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）的突变体SOD 1-G93 A（mutSOD 1）小鼠模型中，突变形式的SOD 1选择性地与脊髓线粒体中的Bcl-2结合并聚集。在这项研究中，我们将测试的假设，定位在脊髓线粒体中的mutSOD 1的部分必须与Bcl-2合作，以表现出毒性。我们还将定义mutSOD 1/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。采用膜片钳技术通过将线粒体（线粒体附着构型）与从双转基因ALS小鼠SOD 1 - 93 A：mitoCFP分离的完整的神经元和非神经元脊髓线粒体连接，我们将系统地表征在疾病的不同阶段中线粒体外膜（OMM）的电导，确定该线粒体表型是否随着疾病进展而特异性改变。我们提出三个具体目标。在目标#1中，我们将在细胞和线粒体水平上研究体外mutSOD 1介导的毒性及其对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SOD 1-G93 A：mitoCFP小鼠的脊髓分离的神经元和非神经元线粒体的生物物理性质。此外，我们将使用从表达Bcl-2的细胞与缺乏Bcl-2的细胞和转基因小鼠中分离的线粒体来研究mutSOD 1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SOD 1-G93 A：Bcl-2（-/-）小鼠并将这些小鼠的疾病表型与Bcl-2未被消除的SOD 1-G93 A小鼠进行比较，在体内测试mutSOD 1与Bcl-2的结合是运动神经元毒性所需的并且是ALS表型的决定因素的假设。通过开发这些目标，我们将了解mutSOD 1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。肌萎缩侧索硬化症（ALS; a.k.a.葛雷克氏症（Lou Gehrig's disease）是最常见的成人运动神经元疾病。这种疾病的特征是脊髓和运动皮质中运动神经元的死亡。这会导致痉挛、反射亢进、全身无力和肌肉萎缩。呼吸肌衰竭通常是致命事件，发生在首次症状发作后1 - 5年内。本研究的主要目的是利用ALS转基因小鼠模型阐明运动神经元死亡的机制。我们的目标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，是体内能量产生的主要来源。它们在维持神经细胞存活方面起着关键作用。病理驱动的这些细胞器的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.