell Biol. 13:499–507.

61. Ida T, et al. (2014) Reactive cysteine persulfidesand S-polythiolation regulate oxidative stressand redox signaling. Proc. Natl. Acad. Sci. U. S. A.111:7606–11.

62. Block ML, Hong J. (2005) Microglia and inflam-mation-mediated neurodegeneration: multipletriggers with a common mechanism. Prog. Neuro-biol. 76:77–98.

63. Xuan A, et al. (2012) Hydrogen sulfide attenuatesspatial memory impairment and hippocampalneuroinflammation in beta-amyloid rat model ofAlzheimer’s disease. J. Neuroinflammation. 9:202.

64. Wang X, et al. (2013) Dysregulation of cystathion-ine gamma-lyase (CSE)/hydrogen sulfide path-way contributes to ox-LDL-induced inflamma-tion in macrophage. Cell. Signal. 25:2255–62.

65. Araujo J, et al. (2011) FOXO4-dependent upregu-

lation of superoxide dismutase-2 in response tooxidative stress is impaired in spinocerebellarataxia type 3. Hum. Mol. Genet. 20:2928–41.

66. Ishii I, et al. (2004) Murine cystathionine gamma-lyase: complete cDNA and genomic sequences,promoter activity, tissue distribution and devel-opmental expression. Biochem. J. 381:113–23.

67. Li L, et al. (2005) Hydrogen sulfide is a novel me-diator of lipopolysaccharide-induced inflamma-tion in the mouse. FASEB J. 19:1196–8.

68. Eto K, Kimura H. (2002) A novel enhancingmechanism for hydrogen sulfide-producing ac-tivity of cystathionine beta-synthase. J. Biol.Chem. 277:42680–5.

69. Kimura H, Eto K. (2002) The production of hy-drogen sulfide is regulated by testosterone andS-adenosyl-L-methionine in mouse brain. J. Neu-rochem. 83:80–6.

70. Kimura H. (2002) Hydrogen sulfide as a neuro-modulator. Mol. Neurobiol. 26:13–9.

71. Sen U, et al. (2008) Cardioprotective role ofsodium thiosulfate on chronic heart failure bymodulating endogenous H2S generation. Phar-macology. 82:201–13.

72. Singh RP, Derendorf H, Ross EA. (2011) Simula-tion-based sodium thiosulfate dosing strategiesfor the treatment of calciphylaxis. Clin. J. Am.Soc. Nephrol. 6:1155–9.

Cite this article as: Snijder PM, et al. (2015) Over-expression of cystathionine γ-lyase suppressesdetrimental effects of spinocerebellar ataxia type 3.Mol. Med. 21:758–68.