Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy.
ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7A(T994I) and ATP7A(P1386S), with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7A(T994I) and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7A(T994I) mislocalization. Flow cytometry documented that non-permeabilized ATP7A(P1386S) fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.
Unit on Human Copper Metabolism, Molecular Medicine Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892-1853, USA.
Ling
L
Yi
Anthony
A
Donsante
Marina L
ML
Kennerson
Julian F B
JF
Mercer
James Y
JY
Garbern
Stephen G
SG
Kaler
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000483:Alleles; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D004705:Endocytosis; D005347:Fibroblasts; D057809:HEK293 Cells; D006801:Humans; D016472:Motor Neuron Disease / Q000235:genetics* / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021381:Protein Transport; D000074405:Valosin Containing Protein; D021601:trans-Golgi Network / Q000378:metabolism
D027682:Cation Transport Proteins; D018797:Cell Cycle Proteins; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases