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

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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 dis- orders 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 2, 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.
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