Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy

Abstract

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.

Overexpression of Endogenous TIMP-2 Increases the Proliferation of BeWo Choriocarcinoma Cells Through the MAPK-Signaling Pathway

“…[22][23][24] In our study, under the biologically relevant condition of TIMP-2, only BeWo cells increased proliferation but not HT-1080 or 293 T cell. This showed the unique characteristic of BeWo cell in terms of the way to respond to TIMP-2.…”

“…The HEK293T has been widely used in studies that involved gene transfection, [22][23][24] and HT-1080 has been used in studies to evaluate the effect of TIMP-2 on cell proliferation. 11,21 Therefore, we used these cell lines as controls to compare the characteristic of BeWo cells in terms of response to TIMP-2 under the biologically relevant condition of TIMP-2.…”

Tandem Duplication of Exons 1–7 Neither Impairs ATP7A Expression Nor Causes a Menkes Disease Phenotype

“…Immunofluorescence Confocal Microscopy: The patient's fibroblasts were examined by confocal microscopy (Zeiss 510) and images captured using META software, as previously described (Yi et al 2012).…”

Inborn errors of copper metabolism

“…These functions are largely fulfilled by a pair of evolutionarily related copper-transporting ATPases, ATP7A and ATP7B. Defects in ATP7B cause a single known phenotype, Wilson disease (Kaler, 2008), whereas mutations in ATP7A are associated with three distinct conditions: (1) Menkes disease, a severe infantile-onset neurodegenerative disorder (Kaler, 1994); (2) occipital horn syndrome, similar to Menkes disease in many clinical and biochemical aspects, with a less severe neurologic phenotype (Kaler et al, 1994); and (3) an isolated distal motor neuropathy, often with adult onset and without overt signs of copper metabolic derangements (Kennerson et al, 2010; Yi et al, 2012). This chapter reviews the neurological and other clinical signs, the biochemical manifestations, and the molecular underpinnings associated with these four entities, as well as treatment considerations for each.…”