Inositol (1,4,5)-trisphosphate receptor proteolysis in ischemic brain injury

缺血性脑损伤中肌醇（1,4,5）-三磷酸受体蛋白水解

• 批准号: 7875589
• 负责人: ROBERT W. NEUMAR
• 金额: $ 20.74万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7875589
• 关键词: Acute; Adult; Affinity; Brain Ischemia; Buffers; C-terminal; Calcium; Calpain; Caspase; Cell Survival; Cells; Cessation of life; Cleaved cell; Cysteine Protease; Data; Dyes; Endoplasmic Reticulum; Exposure to; Face; Family; Heart Arrest; Hippocampus (Brain); Homeostasis; Hour; ITPR1 gene; Immunohistochemistry; In Vitro; Injection of therapeutic agent; Injury; Inositol; Ischemia; Ischemic Brain Injury; LacZ Genes; Length; Ligands; Measures; Mediating; Modification; Motor Cortex; N-Methylaspartate; Nerve Degeneration; Neuronal Injury; Neurons; Nuclear; Pathologic; Peptide Hydrolases; Physiology; Play; Predisposition; Probability; Property; Prosencephalon; Proteins; Proteolysis; Publishing; Rattus; Recombinants; Reperfusion Therapy; Role; Site; Staining method; Stains; Stroke; Testing; Western Blotting; adeno-associated viral vector; base; caspase-3; disability; fluoro jade; genetic regulatory protein; hippocampal pyramidal neuron; in vivo; insight; loss of function; mortality; neuroprotection; new therapeutic target; novel; patch clamp; protein function; public health relevance; receptor; research study; transgene expression; vector

DESCRIPTION (provided by applicant): Ischemic brain injury due to cardiac arrest or stroke represents a major cause of mortality and disability. Although the precise mechanisms of delayed post-ischemic neurodegeneration remain incompletely understood, disruption of Ca2+ homeostasis appears to play a major role. One potential cause of disrupted calcium homeostasis in post-ischemic neurons is proteolytic modification of Ca2+ regulatory proteins. This proposal focuses on caspase 3- and calpain-mediated cleavage of the inositol (1,4,5)-trisphosphate receptor (IP3R), a Ca2+ release channel located on the endoplasmic reticulum (ER). Both published evidence and our own preliminary data suggest that caspase 3- and calpain-mediated cleavage of the type 1 IP3R (IP3R1) generates a constitutively open channel that allows Ca2+ to leak from the ER and impairs the ER capacity to buffer cytosolic calcium overload. The aims of this proposal will test the hypothesis that caspase 3- or calpain- mediated cleavage of IP3R1 generates a constitutively open channel that irreversibly disrupts intracellular Ca2+ homeostasis and contributes to neurodegeneration after excitotoxic and ischemic injury. Specific Aim 1 will measure the channel properties of caspase 3- and calpain-cleaved of IP3R1 and their effect on intracellular calcium homeostasis. Specific Aim 2 will investigate the effect of caspase 3- and calpain-cleaved IP3R1 in primary neuron culture under baseline conditions and after excitotoxic injury. Specific Aim 3 will investigate the effect of caspase 3- and calpain-cleaved IP3R1 on neurons in vivo under baseline and post-ischemic conditions. Overall, the results of these experiments with provide critical insight into the mechanism by which pathologic proteases cause acute neurodegeneration through disruption of intracellular calcium homeostasis. In addition, blocking the caspase- and calpain-cleaved forms of IP3R1 could be a novel therapeutic target for neuroprotection after ischemic brain injury. PUBLIC HEALTH RELEVANCE: A growing body of evidence suggests that sustained disruption of neuronal calcium homeostasis plays a causal role in neuronal death after brain ischemia. This proposal tests the hypothesis that pathologic proteases, caspase-3 and calpains, disrupt neuronal calcium homeostasis through cleavage of the inositol (1,4,5)-trisphosphate receptor, a Ca2+ channel located on the endoplasmic reticulum. The results of these experiments with provide fundamental insight into the mechanism of post-ischemic neurodegeneration and potentially identify a novel therapeutic target for neuroprotection after ischemic brain injury.

描述（由申请人提供）：心脏骤停或中风引起的缺血性脑损伤是死亡和残疾的主要原因。尽管延迟性缺血后神经变性的确切机制仍未完全了解，但 Ca2+ 稳态的破坏似乎起着重要作用。缺血后神经元钙稳态破坏的潜在原因之一是 Ca2+ 调节蛋白的蛋白水解修饰。该提案重点关注 caspase 3 和钙蛋白酶介导的肌醇 (1,4,5)-三磷酸受体 (IP3R) 裂解，IP3R 是位于内质网 (ER) 上的 Ca2+ 释放通道。已发表的证据和我们自己的初步数据都表明，半胱天冬酶 3 和钙蛋白酶介导的 1 型 IP3R (IP3R1) 裂解产生了一个组成性开放通道，允许 Ca2+ 从内质网泄漏，并损害内质网缓冲胞质钙超载的能力。该提案的目的将检验这样的假设：Caspase 3 或钙蛋白酶介导的 IP3R1 裂解会产生一个组成性开放通道，该通道不可逆地破坏细胞内 Ca2+ 稳态，并导致兴奋性毒性和缺血性损伤后的神经退行性变。具体目标 1 将测量 IP3R1 的 caspase 3 和钙蛋白酶裂解的通道特性及其对细胞内钙稳态的影响。具体目标 2 将研究基线条件下和兴奋性毒性损伤后原代神经元培养物中 caspase 3 和钙蛋白酶裂解的 IP3R1 的影响。具体目标 3 将研究基线和缺血后条件下 caspase 3 和钙蛋白酶裂解的 IP3R1 对体内神经元的影响。总体而言，这些实验的结果为病理蛋白酶通过破坏细胞内钙稳态而导致急性神经变性的机制提供了重要的见解。此外，阻断 IP3R1 的 caspase 和 calpain 裂解形式可能成为缺血性脑损伤后神经保护的新治疗靶点。 公共健康相关性：越来越多的证据表明，神经元钙稳态的持续破坏在脑缺血后神经元死亡中起着因果作用。该提案测试了这样的假设：病理蛋白酶、caspase-3 和钙蛋白酶通过裂解肌醇 (1,4,5)-三磷酸受体（位于内质网的 Ca2+ 通道）来破坏神经元钙稳态。这些实验的结果提供了对缺血后神经变性机制的基本见解，并有可能确定缺血性脑损伤后神经保护的新治疗靶点。