Mechanisms of Motor Neuron Disease

ATP7A是一种p型atp酶，通过在反式高尔基网络（TGN）和质膜（PM）上的活性来调节细胞内铜的稳态，其位置通常由细胞内铜浓度决定。ATP7A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变，这是一种新发现的疾病，其确切的病理生理学尚不清楚。我们鉴定了两种ATP7A运动神经病突变（T994I, P1386S）先前与异常细胞内运输相关。在患者成纤维细胞中，全内反射荧光（TIRF）显微镜显示，ATP7AT994I和ATP7AP1386S的稳态平衡发生了变化，过量的PM定位。用金星荧光蛋白标记的突变等位基因转染293T细胞和NSC-34运动神经元也显示出更高的PM定位。突变等位基因从PM到TGN的内吞恢复延迟。免疫沉淀试验显示，ATP7AT994I和p97/VCP之间存在异常相互作用，p97/VCP是一种与其他两种遗传性运动神经病变（包括肌萎缩性侧索硬化症）相关的tgn驻留蛋白。p97/VCP的SiRNA敲低改善了ATP7AT994I的定位。流式细胞术证实，未渗透的ATP7AP1386S成纤维细胞结合了羧基末端的ATP7A抗体，与第8跨膜螺旋的不稳定插入和二亮氨酸内吞恢复信号在PM细胞外表面的重新定位一致。这些发现1)阐明了与ATP7A相关的远端运动神经病变的机制，2)在遗传上不同形式的运动神经元疾病之间建立了共同的联系，3)阐明了ATP7A内吞作用的正常过程，4)强调了ATP7A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) illuminate mechanisms underlying ATP7A-related distal motor neuropathy, 2) establish a common link between genetically distinct forms of motor neuron disease, 3) clarify the normal process of ATP7A endocytosis, and 4) highlight possible functional roles of ATP7A in the peripheral nervous system.