Mechanisms of Motor Neuron Disease

ATP7A是一种P型ATPase，它通过跨高尔基网络(TGN)和质膜(PM)的活性来调节细胞内铜的动态平衡，其位置通常受细胞内铜浓度的控制。ATP7A的缺陷导致Menkes病或其变体，枕角综合征和ATP7A相关的远端运动神经病，这是一种新发现的疾病，其确切的病理生理尚不清楚。我们鉴定了两个ATP7A运动神经病突变(T994I，P1386S)，它们以前与异常的细胞内转运有关。在患者成纤维细胞中，全内反射荧光(TIRF)显微镜显示ATP7AT994I和ATP7AP1386S的稳态平衡发生变化，PM定位过度。用金星荧光蛋白标记的突变等位基因转染293T细胞和NSC-34运动神经元，也显示了较高的PM定位。突变等位基因从PM到TGN的内吞检索被推迟。免疫沉淀分析显示ATP7AT994I和p97/VCP之间存在异常相互作用，p97/VCP是一种驻留在TGN上的蛋白，与另外两种遗传性运动神经病有关，包括肌萎缩侧索硬化症。P97/VCP的siRNA敲除提高了ATP7AT994I的定位。流式细胞术证明，未通透性的ATP7AP1386S成纤维细胞结合了一个羧基末端的ATP7A抗体，这与第八个跨膜螺旋的不稳定插入和二亮氨酸内吞恢复信号重新定位到PM的细胞外面一致这些发现1)阐明了ATP7A相关的远端运动神经病的机制，2)在遗传上不同的运动神经元疾病之间建立了共同的联系，3)阐明了ATP7A内吞的正常过程，4)强调了ATP7A在外周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.