Role of CyPgs UCHL1 in Ischemic Injury and Recovery

 描述(申请人提供)：泛素C末端水解酶L1(UCHL1)是一种多功能大脑蛋白，与帕金森氏症和阿尔茨海默病有关。包括环戊烯酮前列腺素(CyPgs)在内的活性脂类在缺血脑中产生，并与UCHL1的152个半胱氨酸(C152)共价加合，显著改变酶的三维结构，抑制水解酶活性，诱导蛋白质聚集。UCHL1水解酶活性在体外保护神经元免受缺氧性损伤。在新的初步数据中，我们发现UCHL1中半胱氨酸152到丙氨酸的突变(UCHL1C152a)对CyPg结合具有抵抗力，并能保护神经元免受CyPg毒性、缺氧和缺氧-葡萄糖剥夺(OGD)的影响。原代神经元的突起对CyPg损伤非常敏感，与野生型突起相比，UCHL1C152a来源的突起对CyPg诱导的断裂具有抵抗力。与野生型小鼠相比，突变的UCHL1 C152A小鼠在大脑中动脉闭塞(MCAO)后有较小的梗塞和改善的短期运动结果。基于这些数据，我们假设CyPgs和其他活性脂质物种与UCHL1的C152结合，使该酶失活，加重损伤，限制缺血损伤后功能的恢复。主要研究内容如下：1.验证UCHL1 C152A突变对原代培养的神经元具有保护作用的假设。2.确定UCHL1 C152a突变如何改变原代神经元在OGD、缺氧和CyPg处理后的UPP功能、自噬、内质网应激以及靶蛋白的泛素化。3.验证UCHL1 C152A突变可增加小鼠大脑中动脉闭塞后灰质和白质的存活率并改善行为结局的假说。方法：体外方法包括细胞存活/细胞死亡分析，轴突和轴突生长定量，Western blotting，蛋白质泛素化，蛋白质组学分析；体内方法包括小鼠MCAO，长期行为结局，灰质、白质、髓鞘、轴突和突触的定量，Western blotting，UCHL1活性测量和电生理功能比较。拟议的研究将解决大脑从脑缺血中恢复的新机制，并可能提出新的治疗策略，可能会改善中风后的长期运动和认知功能。

 DESCRIPTION (provided by applicant): 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.