Mutation of the caspase-3 cleavage site in the astroglial glutamate transporter EAAT2 delays disease progression and extends lifespan in the SOD1-G93A mouse model of ALS.
Mutation of the caspase-3 cleavage site in the astroglial glutamate transporter EAAT2 delays disease progression and extends lifespan in the SOD1-G93A mouse model of ALS.
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DOI：
10.1016/j.expneurol.2017.03.014
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发表时间：
2017-06
影响因子：
5.3
通讯作者：
Trotti D
中科院分区：
文献类型：
--
作者：
Rosenblum LT;Shamamandri-Markandaiah S;Ghosh B;Foran E;Lepore AC;Pasinelli P;Trotti D
肌萎缩侧索硬化症（ALS）患者和突变型SOD 1小鼠模型中星形胶质细胞谷氨酸转运蛋白EAAT 2的下调被认为是兴奋性毒性导致运动神经元死亡的原因。我们以前报道过，胱天蛋白酶-3切割EAAT 2在一个独特的切割共识网站位于其C-末端结构域，蛋白水解切割，也发生在体内突变的SOD 1小鼠模型的ALS，并导致积累的sumoylated EAAT 2 C-末端片段（CTE-SUMO 1）开始发病。CTE-SUMO 1在星形胶质细胞的PML核体中积累，并导致它们改变其成熟表型并分泌对运动神经元有毒的因子。在这里，我们报告突变的天冬氨酸天冬酰胺突变（D504 N），从而抑制caspase-3切割EAAT 2的EAAT 2序列中的caspase-3的共识网站，赋予保护的SOD 1-G93 A小鼠。产生EAAT 2-D504 N敲入突变小鼠并与SOD 1-G93 A小鼠杂交以评估EAAT 2切割的ALS症状的体内致病相关性。该突变不影响正常EAAT 2功能，也不影响非ALS小鼠。与CTE-SUMO 1积累的时间一致，虽然疾病的发作不受影响，但突变导致进展时间延长，后肢和前肢肌肉无力的发展延迟，以及SOD 1-G93 A小鼠寿命的显著增加。
Downregulation in the astroglial glutamate transporter EAAT2 in amyotrophic lateral sclerosis (ALS) patients and mutant SOD1 mouse models of ALS is believed to contribute to the death of motor neurons by excitotoxicity. We previously reported that caspase-3 cleaves EAAT2 at a unique cleavage consensus site located in its c-terminus domain, a proteolytic cleavage that also occurs in vivo in the mutant SOD1 mouse model of ALS and leads to accumulation of a sumoylated EAAT2 C-Terminus fragment (CTE-SUMO1) beginning around onset of disease. CTE-SUMO1 accumulates in PML nuclear bodies of astrocytes and causes them to alter their mature phenotypes and secrete factors toxic to motor neurons. Here, we report that mutating the caspase-3 consensus site in the EAAT2 sequence with an aspartate to asparagine mutation (D504N), thereby inhibiting caspase-3 cleavage of EAAT2, confers protection to the SOD1-G93A mouse. EAAT2-D504N knock-in mutant mice were generated and crossed with SOD1-G93A mice to assess the in vivo pathogenic relevance for ALS symptoms of EAAT2 cleavage. The mutation did not affect normal EAAT2 function nor non-ALS mice. In agreement with the timing of CTE-SUMO1 accumulation, while onset of disease was not affected, the mutation caused an extension in progression time, a delay in the development of hindlimb and forelimb muscle weakness, and a significant increase in the lifespan of SOD1-G93A mice.