ROLE OF CALCIUM ION ACTIVATED PROTEASES IN AGING AND PATHOLOGY

免疫细胞化学和药理学研究提供了证据 钙激活的硫醇蛋白水解酶(钙调蛋白)在 神经病理学。对这些内源底物的部分消化 酶在几种致病情况下都会发生，在某些情况下是几天 在细胞退化的明显迹象出现之前，一种药物 在两种模式下抑制Calain阻断a=病理的发展 它已经过测试了。根据这些结果，现在是合理的 开始探索卡尔佩恩，与 其他因素也会导致大脑中与年龄相关的变化。四 这类项目构成了本提案。实验1将 测试分解产物的浓度是否来自于 在衰老的小鼠脑中，Calain对血影蛋白的消化增加。引航员 数据指向这一结论；如果得到证实，这将间接提供 有证据表明，随着年龄的增长，钙蛋白酶的活性会更强。实验2将使用 NMDA受体的药理刺激和缺氧以确定是否 老年人的大脑更容易受到致病条件的影响，如果这是 与钙蛋白酶的激活增强有关。前期工作 表明低氧发作时间太短，不会导致脑片老化。这个 拟议的研究将延长这一项目，并测试陈年切片是否 也更容易受到NMDA受体的刺激。血影蛋白的检测 钙蛋白酶保护作用的分解和生理测试 将使用抑制剂来评估过度刺激的可能性 这种蛋白水解酶的活性有助于老年人大脑的病理反应。 这些研究将包括最近推出的钙蛋白酶抑制剂， 似乎更有效力和选择性。实验3将考察 发现的钙蛋白酶底物降解增加的可能性 发病机制和可能的衰老部分是由于促进了 底物与蛋白酶之间的相互作用。膜将被隔离 从短暂性脑缺血后沙土鼠脑组织和不同来源的 老年大鼠脑内不同区域的蛋白分解反应 与外源钙蛋白酶孵育。实验4将确定不同的 淀粉样前体蛋白的成分是钙蛋白和钙蛋白的底物。 如果致病操作触发了蛋白质的部分消化。 这些实验应该提供所需的证据来评估 钙蛋白酶过度激活导致淀粉样蛋白的可能性 在老化过程中形成。

Immunocytochemical and pharmacological studies have provided evidence that calcium activated thiol proteases (calpains) plays am important role in neuropathology. Partial digestion of an endogenous substrate for these enzymes occurs in several pathogenic circumstances, in some cases days before the onset of overt signs of cellular degeneration, and a drug that inhibits calpain blocks the development of a=pathology in two paradigms in which it has been tested. In light of these results, it is now reasonable to begin exploring the possibility that calpain, acting in concert with other factors, also contributes to age-related changes in the brain. Four projects of this type constitute the present proposal. Experiment 1 will test if the concentration of a breakdown product that results from the digestion of spectrin by calpain increases in the aged mouse brain. Pilot data point to this conclusion; if confirmed, this would provide indirect evidence for greater calpain activity, with aging. Experiment 2 will use pharmacological stimulation of NMDA receptors and hypoxia to determine if the aged brain is more vulnerable to pathogenic conditions and if this is associated with an enhanced activation of calpain. Preliminary work suggests that episodes of hypoxia too short to cause aged slices. The proposed studies will extend this project and test if the aged slices are also more vulnerable to NMDA receptor stimulation. Assays of spectrin breakdown and physiological tests of the protective effects of calpain inhibitors will be used to assess the likelihood that excessive stimulation of the protease contributes to pathological responses in the aged brain. These studies will include recently introduced calpain inhibitors that appear to be more potent and selective. Experiment 3 will examine the possibility that the increased degradation of calpain substrates found in pathogenesis and possibly aging is due in part to a facilitation of the interactions between substrates and protease. Membranes will be isolated from the brain of gerbils after transient ischemia and from different regions of the aged rat brain and proteolysis assessed following an incubation with exogenous calpain. Experiment 4 will determine if various components of the amyloid precursor protein are substrates for calpain and if pathogenic manipulations trigger the partial digestion of the protein. These experiments should provide evidence needed to evaluate the possibility that excessive activation of calpain contributes to amyloid formation in aging.