Mitochondrial modulation for neuroprotection in a model of multiple sclerosis

描述（由申请人提供）：本研究的长期目标是确定多发性硬化症（MS）神经保护治疗的分子靶点。现在人们认识到，轴突损伤在多发性硬化症中很常见，是导致永久性残疾的重要原因。开发阻止轴突变性的神经保护疗法是多发性硬化症研究的主要治疗目标。虽然导致轴突变性的确切机制尚不清楚，但它们很可能源于线粒体功能障碍和伴随的细胞能量供应缺陷引发的一系列离子失衡，最终导致线粒体和轴突Ca2+过载。使用小鼠MS模型，实验性自身免疫性脑脊髓炎（EAE），我们已经证明小鼠突变体缺失亲环蛋白D (CyPD-KO)，线粒体渗透过渡（PT）孔的关键调节因子和线粒体Ca2+释放的主要途径，尽管中枢神经系统（CNS）存在炎症，但与野生型（WT）小鼠相比，显著减少轴突损伤。CyPD-KO小鼠发生急性EAE，与WT小鼠相似，但与WT小鼠不同，临床恢复，轴突损伤减少高达80%。重要的是，CyPD-KO小鼠的线粒体抵抗Ca2+介导的PT孔激活，CyPD-KO小鼠的初级皮质神经元抵抗氧和氮自由基诱导的损伤。这些结果表明PT孔在EAE和ms中决定轴突命运的关键作用。该建议的指导假设是，通过CyPD失活来调节PT孔将增强轴突线粒体在病理Ca2+增加时隔离Ca2+的能力，从而延迟PT孔的激活。反过来，抑制PT孔激活将消除ATP消耗，轴浆Ca2+过载，以及导致轴突破坏的分子级联的启动。我们提出以下具体目标，以进一步测试线粒体PT孔及其通过CyPD失活调控在EAE轴突损伤发展中的作用。在Aim 1中，我们将使用CyPDloxP/神经元Cre小鼠来确定CyPD在神经元及其轴突失活，而不是在其他中枢神经系统细胞类型中失活，是否会导致EAE的轴突保护。在Aim 2中，我们将使用WT和CyPD- ko小鼠的原代皮质神经元培养物来确定EAE过程中产生的毒性炎症介质是否1)改变WT神经元的树突稳定性和神经元活力，2)增加线粒体Ca2+水平并激活这些神经元中的PT孔，以及3)CyPD失活是否抑制这些作用。在Aim 3中，我们将确定灭活CyPD的药物是否在体外保护EAE和皮质神经元的轴突。我们的研究结果将扩大我们对线粒体PT孔反应调节如何影响EAE和MS轴突损伤的认识，促进MS治疗的新型神经保护疗法的发展。公共卫生相关性：我们的研究旨在了解如何预防多发性硬化症样疾病小鼠脊髓神经纤维损伤。我们发现，通过阻断线粒体中的一种蛋白质，我们可以显著减少这种多发性硬化症样疾病对神经的损害。这项研究的结果应该会导致多发性硬化症的新治疗方法，通过使用药物来阻断线粒体蛋白。

DESCRIPTION (provided by applicant): The long term objective of this research is to identify molecular targets for neuroprotective therapies in multiple sclerosis (MS). It is now recognized that axon damage occurs commonly in MS and is an important cause of permanent disability. Developing neuroprotective therapies that halt axonal degeneration is a major therapeutic goal of MS research. Although the precise mechanisms leading to axonal degeneration are poorly understood, they most likely stem from a cascade of ionic imbalances initiated by mitochondrial dysfunction and concomitant deficits in cellular energy supply, ultimately resulting in mitochondrial and axonal Ca2+ overload. Using the murine model of MS, experimental autoimmune encephalomyelitis (EAE), we have demonstrated that mouse mutants missing cyclophilin D (CyPD-KO), a key regulator of the mitochondrial permeability transition (PT) pore and the major pathway for Ca2+ release from mitochondria, have dramatically reduced axonal damage compared with wild type (WT) mice despite the presence of inflammation within the central nervous system (CNS). CyPD-KO mice develop acute EAE similar to WT mice but unlike the WT mice, recover clinically and show up to an 80% reduction in axonal damage. Importantly, mitochondria from CyPD-KO mice are resistant to Ca2+-mediated PT Pore activation and primary cortical neurons from CyPD-KO mice resist injury induced by oxygen and nitrogen free radicals, mediators of injury in EAE and MS. These results suggest a critical role for the PT Pore in determining the fate of axons in EAE and MS. The guiding hypothesis of this proposal is that modulation of the PT Pore by inactivation of CyPD will enhance the ability of axonal mitochondria to sequester Ca2+ in response to pathologic increases in Ca2+, thereby delaying activation of the PT Pore. In turn, inhibition of PT Pore activation will abrogate ATP depletion, axoplasmic Ca2+ overload, and the initiation of a molecular cascade that leads to axonal destruction. We propose the following specific aims to further test the role of the mitochondrial PT Pore and its modulation by CyPD inactivation in the development of axonal injury in EAE. In Aim 1, we will use CyPDloxP/neuronal Cre mice to determine whether inactivation of CyPD in neurons and their axons and not in other CNS cell types results in axonal protection in EAE. In Aim 2, we will use primary cortical neuronal cultures from WT and CyPD-KO mice to determine whether toxic inflammatory mediators generated during EAE 1) change dendritic stability and neuronal viability in WT neurons and 2) increase mitochondrial Ca2+ levels and activate the PT Pore in these neurons and whether 3) CyPD inactivation inhibits these effects. In Aim 3, we will determine whether drugs that inactivate CyPD protect axons in EAE and cortical neurons in vitro. Our results will expand our knowledge of how modulation of mitochondrial PT Pore responses influence axonal injury in EAE and MS, facilitating the development of novel neuroprotective therapies for the treatment of MS. PUBLIC HEALTH RELEVANCE: Our research seeks to understand how to prevent damage to nerve fibers in the spinal cord of mice with a multiple sclerosis-like disease. We have found that we can dramatically reduce damage to nerves in this multiple sclerosis-like disease by blocking a protein in mitochondria. The results of this research should lead to new treatment approaches for multiple sclerosis by using drugs to block the mitochondrial protein.