Mitochondrial-Targeted Therapeutics for Treatment of Spinal Cord Injury

描述(申请人提供)：脊髓损伤(SCI)在美国是一个长期的医疗保健问题，除了适度有效的甲基强的松龙外，目前还没有临床上可用的神经保护性干预措施来治疗急性SCI。我们发表的数据和初步结果表明，关键线粒体酶的氧化损伤和随后的线粒体功能障碍是脊髓损伤后神经病理后遗症的关键。这一建议将直接靶向线粒体功能障碍作为治疗挫伤脊髓损伤的一种新的治疗措施，其基本概念是脊髓损伤诱导的兴奋性毒性增加线粒体钙循环/超载和活性氧自由基(ROS)的产生，最终导致线粒体功能障碍和谷胱甘肽(GSH)的耗竭。我们的方法是双管齐下的，旨在利用一种新型的细胞专用抗氧化剂和谷胱甘肽前体NACA(N-乙酰半胱氨酸的酰胺形式)，以及脊髓损伤后用于产生能量的替代生物燃料底物乙酰-L肉碱(ALC)来减少线粒体ROS的产生。我们已发表的初步数据表明，NACA和ALC均可改善大鼠脊髓挫伤后线粒体的生物能量学，而延长NACA或ALC治疗可增加损伤后组织的保存。计划中的实验旨在检验这一新的假设，即减少对关键线粒体蛋白质的氧化损伤将保持线粒体的生物能量学，从而增强神经保护，促进挫伤后脊髓损伤后的功能恢复。具体地说，我们将：1)表征参与生物能量学的特定线粒体蛋白质的氧化损伤，并验证NACA治疗可改善脊髓损伤后线粒体氧化损伤的假设；2)测试NACA和ALC联合治疗将协同作用于保护脊髓损伤后线粒体动态平衡的假设；3)测试NACA和ALC联合治疗将增加脊髓损伤后组织保护和促进长期功能恢复的假设。关键的是，这一应用是建立在利用我们开发的从损伤的脊髓中分离突触(神经元)和非突触(胞体和胶质细胞)线粒体的新技术，以及L1/L2挫伤SCI范例，该范例证明了神经保护和后肢功能恢复的显著改善之间的显著相关性。总的来说，拟议的实验将确定关键的线粒体事件，这些事件可能成为药物干预的潜在新靶点，以更有效地治疗脊髓损伤，或许还有其他中枢神经系统损伤。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is a long-term health care problem in the United States, and with the exception of the modestly effective methylprednisolone, there is currently no neuroprotective intervention clinically available for treatment of acute SCI. Our published data and preliminary results demonstrate that oxidative damage to key mitochondrial enzymes and subsequent mitochondrial dysfunction is key to the neuropathological sequalae following SCI. This proposal focuses on directly targeting mitochondrial dysfunction as a novel therapeutic intervention for contusion SCI, the fundamental concept being that SCI-induced excitotoxicity increases mitochondrial Ca2+ cycling/overload and the production of reactive oxygen species (ROS), ultimately leading to mitochondrial dysfunction and glutathione (GSH) depletion. Our approach is two-pronged, aimed at reducing mitochondrial ROS production utilizing a novel, cell-permeant antioxidant and GSH precursor, NACA (the amide form of N-acetylcysteine), as well as an alternative biofuel substrate for energy production, acetyl-l-carnitine (ALC), following SCI. Our published and preliminary data signify that both NACA and ALC improve mitochondrial bioenergetics following contusion SCI in rats, and that prolonged NACA or ALC treatment increases tissue sparing following injury. The planned experiments are designed to test the novel hypothesis that reducing oxidative damage to key mitochondrial proteins will maintain mitochondrial bioenergetics, thus leading to increased neuroprotection and improved functional recovery following contusion SCI. Specifically we will: 1) Characterize oxidative damage to specific mitochondrial proteins involved in bioenergetics and test the hypothesis that NACA treatment ameliorates mitochondrial oxidative damage following SCI, 2) Test the hypothesis that a combinatorial treatment with NACA and ALC will act synergistically to preserve mitochondrial homeostasis following SCI, and 3) Test the hypothesis that a combinatorial treatment with NACA and ALC will increase tissue sparing and promote long-term functional recovery following SCI. Critically, this application is built around the utilization of several novel techniques we have developed for isolating synaptic (neuronal) and non-synaptic (soma and glia) mitochondria from the injured spinal cord, as well as an L1/L2 contusion SCI paradigm that demonstrates a significant correlation between neuroprotection and remarkable improvements in recovery of hind limb function. Collectively, the proposed experiments will pinpoint key mitochondrial events that could be potential novel targets for pharmacological interventions to more effectively treat SCI and, perhaps, other CNS injuries.