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

突变体 SOD 1 介导的 A 脊髓线粒体毒性机制

• 批准号: 8044026
• 负责人: Davide Trotti
• 金额: $ 33.12万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8044026
• 关键词: ATP Synthesis Pathway; Adult; Amyotrophic Lateral Sclerosis; Apoptotic; Area; BH3 Domain; Binding; Bioenergetics; Cells; Cessation of life; Complex; Consumption; Dependence; Disease; Disease Progression; Energy-Generating Resources; Equilibrium; Event; Failure; Familial Amyotrophic Lateral Sclerosis; Functional disorder; Goals; Health; Impairment; In Situ; In Vitro; Ion Channel; Lead; Life; Light; Link; Measures; Mediating; Membrane; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Molecular; Motor Cortex; Motor Neuron Disease; Motor Neurons; Mus; Muscular Atrophy; Mutate; Mutation; Nerve Degeneration; Neurons; Organelles; Outer Mitochondrial Membrane; Pathogenesis; Pathology; Pathway interactions; Patients; Permeability; Phenotype; Play; Population; Production; Property; Proteins; Publications; Reporting; Respiratory Muscles; Role; Spinal; Spinal Cord; Staging; Structure; Superoxide Dismutase; Symptoms; Techniques; Testing; Toxic effect; Transgenic Mice; Transgenic Organisms; direct application; disease mechanisms study; disease phenotype; fluorescence microscope; in vivo; mitochondrial membrane; motor neuron degeneration; mouse model; mutant; neurotoxicity; patch clamp; prevent; pro-apoptotic protein; uptake

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.

描述（由申请人提供）：在突变的SOD1-G93A（MUTSOD1）小鼠肌萎缩性侧索硬化症（ALS）中，SOD1的突变形式在脊髓线粒体中选择性结合并用Bcl-2结合和聚集。在这项研究中，我们将检验以下假设：脊髓线粒体中的MUTSOD1的一部分必须与Bcl-2合作以表现出毒性。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学的后果。 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随着疾病的发展，线粒体表型被特异性改变。我们提出了三个具体目标。在AIM＃1中，我们将在细胞和线粒体水平及其对Bcl-2的依赖性下研究体外MUTSOD1-介导的毒性。在AIM＃2中，我们将在原位研究从双转基因SOD1-G93A的脊髓分离出的神经元和非神经元线粒体的生物物理特性：携带荧光（青色）标记为蓝色神经元的蓝色神经元线粒体的MitoCFP小鼠。此外，我们将使用从表达Bcl-2与缺乏BCL-2和转基因小鼠的细胞的细胞中分离出的线粒体对MutSod1蛋白对线粒体外膜通道的影响。在AIM＃3中，我们将在体内检验一个假说，即MutSod1与Bcl-2的结合是运动神经元毒性所必需的，并且是通过产生SOD1-G93A：BCl-2（ - / - ）小鼠并将这些小鼠的疾病表型与SOD1-G93A小鼠与该BCL-2的bcl-2进行比较，并将这些小鼠的疾病表型与这些小鼠的疾病表型进行比较，而不是ALS表型的决定因素。通过开发这些目标，我们将通过与抗凋亡蛋白Bcl-2相互作用，MUTSOD1是否会导致线粒体功能障碍以及这种异常机制与ALS小鼠中运动神经元变性的直接相关性。公共卫生相关性肌萎缩性外侧硬化症（ALS；又名Lou Gehrig病）是最常见的成人运动神经元疾病。该疾病的特征是脊髓和运动皮质中运动神经元死亡。这会导致痉挛，超反射，一般无力和肌肉萎缩。呼吸道肌肉的失败通常是致命事件，发生在第一次症状发作后的1 - 5年内。该提案的总体目标是使用ALS的转基因小鼠模型阐明运动神经元死亡的机制。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，也是体内能源产生的主要来源。他们在维持神经元细胞活着中起关键作用。这些细胞器的病理损害可能会改变生与死之间的平衡，并导致神经元变性。