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: 9415109
• 负责人: STEVEN H GRAHAM
• 金额: $ 38.46万
• 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 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9415109
• 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（UCHL 1）是一种多功能脑蛋白，与帕金森病和阿尔茨海默病有关。缺血脑组织中产生的活性脂质包括环戊烯酮类胡萝卜素（CyPgs），它们与UCHL 1的152位半胱氨酸（C152）共价结合，显著改变酶的三维结构，抑制水解酶活性并诱导蛋白质聚集。UCHL 1水解酶活性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UCHL 1（UCHL 1 C152 A）中的半胱氨酸152到丙氨酸突变对CyPgs的结合具有抗性，并保护神经元免受CyPg毒性，缺氧和氧葡萄糖剥夺（OGD）。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，UCHL 1 C152 A衍生的神经突对CyPg诱导的断裂具有抗性。与野生型小鼠相比，突变UCHL 1 C152 A小鼠在大脑中动脉闭塞（MCAO）后具有较小的梗死和改善的短期运动结果。基于这些数据，我们假设CyPgs和其他活性脂质物质与UCHL 1的C152结合并抑制酶，加重损伤，并限制缺血性损伤后功能的恢复。以下目标将被解决：1。测试UCHL 1 C152 A突变在体外保护原代培养的神经元免受缺氧/缺血和CyPgs的假设。 2.确定UCHL 1 C152 A突变如何改变原代神经元中OGD、缺氧和CyPg处理后UPP、自噬、ER应激和靶蛋白泛素化的功能。 3.检验UCHL 1 C152 A突变增加小鼠大脑中动脉闭塞后灰质和白色物质的存活率并改善行为结果的假设。方法学：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、蛋白质组学分析;体内方法包括小鼠中的MCAO、长期行为结果、灰质、白色物质、髓鞘、轴突和突触的定量、蛋白质印迹、UCHL 1活性测量和电生理功能的比较。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，可能改善中风后的长期运动和认知功能。

项目成果

Prostaglandin D2 toxicity in primary neurons is mediated through its bioactive cyclopentenone metabolites.

• DOI: 10.1016/j.neuro.2013.08.001
• 发表时间: 2013-12
• 期刊: NEUROTOXICOLOGY
• 影响因子: 3.4
• 作者: Liu, Hao;Li, Wenjin;Rose, Marie E.;Pascoe, Jordan L.;Miller, Tricia M.;Ahmad, Muzamil;Poloyac, Samuel M.;Hickey, Robert W.;Graham, Steven H.
• 通讯作者: Graham, Steven H.

In vivo transduction of neurons with TAT-UCH-L1 protects brain against controlled cortical impact injury.

• DOI: 10.1371/journal.pone.0178049
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Liu H;Rose ME;Ma X;Culver S;Dixon CE;Graham SH
• 通讯作者: Graham SH

Prolonged opportunity for neuroprotection in experimental stroke with selective blockade of cyclooxygenase-2 activity.

• DOI: 10.1016/j.brainres.2009.05.020
• 发表时间: 2009-07-07
• 期刊: Brain research
• 影响因子: 2.9
• 作者: Ahmad M;Zhang Y;Liu H;Rose ME;Graham SH
• 通讯作者: Graham SH

Increased generation of cyclopentenone prostaglandins after brain ischemia and their role in aggregation of ubiquitinated proteins in neurons.

• DOI: 10.1007/s12640-013-9377-4
• 发表时间: 2013-08
• 期刊: NEUROTOXICITY RESEARCH
• 影响因子: 3.7
• 作者: Liu, Hao;Li, Wenjin;Ahmad, Muzamil;Rose, Marie E.;Miller, Tricia M.;Yu, Mei;Chen, Jie;Pascoe, Jordan L.;Poloyac, Samuel M.;Hickey, Robert W.;Graham, Steven H.
• 通讯作者: Graham, Steven H.

COX2-derived primary and cyclopentenone prostaglandins are increased after asphyxial cardiac arrest.

COX2 衍生的初级前列腺素和环戊烯酮前列腺素在窒息心脏骤停后增加。

• DOI: 10.1016/j.brainres.2013.04.029
• 发表时间: 2013
• 期刊: Brain research
• 影响因子: 2.9
• 作者: Liu,Hao;Rose,MarieE;Miller,TriciaM;Li,Wenjin;Shinde,SunitaN;Pickrell,AliciaM;Poloyac,SamuelM;Graham,StevenH;Hickey,RobertW
• 通讯作者: Hickey,RobertW