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

描述（由申请人提供）：由心脏骤停或中风引起的缺血性脑损伤是导致死亡和残疾的主要原因。尽管延迟后缺血性神经退行性变的确切机制仍不完全清楚，Ca2+稳态的破坏似乎起着主要作用。缺血后神经元钙稳态中断的一个潜在原因是Ca2+调节蛋白的蛋白水解修饰。本研究的重点是caspase 3和calpain介导的肌醇(1,4,5)-三磷酸受体（IP3R）的裂解，这是一种位于内质网（ER）上的Ca2+释放通道。已发表的证据和我们自己的初步数据表明，caspase 3和calpain介导的1型IP3R （IP3R1）的裂解产生了一个组成性开放通道，允许Ca2+从内质网泄漏，并损害内质网缓冲细胞质钙过载的能力。本提案的目的是验证caspase 3或calpain介导的IP3R1的裂解产生一个组成性开放通道的假设，该通道不可逆地破坏细胞内Ca2+稳态，并有助于兴奋毒性和缺血性损伤后的神经变性。特异性Aim 1将测量IP3R1的caspase 3-和calpain-cleaved的通道特性及其对细胞内钙稳态的影响。特异性目的2将研究caspase - 3和calpain-cleaved IP3R1在基线条件下和兴奋毒性损伤后的原代神经元培养中的作用。特异性目的3将研究caspase 3和calpain-cleaved IP3R1在基线和缺血后条件下对体内神经元的影响。总的来说，这些实验的结果为病理蛋白酶通过破坏细胞内钙稳态引起急性神经变性的机制提供了重要的见解。此外，阻断caspase-和calpain-cleaved形式的IP3R1可能是缺血性脑损伤后神经保护的新治疗靶点。

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.