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ALS病例。目前，ALS没有治愈方法，治疗选择很少。90%的病例病因不明，但10%有明确的遗传来源。研究遗传病例使人们对这种疾病有了更深入的了解，这种疾病适用于散发性和家族性病例。基于ALS连锁基因突变的动物模型和散发性ALS的体外模型的持续工作将继续增加对该疾病的了解。 尽管ALS的特征是上下运动神经元变性，但也伴随着胶质细胞功能障碍。特别是，神经胶质谷氨酸转运蛋白EAAT 2的蛋白质水平在ALS患者和动物模型中降低。EAAT 2被半胱天冬酶3切割，产生截短形式的EAAT 2和C末端片段（CTE）。SUMO化形式的CTE（CTE-SUMO 1）在ALS的SOD 1-G93 A小鼠模型的脊髓内积累。 必须在体外和体内分析CTE-SUMO 1产生的机制，以便潜在地发现毒性机制。有两种不同的途径必须被共激活以产生CTE-SUMO 1;导致EAAT 2切割的半胱天冬酶-3的激活和EAAT 2/CTE的SUMO化。拟议的研究将检查ALS的参数内的两个反应的动力学。 此外，检查星形胶质细胞内CTE-SUMO 1表达可能对邻近运动神经元产生的毒性作用，将揭示ALS发病机制的另一个可能领域。先前在共培养模型中的工作表明，星形胶质细胞核内的CTE-SUMO 1积累对邻近的运动神经元是有毒的。这个问题将在体内使用一种新的转基因小鼠模型进一步研究。 公共卫生相关性：在ALS的患者和动物模型中都观察到EAAT 2生理学的改变，因此EAAT 2功能障碍的检查代表了疾病的一个可访问和重要的方面。这里提出的想法是新颖的，因为翻译后修饰结合切割的EAAT 2尚未研究。由星形胶质细胞中受损的EAAT 2和CTE-SUMO 1核积累引起的协同毒性可导致运动神经元死亡;因为EAAT 2的下调是ALS中发生的少数几个确定事件之一，治疗干预的逻辑方法是通过增加EAAT 2的表达水平来抑制（例如，EAAT 2）或遗传地增加谷氨酸的突触摄取。然而，如果CTE-SUMO 1导致毒性，则有限的治疗方法将证明不太有效;相反，组合方法将是必要的。

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.