Mechanisms of Motor Neuron Disease

ATP 7A是一种P型ATP酶，其通过在高尔基体网络（trans-Golgi network，TGN）和质膜（plasma membrane，PM）的活性调节细胞铜稳态，其位置通常由细胞内铜浓度控制。ATP 7A缺陷导致门克斯病或其变体、枕角综合征和ATP 7A相关远端运动神经病，这是一种新发现的疾病，其确切的病理生理学尚不清楚。我们的特点是两个ATP 7A运动神经病突变（T994 I，P1386 S）以前与异常细胞内运输。在患者成纤维细胞中，全内反射荧光（TIRF）显微镜检查表明ATP 7AT 994 I和ATP 7AP 1386 S的稳态平衡发生了变化，PM定位过多。转染293 T细胞和NSC-34运动神经元与标记与金星荧光蛋白的突变等位基因也表现出较高的PM定位。从PM到TGN的突变等位基因的内吞恢复被延迟。免疫沉淀分析显示，ATP 7AT 994 I和p97/VCP，TGN居民蛋白与其他两个遗传性运动神经病变，包括肌萎缩侧索硬化症，异常之间的相互作用。siRNA敲除p97/VCP改善了ATP 7AT 994 I的定位。流式细胞术记录了未透化的ATP 7AP 1386 S成纤维细胞结合羧基末端ATP 7A抗体，这与第8跨膜螺旋的不稳定插入和双亮氨酸内吞回收信号重新定位到PM的细胞外表面一致。这些发现1）阐明了ATP 7A相关远端运动神经病的潜在机制，2）建立了运动神经元疾病遗传学不同形式之间的共同联系，3）阐明了ATP 7A内吞作用的正常过程，4）强调了ATP 7A在周围神经系统中可能的功能作用。

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease, or its variants, occipital horn syndrome and ATP7A-related distal motor neuropathy, a newly discovered condition for which the precise pathophysiology has been obscure. We characterized two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients fibroblasts, total internal reflection fluorescence (TIRF) microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with excess PM localization. Transfection of 293T cells and NSC-34 motor neurons with the mutant alleles tagged with Venus fluorescent protein also showed higher PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was delayed. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, a TGN-resident protein associated with two other inherited motor neuropathies, including amyotrophic lateral sclerosis. SiRNA knockdown of p97/VCP improved ATP7AT994I localization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with destabilized insertion of the 8th transmembrane helix and relocation of a di-leucine endocytic retrieval signal to the extracellular face of the PM. These findings 1) illuminated mechanisms underlying ATP7A-related distal motor neuropathy, 2) established a common link between genetically distinct forms of motor neuron disease, 3) clarified the normal process of ATP7A endocytosis, and 4) highlighted possible functional roles of ATP7A in the peripheral nervous system.