Mechanisms of Mutant SOD 1-Mediated Mitochondria Toxicity in the Spinal Cord of A

描述（由申请人提供）：在肌萎缩性侧索硬化症（ALS）突变型SOD1- g93a （mutSOD1）小鼠模型中，突变形式的SOD1在脊髓线粒体中选择性地与Bcl-2结合并聚集。在本研究中，我们将验证位于脊髓线粒体中的mutSOD1部分必须与Bcl-2合作才能显示毒性的假设。我们还将定义mutSOD1/Bcl-2异常结合对脊髓线粒体离子电导和生物能量学的影响。通过适应和应用膜片钳电生理技术（有丝分裂连接配置）对从双转基因ALS小鼠SOD1-93A: mitoCFP中分离的整体、神经元和非神经元脊髓线粒体进行检测，我们将系统地表征线粒体外膜（OMM）在疾病不同阶段的电导，确定这种线粒体表型是否随着疾病的进展而特异性改变。我们提出了三个具体目标。在目的#1中，我们将在细胞和线粒体水平上研究mutSOD1介导的体外毒性及其对Bcl-2的依赖性。在目标#2中，我们将原位研究从携带荧光（青色）标记的蓝色神经元线粒体的双转基因SOD1-G93A: mitoCFP小鼠脊髓分离的神经元和非神经元线粒体的生物物理特性。此外，我们将利用从表达Bcl-2的细胞、缺乏Bcl-2的细胞和转基因小鼠中分离的线粒体，研究mutSOD1蛋白对线粒体外膜通道的影响。在目标#3中，我们将通过产生SOD1-G93A:Bcl-2（-/-）小鼠，并将这些小鼠的疾病表型与未灭除Bcl-2的SOD1-G93A小鼠进行比较，在体内验证mutSOD1与Bcl-2的结合是运动神经元毒性和ALS表型决定因素所必需的假设。通过发展这些目标，我们将了解mutSOD1是否通过与抗凋亡蛋白Bcl-2相互作用导致线粒体功能障碍，以及这种异常机制与ALS小鼠运动神经元变性的直接相关性。肌萎缩性侧索硬化症（ALS，又名Lou Gehrig's病）是最常见的成人运动神经元疾病。这种疾病的特点是脊髓和运动皮层的运动神经元死亡。这会导致痉挛、反射过度、全身无力和肌肉萎缩。呼吸肌衰竭通常是致命的事件，发生在首次症状出现后的1 - 5年内。这项提议的总体目标是阐明运动神经元死亡的机制使用转基因小鼠ALS模型。我们建议的目的是研究这些小鼠脊髓线粒体的疾病机制。线粒体是亚细胞细胞器，是机体产生能量的主要来源。它们在维持神经元细胞存活方面起着关键作用。病理驱动的这些细胞器损伤可能会改变生与死之间的平衡，并导致神经元变性。

DESCRIPTION (provided by applicant): In the mutant SOD1-G93A (mutSOD1) mouse model of amyotrophic lateral sclerosis (ALS), the mutated form of SOD1 selectively binds and aggregates with Bcl-2 in spinal cord mitochondria. In this study we will test the hypothesis that the portion of mutSOD1 localized in spinal cord mitochondria must partner with Bcl-2 to manifest toxicity. We will also define the consequences of the mutSOD1/Bcl-2 aberrant binding on the spinal cord mitochondria ionic conductances and bioenergetics. By adapting and applying the patch-clamp electrophysiological technique (mito-attached configuration) to integral, neuronal and non-neuronal spinal cord mitochondria isolated from the double transgenic ALS mouse SOD1-93A: mitoCFP, we will systematically characterize the conductances of the outer mitochondrial membrane (OMM) throughout the different stages of disease, determining whether this mitochondria phenotype is specifically altered as the disease progresses. We are proposing three specific aims. In aim #1 we will study in vitro mutSOD1- mediated toxicity at the cellular and mitochondrial levels and its dependence on Bcl-2. In aim #2 we will study in situ the biophysical properties of neuronal and non-neuronal mitochondria isolated from the spinal cord of double transgenic SOD1-G93A: mitoCFP mice carrying fluorescently (cyan) tagged blue neuronal mitochondria. Furthermore, we will study the effect of mutSOD1 proteins on mitochondria outer membrane channels using mitochondria isolated from cells expressing Bcl-2 versus cells lacking Bcl-2 and transgenic mice. In aim #3 we will test in vivo the hypothesis that the binding of mutSOD1 to Bcl-2 is required for motor neuron toxicity and a determinant of the ALS phenotype by generating SOD1-G93A:Bcl-2(-/-) mice and comparing the disease phenotype of these mice to the SOD1-G93A mice in which Bcl-2 was not ablated. By developing these aims, we will understand whether by interacting with the anti-apoptotic protein Bcl-2, mutSOD1 leads to mitochondria dysfunction and the immediate relevance of this aberrant mechanism to motor neuron degeneration in ALS mice. PUBLIC HEALTH RELEVANCE Amyotrophic lateral sclerosis (ALS; a.k.a. Lou Gehrig's disease) is the most common adult motor neuron disease. The disease is characterized by the death of motor neurons in the spinal cord and motor cortex. This leads to spasticity, hyper-reflexia, general weakness and muscle atrophy. Failure of respiratory muscles is generally the fatal event, occurring within 1 - 5 years after the onset of the first symptoms. The overarching goal of this proposal is to shed light on the mechanisms of motor neuron death using transgenic mice model of ALS. The objective of our proposal is to study the disease mechanisms focusing on mitochondria of the spinal cord of these mice. Mitochondria are sub-cellular organelles and the main source of energy production in the body. They play a pivotal role in maintaining neuronal cell alive. A pathology-driven impairment of these organelles may shift the balance between life and death and lead to neuronal degeneration.