Role of CyPgs UCHL1 in Ischemic Injury and Recovery

CyPgs UCHL1 在缺血性损伤和恢复中的作用

• 批准号: 9229586
• 负责人: STEVEN H GRAHAM
• 金额: $ 38.74万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229586
• 关键词: Acute; Address; Alanine; Alzheimer' s Disease; Anoxia; Autophagocytosis; Axon; Axonal Transport; Binding; Biological Assay; Brain; Brain Hypoxia-Ischemia; Cell Death; Cell Survival; Cerebral Ischemia; Cysteine; Cytoskeleton; Data; Dopamine D2 Receptor; Electrophysiology (science); Enzymes; FDA approved; Functional disorder; Funding; Glucose; Gray unit of radiation dose; Hippocampus (Brain); Hour; Human; Hydrolase; Hypoxia; In Vitro; Infarction; Injury; Ischemia; Ischemic Stroke; Knock-in Mouse; Lead; Lipids; Long-Term Potentiation; Measurement; Memory; Methodology; Methods; Middle Cerebral Artery Occlusion; Motor; Mus; Mutation; Myelin; Neocortex; Neurites; Neurons; Outcome; Oxygen; Parkinson Disease; Pathologic; Pathway interactions; Patients; Plasminogen; Play; Property; Prostaglandin D2; Prostaglandins; Proteins; Proteomics; Pyramidal Cells; Recovery; Recovery of Function; Resistance; Role; Site; Slice; Stress; Stroke; Synapses; Testing; Thalamic structure; Tissues; Toxic effect; Translating; Ubiquitin; Ubiquitination; Western Blotting; Wild Type Mouse; adduct; base; behavioral outcome; brain cell; cognitive function; cyclopentenone; deprivation; effective therapy; enzyme structure; excitotoxicity; fetal; functional outcomes; gray matter; improved; in vivo; multicatalytic endopeptidase complex; mutant; neuron loss; novel; novel strategies; novel therapeutic intervention; prevent; protein aggregation; public health relevance; repaired; selective expression; stroke treatment; synaptic function; three dimensional structure; thrombolysis; ubiquitin C-terminal hydrolase; white matter

 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.

 描述（由申请人提供）：泛素 C 末端水解酶 L1 (UCHL1) 是一种多功能脑蛋白，与帕金森病和阿尔茨海默病有关。包括环戊烯酮前列腺素 (CyPgs) 在内的反应性脂质在缺血性脑中产生，并与 UCHL1 的 152 半胱氨酸 (C152) 共价加合，显着改变酶的 3D 结构，抑制水解酶活性并诱导蛋白质聚集。 UCHL1 水解酶活性可在体外保护神经元免受缺氧损伤。在新的初步数据中，我们发现 UCHL1 (UCHL1 C152A) 中的半胱氨酸 152 到丙氨酸的突变能够抵抗与 CyPg 的结合，并保护神经元免受 CyPg 毒性、缺氧和氧葡萄糖剥夺 (OGD) 的影响。原代神经元的神经突对 CyPg 损伤非常敏感，并且与野生型神经突相比，UCHL1 C152A 衍生的神经突对 CyPg 诱导的断裂具有抵抗力。与野生型小鼠相比，突变型 UCHL1 C152A 小鼠的梗死面积更小，并且大脑中动脉闭塞 (MCAO) 后的短期运动结果得到改善。基于这些数据，我们假设 CyPgs 和其他活性脂质与 UCHL1 的 C152 结合并使酶失活，加剧损伤并限制缺血性损伤后功能的恢复。将解决以下目标： 1. 测试 UCHL1 C152A 突变在体外保护原代培养神经元免受缺氧/缺血和 CyPgs 影响的假设。 2. 确定 UCHL1 C152A 突变如何改变原代神经元 OGD、缺氧和 CyPg 处理后的 UPP、自噬、ER 应激和靶蛋白泛素化的功能。 3. 测试以下假设：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果。方法：体外方法包括细胞活力/细胞死亡测定、神经突和轴突生长定量、蛋白质印迹、蛋白质泛素化、蛋白质组分析；体内方法包括小鼠 MCAO、长期行为结果、灰质、白质、髓磷脂、轴突和突触的量化、蛋白质印迹、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，以改善中风后的长期运动和认知功能。