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: 6765780
• 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/6765780
• 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）：肌萎缩性侧索硬化症（ALS，Lou Gehrig病）是一种依赖年龄的运动神经元退化性疾病，其特征是症状发作后五年内进行性无力和死亡。家族性ALS的子集是由Cu/Zn超氧化物歧化酶（SOD1）突变引起的，该突变通过未知机制使这种抗氧化酶有毒。突变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相关的细胞死亡途径。 该项目的目的1是定义将突变体与野生型SOD1区分开的重要生物物理和生化特征。我们实验室中的最新研究表明，突变体SOD1酶在通过贬低影响压力时，都具有增加或失去金属离子的趋势。实验将进行（1）定义优先破坏突变体但不是正常SOD1的条件，（2）确定因通过分析性超细胞化分析来测量的SOD1单体二聚体平衡的因素，（3）对蛋白质的蛋白质区域（3）对蛋白质的蛋白质区域易于蛋白质，这些区域易于蛋白质，这些区域易于蛋白质，可易于蛋白质。 在AIM 2中，将使用一种新型的量热法测量金属与SOD1的亲和力和热力学作为pH或变性剂的函数。还将定义改变突变体SOD1中Cu离子反应性的条件。 AIM 3将采用细胞培养模型来测试与AIM 1和2的物理化学发现相一致的突变SOD1毒性的假设。初步研究将测试是否响应突变体SOD1的特定胁迫，是否发生了早期的溶酶体功能障碍，这是伴有神经细胞瘤细胞中的特定应激而发生的。 AIM 1和2的未来结果可能有利于突变SOD1毒性的替代假设在这些细胞模型中测试，并可能建议在ALS小鼠中评估新的治疗方法。