Calpain mediated injury in post ischemic neurons

钙蛋白酶介导缺血后神经元损伤

• 批准号: 7414551
• 负责人: ROBERT W. NEUMAR
• 金额: $ 33.71万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7414551
• 关键词: Acute; Antibodies; Apoptosis; Brain; Brain Ischemia; C-terminal; Calpain; Cell Cycle Regulation; Cells; Cleaved cell; Condition; Data; Dependovirus; Development; Elevation; Endopeptidases; Engineering; Evaluation; Family; Functional disorder; Gene Expression; Generations; Heart Arrest; Hippocampus (Brain); Hour; Human; ITPR1 gene; Immunoglobulin Fragments; In Vitro; Injection of therapeutic agent; Injury; Investigation; Ischemia; Ischemic Brain Injury; Mediating; Mitosis; Modeling; Modification; N-terminal; Necrosis; Nerve Degeneration; Neurons; Nuclear; Pathologic; Pathway interactions; Patients; Peptide Hydrolases; Perfusion; Pre-Clinical Model; Principal Investigator; Property; Prosencephalon; Protein Isoforms; Protein Overexpression; RNA; RNA Interference; Rattus; Relative (related person); Research Personnel; Rest; Role; RyR2; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Signal Transduction; Slice; Source; Stroke; Synaptic plasticity; Week; Xenopus oocyte; adeno-associated viral vector; calpain inhibitor; calpastatin; cell motility; enhanced green fluorescent protein; feeding; genetic regulatory protein; hippocampal pyramidal neuron; human morbidity; in vivo; inhibitor/antagonist; inositol-1,4,5-triphosphate receptor; insight; m-calpain; mortality; mutant; patch clamp; prevent; programs; vector

DESCRIPTION (provided by applicant): Brain ischemia caused by cardiac arrest and stroke is a significant source of human morbidity and mortality. This proposal focuses on the role of calpains, a family of Ca2+-dependent cytosolic proteases, in delayed necrosis of post-ischemic neurons. Brain calpain activity is pathologically increased after brain ischemia, and calpain inhibitors are neuroprotective in preclinical models. However, the mechanism of calpain-mediated injury is unknown, and the relative roles of the two ubiquitous isoforms, u-calpain and m-calpain, have not been elucidated. Pathologic calpain activity requires sustained cytosolic Ca2+ elevation. Calpain-mediated cleavage of IP3 receptors (IP3R) and ryanodine receptors (RYR) generates stable dysregulated channels that have increased Ca2+ conductance. These observations support the hypothesis that calpains are not only activated by elevated cytosolic Ca2+, but under pathologic conditions contribute to sustained cytosolic Ca2+ overload in a potentially irreversible feed-forward pathway that ultimately causes neuronal necrosis. Specific Aim 1 will use in vivo adeno-associated viral (AAV) vector-mediated overexpression of the specific endogenous calpain inhibitor, calpastatin, to elucidate the causal relationship between post-ischemic calpain activity, cytosolic Ca2+ overload, electrophysiological dysfunction, and delayed necrosis of hippocampal CA1 pyramidal neurons in a rat model of transient forebrain ischemia. In Specific Aim 2, the relative role of u-calpain and m-calpain will be examined using AAV vector-mediated RNA interference in the same model. Specific Aim 3 will examine the role of calpain-cleaved IP3R1. Generation of calpain-cleaved IP3R1 in post ischemic neurons will be immunohistochemically characterized. A truncated IP3R1 mutant corresponding to the stable calpain-derived fragment will be expressed in Xenopus oocytes for nuclear patch clamp analysis of channel function and expressed in CA1 pyramidal neurons in vivo to determine if it causes calpain activity and delayed necrosis. Specific Aim 4 will utilize a similar approach to evaluate the role of calpain-cleaved RYR2. The proposed studies overcome significant obstacles limiting the mechanistic evaluation of calpain's role in in vivo ischemic brain injury. The results will provide fundamental insights into the mechanism of delayed post-ischemic neuronal necrosis, and facilitate the development of effective therapies for patients suffering from cardiac arrest and stroke.

描述（由申请人提供）：心脏骤停和中风引起的脑缺血是人类发病率和死亡率的重要来源。该提案的重点是CALPAIN的作用，Calpains是一个Ca2+依赖性的胞质蛋白酶，在缺血后神经元坏死中。脑缺血后脑钙蛋白酶活性在病理上增加，而钙蛋白酶抑制剂在临床前模型中是神经保护剂。然而，钙蛋白酶介导的损伤的机制尚不清楚，并且尚未阐明两种无处不在的同工型（U-钙蛋白酶和M-钙蛋白）的相对作用。病理性钙蛋白酶活性需要持续的胞质Ca2+升高。 Calpain介导的IP3受体（IP3R）和Ryanodine受体（RYR）的裂解会产生Ca2+电导率增加的稳定失调通道。这些观察结果支持以下假设：Calpain不仅被胞质CA2+升高激活，而且在病理状态下，在潜在的不可逆喂养前途径中有助于持续的胞质Ca2+过载，最终导致神经元坏死。 Specific Aim 1 will use in vivo adeno-associated viral (AAV) vector-mediated overexpression of the specific endogenous calpain inhibitor, calpastatin, to elucidate the causal relationship between post-ischemic calpain activity, cytosolic Ca2+ overload, electrophysiological dysfunction, and delayed necrosis of hippocampal CA1 pyramidal neurons in a rat model of瞬态前脑缺血。在特定的目标2中，将使用AAV矢量介导的RNA干扰在同一模型中检查U-钙蛋白和M-钙蛋白的相对作用。特定的目标3将检查Calpain旋转的IP3R1的作用。在缺血性神经元中产生的钙蛋白酶旋转的IP3R1将以免疫组织化学的特征来表征。与稳定的钙蛋白酶衍生的片段相对应的截短的IP3R1突变体将在爪蟾卵母细胞中表达，以用于通道功能的核贴片夹分析，并在体内CA1锥体神经元中表达，以确定它是否引起钙蛋白酶的活性并延迟坏死。特定的目标4将利用类似的方法来评估Calpain旋转的RYR2的作用。拟议的研究克服了重大障碍，限制了钙蛋白酶在体内缺血性脑损伤中的作用的机理评估。结果将提供有关延迟缺血后神经元坏死机制的基本见解，并促进为患有心脏骤停和中风患者的有效疗法开发。

项目成果

Neuroprotection with delayed calpain inhibition after transient forebrain ischemia.

• DOI: 10.1097/ccm.0b013e31818a8ec8
• 发表时间: 2008-11
• 期刊: Critical care medicine
• 影响因子: 8.8
• 作者: Frederick JR;Chen Z;Bevers MB;Ingleton LP;Ma M;Neumar RW
• 通讯作者: Neumar RW

Comparison of calpain and caspase activities in the adult rat brain after transient forebrain ischemia.

短暂前脑缺血后成年大鼠脑中钙蛋白酶和半胱天冬酶活性的比较。

• DOI: 10.1006/nbdi.2002.0526
• 发表时间: 2002
• 期刊: Neurobiology of disease
• 影响因子: 6.1
• 作者: Zhang,Chen;Siman,Robert;Xu,YAnne;Mills,AngelaM;Frederick,JamesR;Neumar,RobertW
• 通讯作者: Neumar,RobertW