Role of SOD Instability in ALS Motor Neuron Toxicity

SOD 不稳定性在 ALS 运动神经元毒性中的作用

• 批准号: 6894805
• 负责人: LAWRENCE J HAYWARD
• 金额: $ 33.99万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6894805
• 关键词: acidity /alkalinity; amyotrophic lateral sclerosis; analytical ultracentrifugation; apoptosis; biophysics; calorimetry; chemical stability; copper; enzyme activity; gene expression; gene mutation; genetic mapping; genetic susceptibility; lysosomes; motor neurons; mutant; neoplastic cell culture for noncancer research; neuroblastoma; neurogenetics; neurotoxicology; protein denaturation; protein folding; superoxide dismutase; thermodynamics; thermostability; tissue /cell culture; zinc

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is an age-dependent degenerative disorder of motor neurons characterized by progressive weakness and death within five years after the onset of symptoms. A subset of familial ALS is caused by mutations in Cu/Zn superoxide dismutase (SOD1) that render this antioxidant enzyme toxic by an unknown mechanism. Expression of mutant SOD1 in man or mice causes selective degeneration of motor neurons in the brain and spinal cord. The long-term objectives of this study are to understand how mutant SOD1 kills neurons and to identify novel pathways of cell death that may be relevant to ALS. Aim 1 of this project is to define important biophysical and biochemical characteristics that distinguish mutant from wild type SOD1. Recent studies in our lab indicate that mutant SOD1 enzymes all share an increased tendency to unfold or to lose metal ions when stressed by denaturing influences. Experiments will be performed to (1) define conditions that preferentially destabilize mutant but not normal SOD1, (2) identify factors that alter the dynamics of the SOD1 monomer-dimer equilibrium as measured by analytical ultracentrifugation, and (3) map regions of the protein that are susceptible to proteolytic cleavage or partial unfolding. In Aim 2, the affinities and thermodynamics of metal binding to SOD1 as a function of pH or denaturant will be measured using a novel calorimetric approach. Conditions that alter Cu ion reactivity in mutant SOD1 will also be defined. Aim 3 will employ cell culture models to test hypotheses of mutant SOD1 toxicity that are consistent with the physicochemical findings from Aims 1 and 2. Initial investigations will test whether early lysosomal dysfunction occurs in response to specific stresses in mutant SOD1 -bearing neuroblastoma cells or in organotypic spinal cord slice cultures. Future results from Aims 1 and 2 may favor alternative hypotheses of mutant SOD1 toxicity to test in these cellular models and may suggest new therapeutic approaches to evaluate in ALS mice.

描述（由申请人提供）：肌萎缩侧索硬化症（ALS，卢伽雷氏病）是一种年龄依赖性运动神经元退行性疾病，其特征是症状出现后五年内进行性无力和死亡。家族性 ALS 的一部分是由铜/锌超氧化物歧化酶 (SOD1) 突变引起的，这种突变通过未知机制使这种抗氧化酶产生毒性。突变型 SOD1 在人或小鼠中的表达会导致大脑和脊髓中运动神经元的选择性变性。这项研究的长期目标是了解突变体 SOD1 如何杀死神经元，并确定可能与 ALS 相关的新细胞死亡途径。 该项目的目标 1 是确定区分突变型和野生型 SOD1 的重要生物物理和生化特征。我们实验室最近的研究表明，当受到变性影响时，突变型 SOD1 酶都具有增加的展开或失去金属离子的倾向。进行实验的目的是 (1) 确定优先破坏突变体而非正常 SOD1 稳定性的条件，(2) 确定通过分析超速离心测量改变 SOD1 单体-二聚体平衡动态的因素，以及 (3) 绘制蛋白质区域图易于蛋白水解裂解或部分解折叠。 在目标 2 中，将使用一种新颖的量热方法测量金属与 SOD1 结合的亲和力和热力学作为 pH 或变性剂的函数。改变突变体 SOD1 中铜离子反应性的条件也将被定义。 目标 3 将采用细胞培养模型来测试突变型 SOD1 毒性的假设，该假设与目标 1 和 2 的理化结果一致。初步研究将测试早期溶酶体功能障碍是否是由于突变型 SOD1 神经母细胞瘤细胞或突变型 SOD1 神经母细胞瘤细胞中的特定应激而发生的。器官型脊髓切片培养物。目标 1 和 2 的未来结果可能有利于在这些细胞模型中测试突变型 SOD1 毒性的替代假设，并可能提出在 ALS 小鼠中进行评估的新治疗方法。