Rogers Robert S, Tungtur Sudheer, Tanaka Tomohiro, Nadeau Lisa L, Badawi Yomna, Wang Hua, Ni Hong-Min, Ding Wen-Xing, Nishimune Hiroshi
Department of Anatomy and Cell Biology, University of Kansas School of MedicineKansas City, KS, United States.
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas School of MedicineKansas City, KS, United States.
Front Neurosci. 2017 Aug 25;11:473. doi: 10.3389/fnins.2017.00473. eCollection 2017.
Motor neurons in amyotrophic lateral sclerosis (ALS) patients and animal models show degeneration from the nerve terminal, known as dying-back neuropathy. To investigate the mechanism underlying this neuropathy, we analyzed the neuromuscular junctions (NMJs) and motor neuron cell bodies in SOD1 mice using electron microscopy. NMJs of SOD1 mice exhibited significantly higher numbers of autophagosomes and degenerated mitochondria compared to wild-type controls. Mitophagosomes were identified in the NMJ presynaptic terminals of wild-type mice and SOD1 mice. However, the number of mitophagosomes did not increase significantly in SOD1 NMJs indicating a defect in mitophagy, the autophagic process to degrade mitochondria. Consistent with this, proteins essential for mitophagy, p62/SQSTM1, Bnip3, Pink1, and Parkin were down-regulated in motor neurons in SOD1 mice. Importantly, SQSTM1 is one of the genes mutated in familial ALS patients. We evaluated the effect of impaired mitophagy on motor neurons by analyzing the double knockout mice of Pink1 and Parkin, two genes responsible for sensing depolarized mitochondria and delivering degenerated mitochondria to mitophagosomes. The double knockout mice exhibited NMJ degeneration, including axon swelling and NMJ fragmentation at 4 months of age. These phenotypes were rarely observed in wild-type control mice of the same age. The protein level of ATP synthase β subunit increased in the NMJ presynaptic terminals, suggesting the accumulation of mitochondria at NMJs of the double knockout mice. Importantly, NMJ denervation was observed in the double knockout mice. These data suggest that the reduced mitophagy function in motor neurons of SOD1 mice is one of the mechanisms causing degeneration of ALS NMJs.
肌萎缩侧索硬化症（ALS）患者和动物模型中的运动神经元表现出从神经末梢开始的退化，即逆行性神经病变。为了研究这种神经病变的潜在机制，我们使用电子显微镜分析了SOD1小鼠的神经肌肉接头（NMJ）和运动神经元细胞体。与野生型对照相比，SOD1小鼠的NMJ表现出自噬体和退化线粒体的数量显著增加。在野生型小鼠和SOD1小鼠的NMJ突触前末端都发现了线粒体自噬体。然而，SOD1小鼠的NMJ中线粒体自噬体的数量没有显著增加，这表明线粒体自噬存在缺陷，线粒体自噬是一种降解线粒体的自噬过程。与此一致的是，SOD1小鼠运动神经元中线粒体自噬所需的蛋白质p62/SQSTM1、Bnip3、Pink1和Parkin表达下调。重要的是，SQSTM1是家族性ALS患者中发生突变的基因之一。我们通过分析Pink1和Parkin的双敲除小鼠来评估线粒体自噬受损对运动神经元的影响，这两个基因负责感知去极化的线粒体并将退化的线粒体传递到线粒体自噬体。双敲除小鼠在4个月大时表现出NMJ退化，包括轴突肿胀和NMJ碎片化。在相同年龄的野生型对照小鼠中很少观察到这些表型。ATP合酶β亚基的蛋白水平在NMJ突触前末端升高，表明双敲除小鼠的NMJ中线粒体积累。重要的是，在双敲除小鼠中观察到了NMJ失神经支配。这些数据表明，SOD1小鼠运动神经元中线粒体自噬功能的降低是导致ALS患者NMJ退化的机制之一。
