Toxicity of a SUMOylated Fragment of the Glial Glutamate Transporter EAAT2 in ALS

描述(由申请人提供)：在美国，任何时候都有30,000例肌萎缩侧索硬化症病例。目前，肌萎缩侧索硬化症还没有治愈方法，治疗方案也很少。90%的病例病因不明，但10%的病例有明确的遗传来源。对遗传病例的研究使人们对这种疾病有了很好的了解，这既适用于散发病例，也适用于家族病例。基于肌萎缩侧索硬化症相关基因突变的动物模型和散发性肌萎缩侧索硬化症的体外模型的持续工作将继续增加对这种疾病的了解。虽然ALS的特点是上下运动神经元的变性，但也伴随着神经胶质细胞的功能障碍。特别是，在ALS患者和动物模型中，胶质谷氨酸转运体EAAT2的蛋白水平降低。EAAT2被caspase 3切割，产生截短形式的EAAT2和C-末端片段(CTE)。在ALS的SOD1-G93A小鼠模型中，CTE的SUMOylated版本(CTE-SUMO1)在脊髓内积聚。CTE-SUMO1的产生机制必须在体外和体内进行分析，才能潜在地发现其毒性机制。有两条不同的途径必须被共激活才能产生CTE-SUMO1：激活Capase-3导致EAAT2裂解和EAAT2/CTE的SUMO化。拟议的研究将在肌萎缩侧索硬化症的参数范围内检查这两个反应的动力学。此外，对星形胶质细胞内CTE-SUMO1表达可能对邻近运动神经元的毒性影响的研究将揭示ALS发病的另一个可能领域。以前在共培养模型中的研究表明，CTE-SUMO1在星形胶质细胞核内的积聚对邻近的运动神经元是有害的。这个问题将使用一种新的转基因小鼠模型在体内进一步研究。 公共卫生相关性：在ALS的患者和动物模型中都可以看到EAAT2生理学的改变，因此EAAT2功能障碍的检查代表了疾病的一个可接近和重要的方面。这里提出的想法是新颖的，因为结合EAAT2的翻译后修饰还没有被研究过。由于受损的EAAT2和CTE-SUMO1在星形胶质细胞中的核积聚引起的协同毒性可能导致运动神经元死亡；由于EAAT2的下调是ALS中为数不多的坚定事件之一，治疗干预的合乎逻辑的方法是通过增加EAAT2的表达水平来从药物上(如利鲁珠)或从基因上增加突触对谷氨酸的摄取。然而，如果CTE-SUMO1导致毒性，有限的治疗方法将被证明效果较差；相反，联合治疗将是必要的。

DESCRIPTION (provided by applicant): There are 30,000 cases of ALS at any given time in the United States. Presently there is no cure for ALS and few treatment options. 90 percent of cases have unknown etiology, but 10 percent have a clear genetic origin. Studying the genetic cases has allowed great insight into the disease, which is applicable to both sporadic and familial cases. Continued work with animal models based on ALS-linked gene mutations and in vitro models of sporadic ALS will continue to increase knowledge of the disorder. Although ALS is characterized by the degeneration of upper and lower motor neurons there is concomitant dysfunction of glial cells. In particular, protein levels of the glial glutamate transporter EAAT2 decreases in patients and animal models of ALS. EAAT2 is cleaved by caspase 3 creating a truncated form of EAAT2 and a C-terminal fragment (CTE). A SUMOylated version of CTE (CTE-SUMO1) accumulates within the spinal cord of the SOD1-G93A mouse model of ALS. The mechanisms of CTE-SUMO1 creation must be analyzed in vitro and in vivo in order to potentially discover the mechanisms of toxicity. There are two distinct pathways that must be co-activated to create CTE- SUMO1; activation of capase-3 leading to EAAT2 cleavage and the SUMOylation of EAAT2/CTE. The proposed studies will examine the dynamics of both reactions within the parameters of ALS. In addition examination of the possible toxic effects that CTE-SUMO1 expression within astrocytes may have on neighboring motor neurons will expose another possible area of ALS pathogenesis. Previous work in a co-culture model has indicated that CTE-SUMO1 accumulation within astrocyte nuclei is toxic to neighboring motor neurons. This question will be further examined in vivo using a novel transgenic mouse model. PUBLIC HEALTH RELEVANCE: Alterations to EAAT2 physiology have been seen in both patients and animal models of ALS, thus examination of EAAT2 dysfunction represents an accessible and important aspect of disease. The ideas presented here are novel in that a post-translational modification combined with a cleavage of the EAAT2 has not been studied. Concerted toxicity arising from impaired EAAT2 and CTE-SUMO1 nuclear accumulation in astrocytes could cause motor neuron death; because downregulation of EAAT2 is one of the few firm events occurring in ALS, a logical approach for a therapeutic intervention is to pharmacologically (e.g. Riluzule) or genetically increase synaptic uptake of glutamate by increasing the expression levels of EAAT2. However, if CTE- SUMO1 contributes to toxicity, a limited therapeutic approach would prove to be less efficacious; instead a combinatorial approach would be necessary.