Role of Calpastatin in mutant SOD1-mediated motor neuron disease

描述（申请人提供）：肌萎缩性侧索硬化症（ALS）是一种毁灭性的运动神经元疾病，大多数患者在出现疾病症状后3-5年内死亡。大约90%的病例病因不明（散发性肌萎缩侧索硬化症或s -肌萎缩侧索硬化症）。在有家族病史（F-ALS）的患者中，约20%的患者有超氧化物歧化酶1基因（SOD1）突变。来自人类als患者的突变基因已被用于构建过表达突变人类SOD1蛋白的转基因小鼠。这些小鼠的临床症状与ALS患者非常相似，如运动神经元丧失，骨骼肌萎缩，并伴有后肢瘫痪和最终死亡。尽管这些小鼠模型已经存在了十多年，但目前还没有治愈ALS的方法。最近的证据表明神经元蛋白酶calpain异常激活，它可能是ALS运动神经元变性的原因。灭活该蛋白酶的最佳方法是用其自身的内源性抑制剂calpastatin （CAST）进行抑制。我们已经开发了一种转基因小鼠系，在运动神经元中表达非常高水平的CAST。我们将CAST Tg小鼠与ALS小鼠模型hSOD1G93A小鼠杂交，以预防ALS小鼠的运动神经元变性。我们的初步数据表明，CAST在hSOD1G93A小鼠中过表达可提高运动轴突的存活率，双转基因小鼠（hSOD1G93A/CAST）比单独的hSOD1G93A小鼠寿命延长3.4个月。基于这些观察结果，我们提出在SOD1G93A突变小鼠中过表达CAST可以抑制运动神经元的退化，延长动物的寿命。从我们的项目中获得的结果将有助于开发针对ALS的特异性钙蛋白酶抑制剂疗法。我们将使用小鼠育种技术、Southern Blotting、聚合酶链反应、免疫Blotting、免疫细胞化学、电子显微镜和Rotarod测试来实现我们的目标。公共卫生相关性：肌萎缩侧索硬化症（ALS）是一种毁灭性的运动神经元疾病，患者在发病后3-5年内死亡。我们将在ALS小鼠模型中测试一种蛋白酶抑制剂（Calpain-inhibitor， CAST）治疗以预防运动神经元疾病。如果我们的方法取得成功，我们希望开发针对ALS的特异性钙蛋白酶抑制剂疗法。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease and most of the patients die within 3-5 years after disease symptoms are seen. About 90% of the cases the cause of the disease is unknown (sporadic-ALS or S-ALS). About 20% of the cases of the patients that have a family history (F-ALS) have mutations in super oxide dismutase 1 gene (SOD1). Mutant genes from human-ALS patients have been used to construct transgenic mice that overexpress mutant human- SOD1 proteins. These mice develop clinical symptoms very similar to ALS patients such as loss of motor neurons, skeletal muscle atrophy associated with hind limb paralysis and ultimate death. Although, these mouse models have been around for more than a decade the cure is not available for ALS. Recent evidence suggests that neuronal protease calpain is abnormally activated and it may be responsible for degeneration of motor neurons in ALS. The best way to inactivate this protease is to inhibit with its own endogenous inhibitor calpastatin (CAST). We have developed a transgenic mouse line that expresses very high levels of CAST in motor neurons. We will breed CAST Tg mice with mouse model of ALS, hSOD1G93A mice to prevent motor neuron degeneration in ALS mice. Our preliminary data suggests that CAST overexpression in hSOD1G93A mice results in increased survival of motor axons and the double transgenic mice (hSOD1G93A/CAST) live 3.4 months longer than hSOD1G93A mice alone. Based on these observations, we propose that overexpression of CAST in mutant SOD1G93A mice will inhibit motor neuron degeneration and prolong the life span of the animals. The results obtained from our project will help to develop specific calpain inhibitor therapy for ALS. We will use mouse breeding techniques, Southern Blotting, polymerase chain reactions, immunoblotting, immunocytochemistry, electron microscopy and Rotarod tests to achieve our goals. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease and patients die within 3-5 years after disease onset. We will test a protease inhibitor (Calpain-inhibitor, CAST) treatment in mouse model of ALS to prevent motor neuron disease. If we succeed with our approach, we would like to develop specific calpain inhibitor therapy for ALS.