Oxidative stress aggravates brain injury following ischemia/reperfusion (I/R). We previously showed that ubiquilin-1 (Ubqln1), a ubiquitin-like protein, improves proteostasis and protects brains against oxidative stress and I/R-induced brain injury. Here, we demonstrate that a small molecule compound, L-2-oxothiazolidine-4-carboxylic acid (OTC) that functions as a precursor of cysteine, upregulated Ubqln1 and protected cells against oxygen-glucose deprivation–induced cell death in neuronal cultures. Further, the administration of OTC either at 1 h prior to ischemia or 3 h after the reperfusion significantly reduced brain infarct injury and improved behavioral outcomes in a stroke model. Administration of OTC also increased glutathione (GSH) level and decreased superoxide production, oxidized protein, and neuroinflammation levels in the penumbral cortex after I/R in the stroke mice. Furthermore, I/R reduced both Ubqln1 and the glutathione S-transferase protein levels, whereas OTC treatment restored both protein levels, which was associated with reduced ubiquitin-conjugated protein level. Interestingly, in the Ubqln1 knockout (KO) mice, OTC treatment showed reduced neuroprotection and increased ubiquitin-conjugated protein level when compared to the similarly treated non-KO mice following I/R, suggesting that OTC-medicated neuroprotection is, at least partially, Ubqln1-dependent. Thus, OTC is a potential therapeutic agent for stroke and possibly for other neurological disorders and its neuroprotection involves enhanced proteostasis.
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Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet. 2008;371(9624):1612–23.
Kasner SE, Grotta JC. Ischemic stroke. Neurol Clin. 1998;16(2):355–72.
Barber PA, Zhang J, Demchuk AM, Hill MD, Buchan AM. Why are stroke patients excluded from TPA therapy? An analysis of patient eligibility. Neurology. 2001;56(8):1015–20.
Cronin CA. Intravenous tissue plasminogen activator for stroke: a review of the ECASS III results in relation to prior clinical trials. J Emerg Med. 2010;38(1):99–105.
Armstead WM, Ganguly K, Kiessling JW, Riley J, Chen XH, Smith DH, et al. Signaling, delivery and age as emerging issues in the benefit/risk ratio outcome of tPA for treatment of CNS ischemic disorders. J Neurochem. 2010;113(2):303–12.
Shi HL, Liu KJ. Cerebral tissue oxygenation and oxidative brain injury during ischemia and reperfusion. Front Biosci-Landmrk. 2007;12:1318–28.
Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Asp Med. 2009;30(1–2):1–12.
Mari M, Morales A, Colell A, Garcia-Ruiz C, Fernandez-Checa JC. Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal. 2009;11(11):2685–700.
Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta. 2016;1863(12):2977–92.
Schulz JB, Lindenau J, Seyfried J, Dichgans J. Glutathione, oxidative stress and neurodegeneration. Eur J Biochem. 2000;267(16):4904–11.
Choi J, Liu RM, Kundu RK, Sangiorgi F, Wu W, Maxson R, et al. Molecular mechanism of decreased glutathione content in human immunodeficiency virus type 1 Tat-transgenic mice. J Biol Chem. 2000;275(5):3693–8.
Namba K, Takeda Y, Sunami K, Hirakawa M. Temporal profiles of the levels of endogenous antioxidants after four-vessel occlusion in rats. J Neurosurg Anesthesiol. 2001;13(2):131–7.
Park EM, Choi JH, Park JS, Han MY, Park YM. Measurement of glutathione oxidation and 8-hydroxy-2′-deoxyguanosine accumulation in the gerbil hippocampus following global ischemia. Brain Res Protocol. 2000;6(1–2):25–32.
Yabuki Y, Fukunaga K. Oral administration of glutathione improves memory deficits following transient brain ischemia by reducing brain oxidative stress. Neuroscience. 2013;250:394–407.
Smeyne M, Smeyne RJ. Glutathione metabolism and Parkinson’s disease. Free Radic Biol Med. 2013;62:13–25.
Ko HS, Uehara T, Tsuruma K, Nomura Y. Ubiquilin interacts with ubiquitylated proteins and proteasome through its ubiquitin-associated and ubiquitin-like domains. FEBS Lett. 2004;566(1–3):110–4.
Wang H, Monteiro MJ. Ubiquilin interacts and enhances the degradation of expanded-polyglutamine proteins. Biochem Biophys Res Commun. 2007;360(2):423–7.
Stieren ES, El Ayadi A, Xiao Y, Siller E, Landsverk ML, Oberhauser AF, et al. Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein. J Biol Chem. 2011;286(41):35689–98.
Itakura E, Zavodszky E, Shao S, Wohlever ML, Keenan RJ, Hegde RS. Ubiquilins chaperone and triage mitochondrial membrane proteins for degradation. Mol Cell. 2016;63(1):21–33.
Rothenberg C, Srinivasan D, Mah L, Kaushik S, Peterhoff CM, Ugolino J, et al. Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy. Hum Mol Genet. 2010;19(16):3219–32.
Lee DY, Arnott D, Brown EJ. Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery. EMBO Rep. 2013;14(4):373–81.
Liu Y, Lu L, Hettinger CL, Dong G, Zhang D, Rezvani K, et al. Ubiquilin-1 protects cells from oxidative stress and ischemic stroke caused tissue injury in mice. J Neurosci. 2014;34(8):2813–21.
Oppenheimer S. GM1 ganglioside therapy in acute ischemic stroke. Stroke. 1990;21(5):825.
Liu Z, Ruan Y, Yue W, Zhu Z, Hartmann T, Beyreuther K, et al. GM1 up-regulates Ubiquilin 1 expression in human neuroblastoma cells and rat cortical neurons. Neurosci Lett. 2006;407(1):59–63.
Ahmad A, Khan MM, Javed H, Raza SS, Ishrat T, Khan MB, et al. Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat. Mol Cell Biochem. 2012;367(1–2):215–25.
Liu Y, Qiao F, Wang H. Enhanced proteostasis in post-ischemic stroke mouse brains by ubiquilin-1 promotes functional recovery. Cell Mol Neurobiol. 2016;37(7):1325–9.
Lu L, Wang H. Transient focal cerebral ischemia upregulates immunoproteasomal subunits. Cell Mol Neurobiol. 2012;32(6):965–70.
Min JW, Lu L, Freeling JL, Martin DS, Wang H: USP14 inhibitor attenuates cerebral ischemia/reperfusion-induced neuronal injury in mice. J Neurochem 2017, 2017,140(5):826–833.
Chen JL, Zhang CL, Jiang H, Li Y, Zhang LJ, Robin A, et al. Atorvastatin induction of VEGF and BDNF promotes brain plasticity after stroke in mice. J Cerebr Blood F Met. 2005;25(2):281–90.
Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74(1):214–26.
Gilliam LAA, Lark DS, Reese LR, Torres MJ, Ryan TE, Lin CT, et al. Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction. Am J Physiol-Endoc M. 2016;311(2):E293–301.
Liu F, Schafer DP, McCullough LD. TTC, fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion. J Neurosci Methods. 2009;179(1):1–8.
Chen JL, Sanberg PR, Li Y, Wang L, Lu M, Willing AE, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke. 2001;32(11):2682–8.
Terrill JR, Boyatzis A, Grounds MD, Arthur PG. Treatment with the cysteine precursor l-2-oxothiazolidine-4-carboxylate (OTC) implicates taurine deficiency in severity of dystropathology in mdx mice. Int J Biochem Cell Biol. 2013;45(9):2097–108.
Bolling AK, Solhaug A, Morisbak E, Holme JA, Samuelsen JT. The dental monomer hydroxyethyl methacrylate (HEMA) counteracts lipopolysaccharide-induced IL-1beta release-possible role of glutathione. Toxicol Lett. 2017;270:25–33.
Owen JB, Butterfield DA. Measurement of oxidized/reduced glutathione ratio. Methods Mol Biol. 2010;648:269–77.
Chevion M, Berenshtein E, Stadtman ER. Human studies related to protein oxidation: protein carbonyl content as a marker of damage. Free Radic Res. 2000;33(Suppl):S99–108.
Grice GL, Nathan JA. The recognition of ubiquitinated proteins by the proteasome. Cell Mol Life Sci. 2016;73(18):3497–506.
Sommer CJ. Ischemic stroke: experimental models and reality. Acta Neuropathol. 2017;133(2):245–61.
Yang GY, Betz AL. Reperfusion-induced injury to the blood-brain barrier after middle cerebral artery occlusion in rats. Stroke. 1994;25(8):1658–64 discussion 1664-1655.
Yu X, Long YC. Crosstalk between cystine and glutathione is critical for the regulation of amino acid signaling pathways and ferroptosis. Sci Rep. 2016;6:30033.
Ghosh S, Das N, Mandal AK, Dungdung SR, Sarkar S. Mannosylated liposomal cytidine 5′ diphosphocholine prevent age related global moderate cerebral ischemia reperfusion induced mitochondrial cytochrome C release in aged rat brain. Neuroscience. 2010;171(4):1287–99.
Ansari MA, Ahmad AS, Ahmad M, Salim S, Youscuf S, Ishrat T, et al. Selenium protects cerebral ischemia in rat brain mitochondria. Biol Trace Elem Res. 2004;101(1):73–86.
Ansari MA, Joshi G, Huang QZ, Opii WO, Abdul HM, Sultana R, et al. In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: relevance to Alzheimer’s disease and other oxidative stress-related neurodegenerative disorders. Free Radical Bio Med. 2006;41(11):1694–703.
Xie CS, Lovell MA, Markesbery WR. Glutathione transferase protects neuronal cultures against four hydroxynonenal toxicity. Free Radical Bio Med. 1998;25(8):979–88.
Yang Y, Wang JY, Li Y, Fan CX, Jiang S, Zhao L, et al. HO-1 signaling activation by pterostilbene treatment attenuates mitochondrial oxidative damage induced by cerebral ischemia reperfusion injury. Mol Neurobiol. 2016;53(4):2339–53.
Ge P, Luo Y, Liu CL, Hu B. Protein aggregation and proteasome dysfunction after brain ischemia. Stroke. 2007;38(12):3230–6.
Luo T, Park Y, Sun X, Liu C, Hu B. Protein misfolding, aggregation, and autophagy after brain ischemia. Transl Stroke Res. 2013;4(6):581–8.
We would like to thank the Physiology Core Facility at the University of South Dakota (USD) Division of Basic Biomedical Sciences for access to equipment and assistance with data analysis, Mr. Doug Jennewein from the USD-IT Research Computing for help in the database installation and servers operation, and Mrs. Daniela Paez from the USD proteomics Core Facility for assistance in processing and data organization.
New Author Contribution Statement
FQ and EM contributed to some in vitro studies. EC contributed to mass spectrometric analysis of OTC-interacting proteins.
This study was funded by the National Institute of Neurological Disorders and Stroke under research grant NS088084 and by the National Institute of General Medical Sciences under the grant P20GM103443.
Conflict of Interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed in the study. This article does not contain any studies with human participants performed by any of the authors.
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Liu, Y., Min, J., Feng, S. et al. Therapeutic Role of a Cysteine Precursor, OTC, in Ischemic Stroke Is Mediated by Improved Proteostasis in Mice. Transl. Stroke Res. 11, 147–160 (2020). https://doi.org/10.1007/s12975-019-00707-w
- Oxidative stress
- Cysteine precursor