Impairment of the glial glutamate transporter GLT1 in ALS

描述(申请人提供)：胶质细胞导致运动神经元退化，并加重肌萎缩侧索硬化症(ALS)的进展，ALS是人类运动系统的一种特发性、致命的神经退行性疾病。这一贡献的性质尚未完全确定。星形胶质细胞谷氨酸转运蛋白EAAT2(又名：EAAT2)表达和活性低下。GLT-1)在肌萎缩侧索硬化症中已有报道，随后谷氨酸积聚引起的兴奋性毒性是导致运动神经元退化的一种传统机制。我们揭开了EAAT2触发运动神经元毒性的一种非常规方式。我们发现caspase-3选择性地切割ALS中的EAAT2，可能是通过限制星形胶质细胞中caspase-3的非凋亡性激活，产生胞浆EAAT2 C末端SUMO1片段(CTE-SUMO1)。当该片段在星形胶质细胞中外源表达时，聚集在PML核体的细胞核中，并通过增加Netrin-1的表达和释放的机制间接导致运动神经元毒性。少突胶质细胞，而不是星形胶质细胞，在正常的成年中枢神经系统中分泌Netrin-1来维持轴突内环境的稳定。因此，CTE-SUMO1+星形胶质细胞异常的非生理性释放netrin-1也可能是体内运动神经元毒性的一种非细胞自主机制。在这项应用中，我们建议通过在体内测试CTE-SUMO1是否与运动神经元损伤和ALS样表型有关的假说来扩大我们体外研究结果的相关性。为了重申我们在特定目标方面的目标，我们建议：(1)确定CTE-SUMO1是否是体内运动神经元损伤的介质，并评估毒性的方式；(2)确定阻止CTE-SUMO1的积累是否会抑制ALS的表型。目的1在成年小鼠腹角星形胶质细胞中选择性表达CTE-SUMO1，以评价其对C4-C6颈椎节段脊髓内注射AAV1-CTE-SUMO1病毒的整个膈运动神经元池的可能毒性作用。目的2建立一种新的敲入SOD1-G93A小鼠模型，在该模型中，EAAT2中的caspase-3裂解位点发生突变，以阻止内源性CTE-SUMO1的产生。除了这些体内实验，我们建议：(3)确定负责CTE-SUMO1在星形胶质细胞中积聚的疾病相关效应因子。我们希望揭开星形胶质细胞产生CTE-SUMO1的方式，以及家族性ALS相关致病突变的存在是否催化了这一过程。重要的是要了解哪些效应子可以导致EAAT2的切割，从而导致CTE-SUMO1的产生。然后，这些知识可用于设计旨在阻止ALS中CTE-SUMO1产生的药理学方法。最后一个目标的范围可能是关键，特别是如果目标1-2的结果确立了星形胶质细胞CTE-SUMO1片段在运动神经元毒性和ALS发病机制中的作用。

DESCRIPTION (provided by applicant): Glia cells contribute to motor neuron degeneration and aggravate the progression of amyotrophic lateral sclerosis (ALS), an idiopathic, fatal neurodegenerative disease of the human motor system. The nature of this contribution is not yet fully defined. Deficient expression and activity of the astroglia glutamate transporter EAAT2 (a.k.a. GLT-1 in rodents) has been reported in ALS and the excitotoxicity resulting from the ensuing accumulation of glutamate is one conventional mechanism that could contribute to motor neuron degeneration. We unraveled an unconventional way for EAAT2 to trigger motor neuron toxicity. We showed that caspase-3 selectively cleaves EAAT2 in ALS, presumably through a mechanism that involves restricted non-apoptotic caspase-3 activation in astrocytes, generating a cytosolic EAAT2 C-terminus SUMOylated fragment (CTE- SUMO1). This fragment, when exogenously expressed in astrocytes, accumulates in their nuclei in PML- nuclear bodies and indirectly causes motor neuron toxicity via a mechanism that involves increased expression and release of netrin-1. Oligodendrocytes, not astrocytes, secrete netrin-1 in the normal adult CNS to maintain axonal homeostasis. Hence, an abnormal non-physiological release of netrin-1 from CTE-SUMO1+ astrocytes could be one non-cell autonomous mechanism of toxicity to motor neurons in vivo as well. We propose in this application to expand the relevance of our in vitro findings by testing in vivo the hypothesis of whether CTE-SUMO1 is responsible for motor neuron impairment and ALS-like phenotype. To restate our goal in terms of specific aims, we propose: (1) To determine whether CTE-SUMO1 is a mediator of motor neuron impairment in vivo and assess the modalities of toxicity; (2) To determine whether preventing the accumulation of CTE-SUMO1 subsides the ALS phenotype. Aim 1 involves selective expression of CTE-SUMO1in ventral horn astrocytes of adult mice to evaluate its possible toxic effects on the entire phrenic motor neuron pool targeted via intraspinal focal injections of an AAV1-CTE-SUMO1 viral construct at the C4-C6 cervical level. Aim 2 involves the creation of a novel knock-in SOD1-G93A mouse model in which the caspase-3 cleavage site in EAAT2 is mutated to prevent the endogenous production of CTE-SUMO1. In addition to these in vivo experiments, we propose: (3) To determine the disease-relevant effectors responsible for CTE-SUMO1 accumulation in astrocytes. We expect to unravel the modalities of CTE-SUMO1 generation in astrocytes and whether the presence of familial ALS-linked causative mutations catalyzes the process. It is a matter of importance to understand what effectors could lead to the cleavage of EAAT2 and therefore the ensuing CTE-SUMO1 creation. This knowledge can then be applied to design pharmacological approaches aiming at arresting CTE-SUMO1 production in ALS. The scope of this last aim could then be key, in particular if results of aim 1-2 establisha role of the astroglial CTE-SUMO1 fragment in motor neuron toxicity and ALS pathogenesis.