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

基本信息

批准号：
7674172
负责人：
Davide Trotti
金额：
$ 33.8万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
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/7674172
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 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 public health relevance uptake

项目摘要

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（申请人提供）：在肌萎缩侧索硬化症（ALS）突变型SOD1-G93A（mutSOD1）小鼠模型中，突变形式的SOD1选择性地与脊髓线粒体中的Bcl-2结合并聚集。在本研究中，我们将检验以下假设：位于脊髓线粒体中的 mutSOD1 部分必须与 Bcl-2 配合才能表现出毒性。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影响。通过对从双转基因 ALS 小鼠 SOD1-93A: mitoCFP 中分离的完整、神经元和非神经元脊髓线粒体采用膜片钳电生理学技术（线粒体附着配置）并应用，我们将系统地表征外线粒体的电导膜（OMM）贯穿疾病的不同阶段，确定这种线粒体表型是否随着疾病的进展而发生特异性改变。我们提出三个具体目标。在目标#1中，我们将在体外研究mutSOD1介导的细胞和线粒体水平的毒性及其对Bcl-2的依赖性。在目标#2中，我们将原位研究从双转基因SOD1-G93A的脊髓中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 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 mutSOD1 蛋白对线粒体外膜通道的影响。在目标 #3 中，我们将通过生成 SOD1-G93A:Bcl-2(-/-) 小鼠并进行比较，在体内测试以下假设：mutSOD1 与 Bcl-2 的结合是运动神经元毒性所必需的，也是 ALS 表型的决定因素。将这些小鼠的疾病表型与 Bcl-2 未消除的 SOD1-G93A 小鼠进行比较。通过开发这些目标，我们将了解 mutSOD1 是否通过与抗凋亡蛋白 Bcl-2 相互作用而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。公众健康相关性肌萎缩侧索硬化症（ALS；又名卢伽雷氏病）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层运动神经元的死亡。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，发生在首次症状出现后 1 - 5 年内。该提案的总体目标是利用 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，是体内能量产生的主要来源。它们在维持神经元细胞存活方面发挥着关键作用。这些细胞器的病理驱动损伤可能会改变生与死之间的平衡，并导致神经元变性。