Multi-mechanistic protection of mitochondria following traumatic brain injury

目前有500多万人患有创伤性脑损伤(TBI)所致的残疾，每年相关的经济负担高达600亿美元。临床上，脑外伤的治疗选择有限，单一机制药物不太可能预防其毁灭性的神经后果。因此，必须发展多机结合疗法。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脑损伤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，导致线粒体通透性转换孔(MPTP)的形成，并增加了活性氧和氮物种的形成。这些活性物种引发LP，导致神经毒性LP分解产物4-HNE和丙烯醛的形成，它们共价结合导致氧化损伤的线粒体蛋白，进一步增强线粒体呼吸功能障碍，产生活性物种，促进MPTP的形成，其中突触线粒体尤其敏感。当MPTP形成时，线粒体停止产生ATP，并失去其钙缓冲能力。Ca++被释放回胞浆中，在那里它激活了导致细胞骨架降解的蛋白水解酶calain。最终，突触调节失调、神经变性和神经功能障碍随之而来。醛清除剂苯乙肼(PZ)和MPTP抑制剂环孢素A(CsA)分别部分减轻了大鼠大脑皮质撞击伤(CCI)后线粒体功能障碍和神经元损伤。因此，我们的总体假设是，LP诱导的神经毒性醛的清除和MPTP的抑制是互补的，这两种机制的结合将相加或协同地减轻严重受控皮质撞击(CCI)后Lp诱导的大鼠突触和非突触线粒体呼吸功能障碍和LP诱导的氧化损伤，导致细胞骨架降解减少，神经变性减少，运动和认知功能改善。目标1，一种剂量反应，检验了早期给药(15分钟)的假设。其中，PZ和CsA相加或协同作用可减轻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后神经细胞骨架的退化。目的2验证PZ和CsA联合用药改善严重CCI后神经保护治疗窗口的假设。目的3验证这一假设，即在严重CCI后的头28天，PZ和CsA的组合相加或协同减少神经退行性变，改善运动和认知功能。这项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。它改变了FDA批准的两种药物的用途，这两种药物使用免费的神经保护机制，最终确定了一种更具神经保护作用的联合疗法，可以成功地转化为人类脑外伤。

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.