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.
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