Golgi Stress Response Reprograms Cysteine Metabolism to Confer Cytoprotection in Huntington’S Disease

Golgi stress response reprograms cysteine metabolism to confer cytoprotection in Huntington’s disease

Juan I. Sbodioa,1, Solomon H. Snydera,b,c,2, and Bindu D. Paula,1,2

aThe Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and cDepartment of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Contributed by Solomon H. Snyder, November 21, 2017 (sent for review October 11, 2017; reviewed by Rui Wang and X. William Yang) Golgi stress response is emerging as a physiologic process of com- acid deprivation in HD (10). We investigated whether other parable importance to endoplasmic reticulum (ER) and mitochon- forms of stress also influence CSE. We now report that the Golgi drial stress responses. However, unlike ER stress, the identity of the stress response elicited by monensin stimulates CSE by acting via signal transduction pathway involved in the Golgi stress response has ATF4 with characteristics distinguishable from the ER stress been elusive. We show that the Golgi stressor monensin acts via the response. We also show that the Golgi stress response may be PKR-like ER kinase/Activating Transcription Factor 4 pathway. ATF4 is harnessed to stimulate the reverse transsulfuration pathway and the master regulator of amino acid metabolism, which is induced confer cytoprotection in HD. This strategy may be relevant to during amino acid depletion and other forms of stress. One of the mitigation of cytotoxicity in other neurodegenerative condi- the genes regulated by ATF4 is the biosynthetic enzyme for tions involving altered redox homeostasis. cysteine, cystathionine γ-lyase (CSE), which also plays central roles in maintenance of redox homeostasis. Huntington’s disease (HD), a Results neurodegenerative disorder, is associated with disrupted cysteine Monensin Up-Regulates CSE in Mouse Embryonic Fibroblasts via the metabolism caused by depletion of CSE leading to abnormal redox ATF4 Pathway. CSE is the biosynthetic enzyme for cysteine and a balance and stress response. Thus, restoring CSE function and cys- key enzyme in the reverse transsulfuration pathway, which is teine disposition may be beneficial in HD. Accordingly, we har- responsible for generation of glutathione and hydrogen sulfide nessed the monensin-ATF4–signaling cascade to stimulate CSE NEUROSCIENCE (H S) (Fig. 1A). CSE can be induced by a variety of stress stimuli expression by preconditioning cells with monensin, which restores 2 (10–12). We utilized mouse embryonic fibroblasts (MEFs) to cysteine metabolism and an optimal stress response in HD. These findings have implications for treatment of HD and other diseases analyze the response of CSE to different stress conditions. We associated with redox imbalance and dysregulated ATF4 signaling. cultured MEFs in low-cysteine medium, glutamine-depleted medium (−Q), and arginine-depleted medium (−R) or treated Huntington’s disease | Golgi stress response | monensin | ATF4 | CSE them with erastin (ERA), an inhibitor of cystine transport that elicits amino acid starvation (Fig. 1B). As reported previously (10), low-cysteine medium or erastin treatment markedly in- n addition to its essential function in the transport, processing, B and targeting of proteins through the secretory pathway, the creases levels of CSE in MEFs (Fig. 1 ). Interestingly, omission of I other amino acids also influences CSE, as removal of glutamine Golgi complex has been proposed to be a sensor of stress (1). While B the signal transduction cascade activated during endoplasmic re- and arginine from the medium augments CSE levels (Fig. 1 ). ticulum (ER) stress is well characterized, the Golgi stress response is relatively unexplored (2). The Golgi complex has been implicated Significance in sensing and transducing death signals in neurodegenerative disorders such as Alzheimer’s disease, amyotrophic lateral sclerosis, Golgi stress response is emerging as a major stress response and Huntington’s disease (HD) (3–5). In this study we have iden- pathway although molecular players in the process have not tified the key molecular components mediating the Golgi stress been identified. We show that Golgi stress response induced by response and its relevance to amino acid metabolism in HD. the ionophore monensin is mediated by the PERK-ATF4 pathway. HD is an autosomal-dominant neurodegenerative disorder We further show that subtoxic levels of monensin precondition triggered by expansion of CAG repeats in the gene encoding cells to respond to future damaging insults. Monensin stimulates huntingtin (6). The precise molecular mechanisms whereby the reverse transsulfuration pathway via cystathionine γ-lyase, mutant huntingtin (mHtt) causes neurodegeneration have been the biosynthetic enzyme for cysteine, important for redox ho- elusive. Recently, we demonstrated a major depletion of cys- meostasis. We have harnessed this pathway to mitigate toxicity ’ tathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in associated with cysteine deprivation in Huntington sdisease HD (7). Cysteine supplements reverse abnormalities of HD tis- (HD). These findings are relevant to not only HD, but also other sues and improve survival in mouse models of HD. The CSE diseases involving redox imbalance. Targeting the molecular depletion is caused by sequestration of the basal transcription controls for restoration of cysteine balance may offer more ro- factor regulating CSE, specificity protein 1 (SP1), by mHtt (7–9). bust therapeutic avenues for disease management. Under conditions of cysteine stress, activating transcription fac- Author contributions: J.I.S., S.H.S., and B.D.P. designed research; J.I.S. and B.D.P. per- tor 4 (ATF4) controls expression of CSE. ATF4 is also dysre- formed research; J.I.S. and B.D.P. contributed new reagents/analytic tools; J.I.S., S.H.S., gulated in HD, leading to elevated oxidative stress and and B.D.P. analyzed data; and J.I.S., S.H.S., and B.D.P. wrote the paper. cytotoxicity (10). The aberrant ATF4 response stems from oxi- Reviewers: R.W., Laurentian University; and X.W.Y., University of California, Los Angeles. dative stress associated with abnormalities of cysteine bio- The authors declare no conflict of interest. synthesis and transport. Accordingly, antioxidants reverse the Published under the PNAS license. abnormal ATF4 response to nutrient stress (10). 1J.I.S. and B.D.P. contributed equally to this work. CSE is a highly inducible protein and is regulated at multiple 2To whom correspondence may be addressed. Email: [email protected] or bpaul8@jhmi. levels. It mediates cytoprotection when induced by the in- edu. α flammatory stimulus TNF as well as by LPS and ER stress (11, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 12). Earlier we reported that CSE is markedly induced by amino 1073/pnas.1717877115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1717877115 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Monensin (Mone) induces CSE in MEFs. (A) Schematic representation of the reverse transsulfuration pathway via which cysteine is generated by CSE. (B) CSE is induced by a variety of stress stimuli. Low cysteine (LC) at 0.05 mM, glutamine depletion (−Q) and arginine depletion (−R), and ERA (1 μM), an inhibitor of cystine transport, induce CSE in wild-type MEFS. (C) Structure of the ionophore Mone. (D) Induction of CSE by ER and Golgi stress. TG (0.5 μM), an ER stress agent and the Golgi stress agents Mone, BFA (0.5 μg/mL), and Nig (1 μM) induce CSE in MEFs after 18 h. DMSO (D) was used as a vehicle control. (E) Mone induces ATF4, the transcription factor for CSE in MEFs. MEFs were treated with 5 μM Mone or 1 μM TG or DMSO (D, vehicle control) for 18 h and analyzed by Western blotting. (F) Immunofluorescence analysis showing the induction and nuclear localization of ATF4 in response to different stress stimuli. Wild-type MEFs were cultured on coverslips in medium lacking glutamine (−Q) or grown in full medium and treated with Mone (5 μM) or TG (1 μM) and stained for ATF4 (green) and DNA (DAPI). (Magnification: 63×.) (G) The up-regulation of ATF4 occurs at the transcriptional level. MEF cells were incubated as in E. Cells were scraped, and the RNA was isolated and analyzed by real-time qPCR. n = 3 (means ± SD). ***P < 0.001. (H) Mone induces ATF4 in a concentration-dependent manner. MEFs were incubated with increasing concentrations of Mone (0.1–10 μM) or the vehicle (D, DMSO) control and analyzed for induction of ATF4 and CSE.

ER stress can also induce CSE in MEFs (10, 12). Because of increased stimulation at concentrations from 5 to 10 μM. CSE is the cross-talk between ER and Golgi, we wondered whether also induced in response to monensin treatment (Fig. 1H). Golgi stress might influence CSE in a fashion similar to that elicited by ER stress. We utilized monensin, which impairs Golgi Activation of ATF4 by Monensin Involves PERK in MEFs. PERK is a function, to induce Golgi stress (13). Monensin is an ionophore well-characterized inhibitor of protein translation that acts by α that binds sodium and protons and, when incorporated into phosphorylating eIF2 to inhibit protein translation (15). Under cholesterol-rich membranes, elicits proton leakage from acidic conditions of global translational arrest, elicited by stress stimuli organelles including the Golgi apparatus (Fig. 1C). We also ex- such as ER stress, selected proteins, such as ATF4, undergo increased expression to enable cell survival (16, 17). Since ER amined induction of CSE in response to nigericin (Nig), another stress activates ATF4 via PERK (18), we wondered whether Golgi stressor (13), as well as brefeldin A (BFA) (14), which monensin similarly influences PERK. Accordingly, we compared interrupts trafficking between ER and Golgi. All three stressors actions of monensin and thapsigargin in efforts to contrast reg- markedly increase CSE levels comparable to the actions of D ulation of ATF4 and PERK. thapsigargin (TG), a classic stimulus to ER stress (Fig. 1 ). Both monensin and thapsigargin enhance levels of CSE, Induction of CSE by TG is mediated by the transcription ATF4, and phospho-PERK (Fig. 2A). While thapsigargin ro- factor ATF4 (10). Monensin also stimulates ATF4 levels to an bustly increases levels of binding Ig protein (BiP), which accu- E extent similar to thapsigargin (Fig. 1 ). Immunofluorescent ex- mulates in response to ER stress, monensin does not influence amination reveals enhancement of ATF4 by monensin selectively BiP levels. This finding indicates that the stress response of in the nuclei of MEFs (Fig. 1F). Both monensin and thapsigargin monensin differs from that induced by thapsigargin (19). We increase mRNA levels of ATF4, indicating that their inductive also demonstrated that Golgi stress differs in its properties influences are at the transcriptional level (Fig. 1G). The en- fromaminoaciddeprivation(Fig.2B). Glutamine deprivation, hancement of ATF4 by monensin is concentration-dependent like monensin, enhances ATF4 levels, but whereas monensin with a modest augmentation evident at 0.1 μM monensin and augments phospho-PERK, no such increase occurs with glutamine

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1717877115 Sbodio et al. Downloaded by guest on October 1, 2021 Fig. 2. Induction of ATF4 by monensin (Mone) involves the PERK pathway. (A) Mone promotes phosphorylation of PERK. Wild-type MEFs were incubated with either TG (1 μM) or Mone (5 μM) for 18 h, and lysates were analyzed by Western blotting using antibodies against CSE, ATF4, BiP, phospho-PERK, PERK, and actin (loading control). (B) Induction of ATF4 by Mone differs from amino acid deprivation and ER stress. Wild-type MEFs were cultured in full medium or − μ μ medium lacking glutamine ( Q) or medium containing DMSO (D, vehicle), Mone (5 M), or TG (1 M), and lysates were analyzed by Western blotting using NEUROSCIENCE + + − − antibodies against ATF4, BiP, phospho-PERK, PERK, and actin (loading control). (C) Induction of ATF4 by Mone requires PERK. Wild-type (PERK / ) and PERK / MEFs were treated with DMSO (D, vehicle), Mone (5 μM), or TG (1 μM) for 18 h, and lysates were analyzed by Western blotting using antibodies against ATF4, BiP, phospho-PERK, PERK, and actin (loading control). (D) Schematic representation and Western blot analysis of ATF4 and CSE induction by Mone. Treatment with Mone promotes phosphorylation of PERK, which phosphorylates the eukaryotic translation initiation factor 2α (eIF2α), resulting in up-regulation of ATF4 and consequent induction of CSE. Cells were treated with either DMSO (D) or 5 μM Mone for 18 h and analyzed by Western blotting.

depletion. To establish definitively that monensin acts via PERK, of ATF4 by monensin in striatal cells does not involve phos- we utilized PERK-deleted MEFs and analyzed the induction of phorylation of GCN2. ATF4. Both monensin and thapsigargin fail to augment ATF4 in We analyzed the response of ATF4 at various time points in PERK-deleted cell lines indicating that PERK phosphorylation is the Q111 striatal cells. Enhancement of ATF4 by monensin in involved in ATF4 up-regulation (Fig. 2C). Thus, monensin acts via Q111 cells is first evident at 4 h and increases progressively at the PERK pathway wherein phosphorylation of PERK leads to 8 and 16 h (Fig. 3C). Levels of CSE protein also increase in phosphorylation of eIF2α to promote up-regulation of ATF4 and its parallel with ATF4. The phosphorylation of PERK also in- downstream targets such as CSE (Fig. 2D). creases as a function of time (Fig. 3C). Next we examined concentration-response relationships at Monensin Induces ATF4 in Striatal Cell Lines. Previously we reported different time points for monensin’s actions on CSE, ATF4, and that induction of ATF4 is compromised in striatal HD cell lines phospho-PERK to determine the lowest concentration of monensin in response to low-cysteine conditions (10). We wondered that can induce ATF4 in Q111 cell lines (Fig. 3D). Only negligible whether monensin can induce ATF4 in HD cell lines. We ex- levels of these targets are elicited by 0.01 μM monensin, whereas amined induction of ATF4 by monensin in the striatal cell-line Q7/Q7 Q111/Q111 0.05 μM monensin produces robust enhancements evident at 3 d models STHdh (Q7) and STHdh (Q111) harboring with levels of CSE, ATF4, and phospho-PERK similar at 5 and 7 d 7 and 111 glutamine repeats, respectively. Immunofluorescence (Fig. 3D). We did not observe any cytotoxicity and/or adverse studies show that monensin can induce ATF4 in both Q7 and effects in these cell lines after prolonged monensin treatment Q111 striatal cell lines (Fig. 3A). To explore whether PERK’s influence upon ATF4 is altered in at these concentrations. Nuclear staining with DAPI revealed HD, we monitored phosphorylation of PERK in Q7 and intact nuclear morphology. In addition, the integrity of the Q111 striatal cell lines in response to thapsigargin or monensin Golgi apparatus under conditions of prolonged low-dose treatment. Phospho-PERK levels are similar in Q7 and Q111 cell monensin treatment was not significantly altered as assessed lines treated with these stressors. As in the MEFs, BiP is not by immunostaining with known the Golgi markers giantin and – E induced by monensin in Q7 or Q111 striatal cell lines, contrasted acyl-CoA binding containing domain 3 (ACBD3) (Fig. 3 ). with robust induction of BiP by thapsigargin in both Q7 and Since CSE is one of the biosynthetic enzymes for H2S, we also Q111 cell lines (Fig. 3B). measured the production of this gasotransmitter in Q7 and General control nonderepressible 2 (GCN2) is a kinase which, Q111 cell lines. As observed previously, the production of H2Sis like PERK, can mediate ATF4 expression via eIF2α phosphoryla- almost twofold lower in Q111 cell lines (Fig. S1) (7). H2S pro- tion (20). GCN2 undergoes autophosphorylation under conditions duction is increased in Q111 cell lines upon monensin treatment; of amino acid starvation so that accumulation of phospho-GCN2 however, treatment of Q111 cell lines with propargylglycine, an is an indicator of amino acid deprivation (20). While arginine inhibitor of CSE, prevents this increase (Fig. S1). These findings depletion promotes phosphorylation of GCN2, thapsigargin or indicate that monensin stimulates H2S production via the reverse monensin treatment fail to do so (Fig. 3B). Thus, the induction transsulfuration pathway.

Sbodio et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 transporter for cystine), Cat1 (cationic amino acid transporter 1), Lat1 (large amino acid transporter 1), and Cse (Fig. 4C). This response was not observed in Q111 cells pretreated with vehicle (DMSO). Unlike ER stress, the molecular markers of Golgi stress response have not been as well characterized. There have been reports of proteins that are up-regulated during Golgi stress response including ACBD3 (19). Accordingly, we monitored the induction of Acbd3 transcripts after monensin treatment, which was induced under these conditions (Fig. 4C). Immunofluores- cence analysis also revealed an increase of nuclear ATF4 upon monensin treatment. ACBD3 was used as a marker for Golgi (Fig. 4D). To determine whether the rescue of the ATF4 response by monensin would enhance cell viability in low-cysteine medium, we pretreated the Q7 and Q111 cells with either monensin or vehicle (DMSO), followed by cysteine deprivation. Under low- cysteine conditions without monensin, Q7 cells display a modest reduction in cell viability (Fig. 4 E and F). By contrast, for Q111 cells, viability in a low-cysteine medium is reduced by about 75%. However, pretreatment with monensin dramatically enhances cell viability of Q111 cells in low-cysteine medium to levels comparable to Q7 cells (Fig. 4 E and F). Thus, low concentrations of monensin prevent toxicity associated with cysteine deprivation in HD cells by up-regulating the reverse transsulfuration pathway via ATF4 and its targets including CSE (Fig. 4G). The protective effect of monensin is dependent on CSE expression, as MEFs lacking CSE fail to grow robustly under cysteine-deprived conditions even after monensin pretreatment, unlike the wild-type cells. The small increase in viability in the CSE-deleted cells may reflect actions of monensin on the cystine Fig. 3. Monensin (Mone) can induce ATF4 in a striatal cell culture model of transporter (xCT), which is also regulated by ATF4 (Fig. S2). Huntington’s disease. (A) Immunofluorescence analysis showing the in- These results show that full functionality of CSE is required for duction and nuclear localization of ATF4 in response to Mone treatment. growth under cysteine-deprived conditions. Q7 and Q111 cells were plated on coverslips and incubated with either μ DMSO or 5 M Mone for 18 h and the cells were fixed, permeabilized, and Discussion stained for ATF4 (green) and DNA (DAPI). (Magnification: 63×.) (B) Mone- mediated ATF4 induction is distinct from that induced by amino acid dep- The identity of the molecular components involved in the Golgi rivation or ER stress. Q7 and Q111 cells were cultured in full medium or stress response has been elusive. The principal finding of this medium lacking arginine (−R) or medium containing DMSO (D, vehicle), study is that monensin, a Golgi stress agent, signals via the Mone (5 μM), or TG (1 μM) for 18 h, and lysates were analyzed by Western PERK-ATF4 pathway and intersects with pathways responsible blotting using antibodies against ATF4, phospho-GCN2, GCN2, phospho- for maintenance of redox homeostasis. Stimulation of ATF4 by PERK, PERK, BiP, and actin (loading control). (C) Time course of induction of monensin occurs through PERK, as ATF4 is not induced by μ ATF4 in Q111 cells. Q111 cells were incubated in medium containing 5 M monensin in PERK knockout MEFs. A consequence of ATF4 Mone for various time points up to 16 h with a vehicle (D, DMSO) control at induction is the stimulation of the reverse transsulfuration the longest time point (16D) and analyzed for induction of ATF4, CSE, phosphor-PERK, PERK, and actin (loading control). (D) Determination of the pathway, which plays a central role in the maintenance of redox lowest concentration of Mone that can induce CSE in Q111 cells. Striatal homeostasis in cells. Low concentrations of monensin up- Q111 cells were incubated in medium containing low doses of Mone regulate CSE, a key enzyme of the reverse transsulfuration (0.01 μM and 0.05 μM) or DMSO for long periods of time. Lysates analyzed by pathway and the biosynthetic enzyme for cysteine, via ATF4, Western blotting using antibodies against CSE, ATF4, phospho-PERK, PERK, which regulates CSE during stress. This pathway is disrupted in and actin (loading control). (E) Prolonged treatment with Mone does not Huntington’s disease, as a result of which the HD cell lines are disrupt the Golgi apparatus. Striatal Q111 cells plated on coverslips were sensitive to cysteine deprivation. The cell death elicited by cys- μ incubated in the presence of Mone (0.05 M) or DMSO for up to 7 d. The cells teine starvation in these cell lines is prevented by monensin. were fixed, permeabilized, and stained for Giantin (green), ACBD3 (red), and These findings support the notion that enhancement of the reverse DNA (DAPI). (Magnification: 63×.) transsulfuration pathway as a whole may be therapeutic in HD. Earlier, we reported that treatment with cysteine and N-ace- Protective Effect of Monensin Against Cysteine Deprivation in HD Cell tylcysteine rescues abnormalities in HD cell lines and mice (7). Lines. We previously reported that cysteine deprivation is lethal However, supplementation with cysteine does not fully restore in HD cell lines due to depletion of CSE, while supplementation the functionality of CSE. The use of monensin to activate the with cysteine is beneficial (7, 10). While Q7 cells robustly up- reverse transsulfuration pathway provides a more complete rescue of ATF4 and its targets including CSE, the biosynthetic regulate ATF4 and CSE when grown in low-cysteine medium, enzyme for cysteine and H S. Q111 cells are highly compromised in their ability to respond to 2 HD is associated with elevated oxidative stress (21, 22), which low cysteine. The expression of ATF4 or CSE under these con- is responsible for the aberrant ATF4 response to cysteine deficits A B ditions is minimal (Fig. 4 and ). However, when Q111 cells (10). Low-cysteine levels due to CSE depletion contribute to are pretreated with monensin before growth in low-cysteine oxidative stress, as cysteine is a building block for the major medium, ATF4 induction is rescued (Fig. 4 A and B). This res- antioxidant glutathione and is itself an antioxidant. In addition, cue occurs at the transcriptional level, as monensin markedly cysteine is also the substrate for the generation of hydrogen sulfide, stimulates transcription of ATF4 and its targets, xCT (the which regulates cytoprotective pathways (Fig. 1A)(8,23–25). The

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Fig. 4. Monensin (Mone) can restore the expression of ATF4 and prevent cell death of HD cells in low-cysteine medium. (A) Striatal cells Q7 and Q111 were pretreated with DMSO (vehicle) or a low concentration of Mone (0.05 μM) for 18 h. After this preincubation, cells were cultured in regular or low-cysteine medium for 24 h containing either DMSO or Mone (0.05 μM). Cells were analyzed by Western blotting using antibodies against ATF4 and CSE levels. (B) Quantitation for ATF4 expression: n = 4 (means ± SE); *P < 0.05. (C) Restoration of ATF4 and its targets (Cse, xCT, Cat1, Lat1) by Mone in low-cysteine medium occurs at the transcriptional level. Striatal Q7 and Q111 cells were treated as in A. Transcript levels of Atf4, Cse, xCT, Cat1, and Lat1 were monitored by real-time qPCR. Acbd3, a known target of Mone, was used as a control. n = 3 (means ± SD); ***P < 0.001. (D) Nuclear localization of ATF4 induced by Mone during cysteine deprivation. Striatal cells Q7 and Q111 were plated on coverslips, treated as in A, and analyzed by immunofluorescence using antibodies against ATF4 (green) and ACBD3 (red); DAPI (blue) was used to stain DNA. (Magnification: 63×.) (E and F) Viability of Q111 cells in low-cysteine medium is restored by Mone treatment. Q7 and Q111 striatal cells were treated as in A, and viability was monitored by light microscopy (magnification: 10×)(E) and quantitated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. n = 3 (means ± SD); ***P < 0.001 (F). (G) Model for the protective function of mild Golgi stress in Q111 striatal cells. Treatment of striatal HD cells with Mone up-regulates ATF4 via the PERK pathway, leading to increased expression of CSE and cysteine synthesis via the reverse transsulfuration pathway. Moreover, the cystine transporters (xCT) may also be up-regu-

lated by ATF4, in addition to other targets. This would lead to an increase in both cysteine and H2S levels, promoting growth in cysteine-deprived conditions.

increased oxidative stress compromises the response of the stress- The use of monensin as a stimulant of the reverse trans- sensitive transcription factor ATF4, which also includes up- sulfuration pathway has implications for other disorders in- regulation of CSE. This abnormality culminates in a vicious cycle volving redox imbalance that may also display a suboptimal that mediates cell death. Mitigating oxidative stress in HD cell lines ATF4 response. For example, Alzheimer’s disease is associated reverses this abnormality (10). In the present study we have shown with generalized oxidative stress as well as specific deficits in that low concentrations of monensin correct these deficits by re- levels of H2S (26). HIV patients have depleted levels of CSE as storing ATF4 function in response to cysteine deprivation via the well as elevated oxidative stress (27). Interestingly, N-acetylcys- PERK pathway. teine (NAC) is employed in treating the cachexia of HIV

Sbodio et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 infection (28). Oxidative stress has been linked to an increase in amino acid homeostasis, which would normally be up-regulated muscle fatigue, which is relieved by NAC treatment (29). There under this stress, are impaired in HD cell lines. Thus, novel has been very little exploration of CSE levels in association with molecules that can stimulate the reverse transsulfuration path- various disease states. Recently, CSE has been reported to be way may have adaptive benefits in HD as well as under diverse depleted in spinocerebellar-degeneration patients (30). In addi- conditions involving altered oxidative stress. Although low con- tion, glutathione and cysteine levels decline with normal aging centrations of monensin afforded protective effects in cell-line (31, 32) and in cancers (33). Mice in which the neuronal cysteine models of HD, their effects in mouse models of HD and the transporter EAAC1 has been deleted undergo age-dependent minimal effective dose required to mitigate symptoms remain to neurodegeneration (34). Thus, dietary supplementation with be evaluated. In summary, we utilized a cell-line model of HD to cysteine or cysteine-rich foods has proved beneficial in several establish that stimulating the reverse transsulfuration pathway neurodegenerative diseases involving oxidative stress. For ex- protects cells. Low-grade Golgi stress induced by monensin ample, intake of whey protein, which has high levels of cysteine, preconditions cells to tolerate cysteine deficiency associated has been linked to decreased oxidative stress, increased gluta- with Huntington’s disease. These findings may be relevant to thione content, and improved antioxidant status in patients with other neurodegenerative diseases associated with deficits in ’ Parkinson s disease (35). Supplementation with NAC, a more cysteine metabolism. stable cysteine derivative, has also proved beneficial in elderly subjects, eliciting improved motor function (36, 37). Materials and Methods The reverse transsulfuration pathway is a central hub in redox Cell Lines. MEFs, STHdhQ7/Q7 (Q7), and STHdhQ111/Q111 (Q111) (a gift from control so that stimulating this pathway may impart therapeutic Marcy MacDonald, Harvard Medical School, Boston) were maintained in benefits in diverse disorders. Our findings reveal that low-grade normal DMEM containing 10% FBS, 5 mM glutamine, and antibiotics. Golgi stress, which does not elicit toxicity, can up-regulate cytoprotective defense mechanisms and may prime or pre- Monensin Rescue. Cells were plated on 6-cm plates at 50% confluency. The condition cells to withstand additional insults (Fig. 4 E–G). In following day, monensin or DMSO were added to 0.05 μM concentration. HD cell lines, ATF4 up-regulation in response to cysteine After 18 h, cells were rinsed with PBS, and either regular medium or low- μ deficits is halted due to high-oxidative-stress levels (10). Sup- cysteine medium with either DMSO or monensin (0.05 M) was added. Cells plementing diets with NAC compensates for low levels of cys- were harvested and analyzed after 18 h. Additional details of reagents and methods are available in SI Materials and Methods. teine and reduces oxidative stress, but does not restore the full functionality of CSE with respect to its enzymatic activity in ACKNOWLEDGMENTS. This work was supported by grants from the US Pub- generating H2S, cysteine, and other thiol products such as cys- lic Health Service (MH18501) (to S.H.S.) and the CHDI Foundation (to S.H.S. teamine and CoA. Moreover, ATF4 signaling and regulation of and B.D.P.).

1. Machamer CE (2015) The Golgi complex in stress and death. Front Neurosci 9:421. 20. Harding HP, et al. (2000) Regulated translation initiation controls stress-induced gene 2. Sasaki K, Yoshida H (2015) Organelle autoregulation-stress responses in the ER, Golgi, expression in mammalian cells. Mol Cell 6:1099–1108. mitochondria and lysosome. J Biochem 157:185–195. 21. Browne SE, Beal MF (2006) Oxidative damage in Huntington’s disease pathogenesis. 3. Nakagomi S, et al. (2008) A Golgi fragmentation pathway in neurodegeneration. Antioxid Redox Signal 8:2061–2073. Neurobiol Dis 29:221–231. 22. Ayala-Peña S (2013) Role of oxidative DNA damage in mitochondrial dysfunction and 4. Gonatas NK, Gonatas JO, Stieber A (1998) The involvement of the Golgi apparatus in Huntington’s disease pathogenesis. Free Radic Biol Med 62:102–110. the pathogenesis of amyotrophic lateral sclerosis, Alzheimer’s disease, and ricin in- 23. Paul BD, Snyder SH (2012) H2S signalling through protein sulfhydration and beyond. – toxication. Histochem Cell Biol 109:591–600. Nat Rev Mol Cell Biol 13:499 507. 5. Sbodio JI, Paul BD, Machamer CE, Snyder SH (2013) Golgi protein ACBD3 mediates 24. Paul BD, Snyder SH (2015) Modes of physiologic H2S signaling in the brain and pe- – neurotoxicity associated with Huntington’s disease. Cell Rep 4:890–897. ripheral tissues. Antioxid Redox Signal 22:411 423. – 6. Anonymous; The Huntington’s Disease Collaborative Research Group (1993) A novel 25. Paul BD, Snyder SH (2015) Protein sulfhydration. Methods Enzymol 555:79 90. gene containing a trinucleotide repeat that is expanded and unstable on Hunting- 26. Liu XQ, Liu XQ, Jiang P, Huang H, Yan Y (2008) [Plasma levels of endogenous hydrogen sulfide and homocysteine in patients with Alzheimer’s disease and vascular ton’s disease chromosomes. Cell 72:971–983. dementia and the significance thereof]. Zhonghua Yi Xue Za Zhi 88:2246–2249. Chinese. 7. Paul BD, et al. (2014) Cystathionine γ-lyase deficiency mediates neurodegeneration in 27. Martin JA, Sastre J, de la Asunción JG, Pallardó FV, Viña J (2001) Hepatic gamma- Huntington’s disease. Nature 509:96–100. cystathionase deficiency in patients with AIDS. JAMA 285:1444–1445. 8. Paul BD, Snyder SH (2015) H S: A novel gasotransmitter that signals by sulfhydration. 2 28. Roederer M, Ela SW, Staal FJ, Herzenberg LA, Herzenberg LA (1992) N-acetylcysteine: Trends Biochem Sci 40:687–700. A new approach to anti-HIV therapy. AIDS Res Hum Retroviruses 8:209–217. 9. Paul BD, Snyder SH (2014) Neurodegeneration in Huntington’s disease involves loss of 29. Reid MB, Stokic DS, Koch SM, Khawli FA, Leis AA (1994) N-acetylcysteine inhibits cystathionine γ-lyase. Cell Cycle 13:2491–2493. muscle fatigue in humans. J Clin Invest 94:2468–2474. 10. Sbodio JI, Snyder SH, Paul BD (2016) Transcriptional control of amino acid homeo- 30. Snijder PM, et al. (2015) Overexpression of cystathionine γ-lyase suppresses detri- stasis is disrupted in Huntington’s disease. Proc Natl Acad Sci USA 113:8843–8848. mental effects of spinocerebellar ataxia type 3. Mol Med 21:758–768. 11. Sen N, et al. (2012) Hydrogen sulfide-linked sulfhydration of NF-κB mediates its an- 31. Chen TS, Richie JP, Jr, Lang CA (1989) The effect of aging on glutathione and cysteine – tiapoptotic actions. Mol Cell 45:13 24. levels in different regions of the mouse brain. Proc Soc Exp Biol Med 190:399–402. 12. Dickhout JG, et al. (2012) Integrated stress response modulates cellular redox state via 32. Dröge W (2005) Oxidative stress and ageing: Is ageing a cysteine deficiency syn- γ induction of cystathionine -lyase: Cross-talk between integrated stress response and drome? Philos Trans R Soc Lond B Biol Sci 360:2355–2372. – thiol metabolism. J Biol Chem 287:7603 7614. 33. Hack V, et al. (1997) Cystine levels, cystine flux, and protein catabolism in cancer – 13. Dinter A, Berger EG (1998) Golgi-disturbing agents. Histochem Cell Biol 109:571 590. cachexia, HIV/SIV infection, and senescence. FASEB J 11:84–92. – 14. Pelham HR (1991) Multiple targets for brefeldin A. Cell 67:449 451. 34. Aoyama K, et al. (2006) Neuronal glutathione deficiency and age-dependent neu- 15. Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an rodegeneration in the EAAC1 deficient mouse. Nat Neurosci 9:119–126. endoplasmic-reticulum-resident kinase. Nature 397:271–274. 35. Tosukhowong P, et al. (2016) Biochemical and clinical effects of Whey protein sup- 16. Vattem KM, Wek RC (2004) Reinitiation involving upstream ORFs regulates plementation in Parkinson’s disease: A pilot study. J Neurol Sci 367:162–170. ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA 101:11269–11274. 36. Hauer K, et al. (2003) Improvement in muscular performance and decrease in tumor 17. Rutkowski DT, Kaufman RJ (2003) All roads lead to ATF4. Dev Cell 4:442–444. necrosis factor level in old age after antioxidant treatment. J Mol Med (Berl) 81: 18. Harding HP, et al. (2003) An integrated stress response regulates amino acid me- 118–125. tabolism and resistance to oxidative stress. Mol Cell 11:619–633. 37. McCarty MF, DiNicolantonio JJ (2015) An increased need for dietary cysteine in sup- 19. Oku M, et al. (2011) Novel cis-acting element GASE regulates transcriptional induction port of glutathione synthesis may underlie the increased risk for mortality associated by the Golgi stress response. Cell Struct Funct 36:1–12. with low protein intake in the elderly. Age (Dordr) 37:96.

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