Ubiquitin C-terminal hydrolase L1 (UCHL1) is a multifunctional brain protein that has been implicated in Parkinson's and Alzheimer's Diseases. Reactive lipid species including cyclopentenone prostaglandins (CyPgs) are produced in ischemic brain and covalently adduct the 152 cysteine (C152) of UCHL1, significantly alter the 3D structure of the enzyme, inhibit hydrolase activity and induce aggregation of the protein. UCHL1 hydrolase activity protects neurons against hypoxic injury in vitro. In new preliminary data, we have found that a cysteine 152 to alanine mutation in UCHL1 (UCHL1 C152A) is resistant to binding to CyPgs and protects neurons from CyPg toxicity, anoxia and oxygen-glucose deprivation (OGD). Neurites of primary neurons are very sensitive to CyPg injury, and the UCHL1 C152A-derived neurites are resistant to CyPg induced fragmentation compared to wild type neurites. Mutant UCHL1 C152A mice have smaller infarctions and improved short term motor outcome after middle cerebral artery occlusion (MCAO) compared to wild type mice. Based on these data, we hypothesize that CyPgs and other reactive lipid species bind to C152 of UCHL1 and inactivate the enzyme, exacerbate injury, and limit recovery of function after ischemic injury. The following aims will be addressed: 1. Test the hypothesis that the UCHL1 C152A mutation protects primary cultured neurons from hypoxia/ischemia and CyPgs in vitro. 2. Determine how the UCHL1 C152A mutation alters the function of the UPP, autophagy, ER stress, and ubiquitination of target proteins after OGD, hypoxia and CyPg treatment in primary neurons. 3. Test the hypothesis that the UCHL1 C152A mutation increases survival of both gray and white matter and improves behavioral outcome after middle cerebral artery occlusion in mice. Methodology: In vitro methods include cell viability/cell death assays, neurite and axon outgrowth quantification, Western blotting, protein ubiquitination, proteomic analysis; in vivo methods include MCAO in mice, long term behavioral outcome, quantification of gray, white matter, myelin, axons and synapses, Western blotting,UCHL1 activity measurement and comparison of electrophysiological function. The proposed studies will address a novel mechanism by which the brain recovers from cerebral ischemia and may suggest new therapeutic strategies that may improve long term motor and cognitive function after stroke.

Public Health Relevance

Thrombolysis with tissue plasminogen activating factor is the only FDA-approved therapy for acute ischemic stroke, but <10% of patients with ischemic stroke benefit from this treatment. The current project aims to identify mechanisms by which brain cells repair damage after stroke and augment cognitive function. These results could lead to new treatments for stroke that could be given hours or days after stroke and improve functional outcome.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS037459-17
Application #
9415109
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Bosetti, Francesca
Project Start
1999-06-01
Project End
2019-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
17
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Neurology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Liu, Hao; Rose, Marie E; Ma, Xiecheng et al. (2017) In vivo transduction of neurons with TAT-UCH-L1 protects brain against controlled cortical impact injury. PLoS One 12:e0178049
Graham, Steven H; Liu, Hao (2017) Life and death in the trash heap: The ubiquitin proteasome pathway and UCHL1 in brain aging, neurodegenerative disease and cerebral Ischemia. Ageing Res Rev 34:30-38
Graham, Steven H (2016) Modification of ubiquitin C-terminal hydrolase L1 by reactive lipid species: role in neural regeneration and diseases of aging. Neural Regen Res 11:908-9
Liu, H; Li, W; Rose, M E et al. (2015) The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury. Cell Death Dis 6:e1966
Liu, Hao; Chen, Jie; Li, Wenjin et al. (2015) Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury. FEBS J 282:2045-59
Ahmad, Muzamil; Dar, Nawab J; Bhat, Zubair S et al. (2014) Inflammation in ischemic stroke: mechanisms, consequences and possible drug targets. CNS Neurol Disord Drug Targets 13:1378-96
Shaik, Jafar Sadik B; Miller, Tricia M; Graham, Steven H et al. (2014) Rapid and simultaneous quantitation of prostanoids by UPLC-MS/MS in rat brain. J Chromatogr B Analyt Technol Biomed Life Sci 945-946:207-16
Liu, Hao; Rose, Marie E; Miller, Tricia M et al. (2013) COX2-derived primary and cyclopentenone prostaglandins are increased after asphyxial cardiac arrest. Brain Res 1519:71-7
Liu, Hao; Li, Wenjin; Ahmad, Muzamil et al. (2013) Increased generation of cyclopentenone prostaglandins after brain ischemia and their role in aggregation of ubiquitinated proteins in neurons. Neurotox Res 24:191-204
Liu, Hao; Li, Wenjin; Rose, Marie E et al. (2013) Prostaglandin D2 toxicity in primary neurons is mediated through its bioactive cyclopentenone metabolites. Neurotoxicology 39:35-44

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