Sasaki Shoichi, Yamashita Takenari, Kwak Shin
Department of Neurology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
Department of Neurology, Graduate School of Medicine, University of Tokyo, Japan; Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Japan.
Neurosci Lett. 2015 Jun 26;598:79-84. doi: 10.1016/j.neulet.2015.05.025. Epub 2015 May 14.
In the motor neurons of amyotrophic lateral sclerosis (ALS) patients, an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) is down-regulated and consequently GluA2 mRNAs unedited at the Q/R site is expressed in contrast to normal motor neurons that express only GluA2 edited at this site. Motor neurons of the mice lacking ADAR2 undergo Ca(2+)-permeable AMPA receptor-mediated slow death. We investigated the spinal cords of conditional ADAR2-knockout mice modeling ALS for the involvement of autophagy. In the motor neurons of the early- and late-symptomatic-stage mice, LC3-immunopositivity or immunoreactivity for both LC3- and p62 was observed, whereas the presymptomatic-stage mice showed no LC3- or p62-immunoreactivity. Western blot analyses showed increased expression of autophagy associated proteins in the anterior horn of the early symptomatic-stage mice. Electron-microscopically, autophagy was observed in the motor neurons most frequently in the early-symptomatic-stage mice which showed the severest motor neuron degeneration. Increased autophagy flux was not recognized in the wild-type mice or AR2res (ADAR2(flox/flox)/VAChT-Cre. Fast/GluR-B(R)(/)(R)) mice having motor neurons genetically engineered to express normally edited GluA2 in the absence of ADAR2, which show normal Ca(2+)-permeability of the AMPA receptors in motor neurons. Significantly increased autophagy flux in the degenerating motor neurons of ADAR2-knockout mice likely resulted from Ca(2+) overload.
在肌萎缩侧索硬化症（ALS）患者的运动神经元中，一种名为作用于RNA 2的腺苷脱氨酶（ADAR2）的RNA编辑酶表达下调，因此与仅表达在此位点编辑的GluA2的正常运动神经元相反，在Q/R位点未编辑的GluA2 mRNA得以表达。缺乏ADAR2的小鼠的运动神经元会经历钙（Ca2+）通透性AMPA受体介导的缓慢死亡。我们研究了模拟ALS的条件性ADAR2基因敲除小鼠的脊髓，以探究自噬的参与情况。在早发性和晚发性症状期小鼠的运动神经元中，观察到LC3免疫阳性或LC3和p62的免疫反应性，而症状前期小鼠未显示LC3或p62免疫反应性。蛋白质印迹分析表明，早发性症状期小鼠前角中自噬相关蛋白的表达增加。电子显微镜观察发现，在运动神经元中，自噬最常出现在早发性症状期小鼠中，这些小鼠的运动神经元退化最为严重。在野生型小鼠或AR2res（ADAR2（flox/flox）/VAChT-Cre.Fast/GluR-B（R）（/）（R））小鼠中未发现自噬通量增加，这些小鼠的运动神经元经过基因工程改造，在没有ADAR2的情况下正常表达编辑后的GluA2，其运动神经元中的AMPA受体具有正常的钙（Ca2+）通透性。ADAR2基因敲除小鼠退化的运动神经元中自噬通量显著增加可能是由钙（Ca2+）过载导致的。
