Multi-mechanistic protection of mitochondria following traumatic brain injury

目前有超过500万人患有创伤性脑损伤（TBI）导致的残疾，每年带来600亿美元的相关经济负担。临床上对创伤性脑损伤的治疗选择有限，单一机制的药物不太可能预防其破坏性的神经系统后果。因此，必须开发多机制联合疗法。突触和非突触线粒体功能障碍和脂质过氧化（LP）诱导的氧化损伤是与TBI相关的下游病理的关键因素，包括细胞骨架降解、神经变性和行为障碍。损伤后，增加的Ca++被线粒体隔离，导致线粒体通透性过渡孔（mPTP）的形成，活性氧和活性氮的形成增加。这些活性物质引发LP，导致LP分解产物4- HNE和丙烯醛形成神经毒性，它们与线粒体蛋白共价结合，引起氧化损伤，进一步增强线粒体呼吸功能障碍，产生活性物质，增强mPTP的形成，其中突触线粒体特别容易受到影响。mPTP形成后，线粒体停止产生ATP并失去钙离子缓冲能力。钙离子被释放回细胞质中，在那里它激活蛋白酶钙蛋白酶，导致细胞骨架降解。最终，突触失调、神经退行性变和神经功能损害随之而来。醛清除剂苯elzine （PZ）和mPTP抑制剂cyclosporine A （CsA）可部分减轻大鼠控制性皮质撞击损伤（CCI）后的线粒体功能障碍和神经元损伤。因此，我们的总体假设是LP诱导的神经毒性醛清除和mPTP抑制是互补的，这两种机制的结合将增加或协同减轻大鼠严重控制性皮质影响（CCI）后突触和非突触线粒体呼吸功能障碍和LP诱导的氧化损伤，从而减少细胞骨架降解，减少神经变性，改善运动和认知功能。目的1，剂量反应，验证了早期给药（15分钟）PZ和CsA联合用药，可加加或协同减弱突触和非突触线粒体呼吸功能障碍和lp诱导的氧化损伤，以及严重CCI后神经元细胞骨架降解的假设。目的2验证了PZ和CsA联合治疗改善严重CCI后神经保护的治疗窗口期的假设。目的3验证了PZ和CsA联合治疗在严重CCI后的前28天内可加性或协同性地减少神经变性并改善运动和认知功能的假设。本研究将确定损伤后突触和非突触线粒体对药物治疗的不同反应。它重新利用了两种FDA批准的药物，这两种药物使用互补的神经保护机制，最终确定了一种更具有神经保护作用的联合疗法，可以成功地转化为人类TBI。

Over five million people currently live with a disability resulting from a traumatic brain injury (TBI), with an associated economic burden of 60 billion dollars annually. Clinically there are limited treatment options for TBI, and single mechanism agents are unlikely to prevent its devastating neurologic consequences. Therefore, multi-mechanistic combinational therapies must be developed. Synaptic and non-synaptic mitochondrial dysfunction and lipid peroxidation (LP) induced oxidative damage are key contributors to the downstream pathology associated with TBI, including cytoskeletal degradation, neurodegeneration and behavioral impairments. Following injury, increased Ca++ is sequestered by mitochondria resulting in formation of the mitochondrial permeability transition pore (mPTP) and increased formation of reactive oxygen and nitrogen species. These reactive species initiate LP, leading to formation of the neurotoxic LP-breakdown products 4- HNE and acrolein, which covalently bind mitochondrial proteins causing oxidative damage, further enhancing mitochondrial respiratory dysfunction, generation of reactive species, and enhancing formation of mPTP, with synaptic mitochondria being particularly susceptible. Upon mPTP formation, the mitochondria stop producing ATP and lose their Ca++ buffering capacity. Ca++ is released back into the cytosol where it activates the protease calpain causing cytoskeletal degradation. Ultimately, synaptic dysregulation, neurodegeneration and neurologic impairment ensue. Individually, the aldehyde scavenger phenelzine (PZ) and the mPTP inhibitor cyclosporine A (CsA) partially attenuate mitochondrial dysfunction and neuronal damage following controlled cortical impact injury (CCI) in rats. Therefore, our overall hypothesis is that LP-induced neurotoxic aldehyde scavenging and inhibition of mPTP are complimentary and that combining these two mechanisms will additively or synergistically attenuate synaptic and non-synaptic mitochondrial respiratory dysfunction and LP- induced oxidative damage following severe controlled cortical impact (CCI) in rats, leading to decreased cytoskeletal degradation, decreased neurodegeneration, and improved motor and cognitive function. Aim 1, a dose response, tests the hypothesis that early administration (15min.) of the combination, PZ & CsA, additively or synergistically attenuates synaptic and non-synaptic mitochondrial respiratory dysfunction and LP-induced oxidative damage, as well as neuronal cytoskeletal degradation following severe CCI. Aim 2 tests the hypothesis that the combination, PZ & CsA, improves the therapeutic window for neuroprotection following severe CCI. Aim 3 tests the hypothesis that the combination, PZ & CsA, additively or synergistically decreases neurodegeneration and improves motor and cognitive function over the first 28 days following severe CCI. This study will identify differential responses of synaptic and non-synaptic mitochondria to drug therapy following injury. It re-purposes two FDA approved drugs, which use complimentary neuroprotective mechanisms to ultimately identify a more neuroprotective combinational therapy that can successfully translate to human TBI.