The contribution of cytoskeleton disruptions in ALS disease pathology

 描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种致命的神经退行性疾病，其特征在于大脑和脊髓运动神经元的选择性丧失。疾病进展迅速，患者通常在发病后3-5年死于呼吸衰竭。已经鉴定了许多导致ALS的突变基因，但运动神经元变性的根本原因尚不清楚。最近对含有可遗传形式的ALS的家族的基因组分析已经确定了导致ALS的两个细胞骨架基因profilin 1和TUBA 4A中的多个突变;将细胞骨架功能障碍与ALS病理联系起来。在这个建议中，我将研究ALS突变基因是否破坏了两个对运动神经元健康至关重要的细胞神经依赖性功能。运动神经元从细胞体延伸出很远的结构，并严重依赖微管细胞骨架将细胞物质运输到这些远端结构。在第一部分中，我将研究突变体TUBA 4A的表达是否影响必需的微管为基础的货物运输和从远端结构。我首先将使用活细胞成像来追踪表达突变TUBA 4A的原代培养神经元中各种必需货物的运输速率。接下来，我将通过检测突变型TUBA 4A表达小鼠坐骨神经中运动蛋白运动的变化来研究发育或细胞相互作用是否起作用。最后，我将评估天气突变体TUBA 4A表达改变运动神经元对微管不稳定神经毒性损伤的耐受性。运动神经元的损伤导致运动神经元轴突与神经肌肉接头（NMJ）分离。在最初的NMJ去神经支配后，运动神经元可以遵循两条路径之一：轴突再生和重新附着到NMJ或继续轴突死亡和细胞死亡。受损轴突顶端微管的稳定性将影响这两条路径中哪一条将是最终结果。在第二部分的建议，我将检查是否突变TUBA 4A表达增加回缩球的形成和减少损伤后轴突再生。我将首先使用活细胞成像来测量表达突变TUBA 4A的原代运动神经元中切断的轴突的再生长速率。接下来，我将研究突变体TUBA 4A表达是否改变突变体TUBA 4A表达小鼠坐骨神经损伤后生长锥形成和微管组织的速率。最后，我将研究突变TUBA 4A是否影响神经损伤后表达突变TUBA 4A的小鼠肌肉组织的神经再支配。该提案的目标是进一步了解ALS疾病病理学中细胞因子依赖性功能的作用。从这项提案中获得的结果将为ALS中细胞死亡的原因提供新的见解，并将为药物研究和治疗开辟新的领域。

 DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons from the brain and spinal cord. Disease progression is rapid and patients typically die from respiratory failure 3-5 years following disease onset. Numerous ALS causing mutant genes have been identified but the underlying cause of motor neuron degeneration is not known. Recent genome analysis from families that contain a heritable form of ALS have identified multiple mutations in two cytoskeletal genes, profilin 1 and TUBA4A, that cause ALS; linking cytoskeletal dysfunction to ALS pathology. In this proposal I will examine whether an ALS mutant gene disrupts two cytoskeletal-dependent functions that are crucial for motor neuron health. Motor neurons extend structures great distances from the cell body and rely heavily on the microtubule cytoskeleton for the transport of cellular materials to these distal structures. In the first part of the proposl, I will examine whether expression of mutant TUBA4A affects the transport of essential microtubule based cargos to and from the distal structures. I first will use live cell imaging to track the rate of transport of various essential cargos in primary cultured neurons expressing mutant TUBA4A. Next I will examine if developmental or cellular interactions play a contributing role by examining changes in motor protein movement in the sciatic nerve of mutant TUBA4A expressing mice. Finally, I will evaluate weather mutant TUBA4A expression changes the motor neuron tolerance to microtubule-destabilizing neurotoxic insult. Injury to motor neurons causes separation of the motor neuron axon from the neuromuscular junction (NMJ). After the initial denervation of the NMJ the motor neuron can follow one of two paths: axon regrowth and reattachment to the NMJ or continued axon dieback and cell death. The stability of the microtubules in the tip of the injured axon will influence which of these two paths will be the finl outcome. In the second part of the proposal I will examine if mutant TUBA4A expression increases retraction bulb formation and decreases axon regeneration following injury. I will first use live cell imaging to measure the rate of regrowth of severed axons in primary motor neurons expressing mutant TUBA4A. I will next examine whether mutant TUBA4A expression changes the rate of growth cone formation and microtubule organization after sciatic nerve lesion in mutant TUBA4A expressing mice. Finally, I will examine whether mutant TUBA4A affects the reinnervation of muscle tissue following nerve crush in mutant TUBA4A expressing mice. The goal of this proposal is to further knowledge on how cytoskeleton-dependent functions are involved in ALS disease pathology. The results gained from this proposal will offer new insight into the cause of cell death in ALS and will open new areas for drug research and therapy.