CYTOPROTECTION BY INHIBITION OF CALPAIN PROTEASES

在啮齿动物模型中，长期饮酒后发生的N-甲基-D-天冬氨酸受体上调与神经细胞毒性有关，这是通过L-谷氨酸激活这些钙通道而导致的钙通量增加而实现的。钙蛋白酶是一类钙激活的半胱氨酸蛋白酶，在神经细胞中介导钙介导的损伤。本项目的重点是a)利用PC12细胞模型检测L-谷氨酸介导的细胞损伤的机制；b)确定抑制Calain活性是否会导致显著的细胞保护；c)在L-谷氨酸的细胞毒性模型中测试各种作为细胞保护剂的Calain抑制剂；d)将这些观察扩展到啮齿动物的神经细胞损伤模型。此前在这个实验室中已经证明，利托那韦是一种HIV蛋白酶抑制剂，也是一种具有竞争性的钙蛋白酶活性抑制剂，其Ki为~10微米，完全在临床给药期间发现的范围内，当用作抗逆转录病毒时。利用Fura-2，发现L-谷氨酸显著增加细胞内游离钙浓度。L-谷氨酸引起的细胞内游离钙浓度的增加也使钙蛋白酶的活性显著增加。在这个模型中，发现L谷氨酸暴露导致肌动蛋白、tau和NF68的显著降解，这一过程可被MK801、Calain抑制剂I和利托那韦阻断。细胞毒性实验发现，MK801、钙蛋白酶抑制剂I和利托那韦均能抑制L谷氨酸诱导的细胞死亡。Caspase抑制剂(Z-DEVD-FMK)和DNQX(AMPA抑制剂)均无保护作用，也不能阻止L谷氨酸诱导的细胞骨架蛋白降解。从这项工作的初始阶段，我们得出结论：L-谷氨酸介导的PC12细胞毒性是一个钙蛋白酶依赖的过程。在未来的工作中，这些药物的神经保护作用将在啮齿动物模型中进行检验。如果这是成功的，这将增加利托那韦或其类似物作为神经保护剂的可能性，因为细胞损伤被认为是通过钙介导的过程发生的。

NMDA receptor upregulation occurring after prolonged alcohol administration in rodent models has been implicated in neural cytotoxicity, via an increase in calcium-flux resulting from l-glutamate activation of these calcium channels. Calpains are a class of calcium-activated cysteine proteases that are known to mediate calcium-mediated injury in neural cells. The focus of this project is to a) examine the mechanism of l-glutamate mediated cell injury using a PC12 cell model; b) determine whether inhibition of calpain activity would result in significant cytoprotection; c) test a variety of calpain inhibitors as cytoprotectants in an l-glutamate model of cytotoxicity; d) extend these observations to a rodent model of neural cell injury. It was previously shown in this laboratory that ritonavir, an HIV protease inhibitor, is also a competitive inhibitor of calpain activity, with a Ki of ~10 microM, well within range found during clinical dosing of the drug in humans when used as an anti-retroviral. Using fura-2, it was found that l-glutamate singificantly increased intracellular concentrations of free calcium. The increase in intracellular free calcium induced by l-glutamate was also shown to result in a signficant increase in calpain protease activity. Calpain activation is followed by degradation of a variety of cytoskeletal components, and in this model it was found that l-glutamate exposure resulted in significant degradation of actin, tau, and NF68, which was blocked by MK801, calpain inhibitor I and ritonavir. In cytotoxicity tests, it was found that MK801, calpain inhibitor I, and ritonavir also all inhibited l-glutamate mediated cell death. Neither a caspase inhibitor (Z-DEVD-FMK) nor DNQX (AMPA inhibitor) had any protectant effects, nor did they prevent l-glutamate induced breakdown of cytoskeletal proteins. We conclude from the initial phases of this work that l-glutamate mediated cytotoxicity in PC12 cells is a calpain-dependent process. In future work, the neuroprotective effects of these agents will be examined in a rodent model. If this is successful, this would raise the possibility that ritonavir or its analogues might be useful as neuroprotective agents when cellular injury is thought to occur via a calcium-mediated process.