Mitochondrial modulation for neuroprotection in a model of multiple sclerosis

描述(申请人提供)：这项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。现已认识到，轴突损伤在多发性硬化症中常见，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，但它们很可能源于一系列由线粒体功能障碍和伴随的细胞能量供应不足引发的离子失衡，最终导致线粒体和轴突钙超载。利用实验性自身免疫性脑脊髓炎(EAE)的小鼠模型，我们证明了缺失亲环素D(CyPD-KO)的小鼠突变体尽管中枢神经系统(CNS)内存在炎症，但与野生型(WT)小鼠相比，尽管存在中枢神经系统(CNS)内的炎症，但缺失亲环素D(CyPD-KO)的小鼠显著减少了轴突损伤。CyPD-KO小鼠发生急性EAE类似于WT小鼠，但与WT小鼠不同，临床恢复，轴突损伤减少80%。重要的是，CyPD-KO小鼠的线粒体对钙离子介导的PT孔激活具有抵抗力，CyPD-KO小鼠的原代皮质神经元可以抵抗氧和氮自由基引起的损伤，这些损伤是EAE和MS中的损伤介质。这些结果表明PT孔在决定EAE和MS中轴突的命运中发挥了关键作用。该提议的指导假设是，通过灭活CyPD对PT孔的调节，将增强轴突线粒体在应对病理性钙离子增加时隔离钙离子的能力，从而延迟PT孔的激活。反过来，抑制PT孔的激活将消除ATP耗竭、轴浆钙超载，以及导致轴突破坏的分子级联反应的启动。我们提出了以下具体目标，以进一步测试线粒体PT孔的作用以及CyPD失活对其在EAE轴突损伤发展中的调节作用。在目标1中，我们将使用CyPDloxP/神经元CRE小鼠来确定神经元及其轴突中CyPD的失活是否会导致EAE中轴突的保护。在目标2中，我们将使用WT和CyPD-KO小鼠的原代皮质神经元培养来确定在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)增加线粒体钙水平并激活这些神经元的PT孔，以及3)CyPD灭活是否抑制这些作用。在目标3中，我们将确定使CyPD失活的药物是否在体外保护EAE和皮质神经元中的轴突。我们的结果将扩大我们对线粒体PT孔反应的调节如何影响EAE和MS中的轴突损伤的知识，促进开发治疗MS的新型神经保护疗法。与公共卫生相关：我们的研究试图了解如何防止患有多发性硬化症的小鼠脊髓神经纤维受到损害。我们发现，通过阻断线粒体中的一种蛋白质，我们可以极大地减少这种多发性硬化症对神经的损害。这项研究的结果应该会导致通过使用药物来阻断线粒体蛋白来治疗多发性硬化症的新方法。

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.