Rad and amyotrophic lateral sclerosis (ALS)

肌萎缩侧索硬化症(ALS)是一种成人起病的神经退行性疾病，通常在 三到五年出现症状。这种疾病的特点是进行性丧失 运动神经系统和随意肌肉功能，对中枢或感觉神经功能没有太大影响。 出于这个原因，受苦的人在屈服于 呼吸麻痹。到目前为止，还没有真正有效的方法来减缓或逆转ALS的进展。 虽然运动神经元死亡是ALS最公认的特征，但ALS背后的分子事件 并不限于神经系统。对肌萎缩侧索硬化症病理的更准确的描述是瘫痪发生 由于电机部件的损失。在这方面，运动神经元和神经元之间脆弱联系的保真度 骨骼肌(神经肌肉接头)不仅依赖于神经元的输入，而且还依赖于 效应器肌肉。因此，这项研究的长期目标是了解骨骼肌是如何 功能障碍导致神经肌肉接头不稳定和运动神经元死亡 多种形式的肌萎缩侧索硬化症。在本提案规定的持续时间内，单体G蛋白Rad(RAS- 与糖尿病相关)在肌萎缩侧索硬化症发病机制中的作用将被研究。RAD特别值得关注 因为：1)散发性肌萎缩侧索硬化症患者在出现症状之前，肌肉中Rad的表达增强 患者和两个已建立的家族性ALS小鼠模型(SOD1G93A和SOD1G86R)，2)Rad是一种有效的抑制剂 骨骼肌L型钙通道(CaV1.1)；3)Rad的慢性上调引起明显的肌肉 萎缩。初步数据还表明，CaV1.1-L型钙通道和钙通道的两种生理功能 兴奋-收缩(EC)偶联的电压传感器在全球范围内的小鼠肌肉中逐渐受损 在表达SOD1G93A的小鼠和SOD1G93A仅限于骨骼肌表达的小鼠中。目标1 将决定肌肉中Rad的慢性上调是否会导致NMJ不稳定和随后 运动神经元死亡。在这些实验中，AAV1介导的肌肉特异性Rad过度表达的影响 对运动神经元活性的纵向评估将使用免疫组织学和 电生理学方法。在Aim 2中，将创建一个Rad Null-SOD1G93A鼠系，以揭示 Rad直接参与促进肌肉萎缩、NMJ失稳和运动神经元死亡。真恶心， 然后将使用运动、组织学、超微结构和电生理技术来确定 基因消融Rad是否可以限制SOD1G93A对肌肉完整性和 运动神经元的生存能力。利用qrt-pcr、免疫印迹和膜片钳电生理学，Aim 3将检测 Rad表达增强和CaV1.1功能下调是否在多种类型的ALS中共同存在。

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within three to five years of the appearance of symptoms. The disease is characterized by the progressive loss of the motor nervous system and voluntary muscle function without great effect on central or sensory neural function. For this reason, afflicted individuals effectively become prisoners within their own bodies before succumbing to respiratory paralysis. To date, there is no truly effective means to slow or reverse the progression of ALS. While motoneuron death is the most recognized characteristic of ALS, the molecular events that underlie ALS are not restricted to the nervous system. A more accurate description of ALS pathology is that paralysis occurs as a result of motor unit loss. In this regard, the fidelity of the fragile connections between motoneurons and skeletal muscle (neuromuscular junctions) is dependent not only on neuronal input but also on the integrity of the effector muscle. Thus, the long-term goal of this research is to understand how skeletal muscle dysfunction contributes to the destabilization of neuromuscular junctions and motoneuron death in multiple forms of ALS. In the defined duration of this proposal, a role for the monomeric G protein Rad (Ras- related Associated with Diabetes) in ALS pathogenesis will be investigated. Rad is of particular interest because: 1) Rad expression is enhanced just prior to presentation of symptoms in muscle of sporadic ALS patients and two established familial ALS mouse models (SOD1G93A and SOD1G86R), 2) Rad is a potent inhibitor of skeletal muscle L-type Ca2+ channels (CaV1.1), and 3) chronic upregulation of Rad causes marked muscle atrophy. Preliminary data also indicate that the two physiological functions of CaV1.1 - L-type Ca2+ channel and voltage-sensor for excitation-contraction (EC) coupling - are progressively impaired in muscle of mice globally expressing SOD1G93A and in mice in which SOD1G93A expression has been restricted to skeletal muscle. Aim 1 will determine whether chronic upregulation of Rad in muscle can cause NMJ destabilization and subsequent motoneuron death. In these experiments, the impact of AAV1-mediated, muscle-specific Rad overexpression on motoneuron viability will be assessed longitudinally using a combination of immunohistological and electrophysiological methods. In Aim 2, a Rad null-SOD1G93A mouse line will be created in order to reveal the direct involvement of Rad in promoting muscle atrophy, NMJ destabilization and motoneuron death. Gross, locomotor, histological, ultrastructural and electrophysiological techniques will then be employed to determine whether genetic ablation of Rad can limit the deleterious effects of SOD1G93A on muscle integrity and motoneuron viability. Using qRT-PCR, immunoblotting and patch-clamp electrophysiology, Aim 3 will test whether enhanced Rad expression and depression of CaV1.1 function are common to multiple forms of ALS.