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. 2009 May 1;284(18):12384-98.
doi: 10.1074/jbc.M900992200. Epub 2009 Feb 23.

VCP mutations causing frontotemporal lobar degeneration disrupt localization of TDP-43 and induce cell death

Michael A Gitcho  1 , Jeffrey Strider, Deborah Carter, Lisa Taylor-Reinwald, Mark S Forman, Alison M Goate, Nigel J Cairns
Affiliations

VCP mutations causing frontotemporal lobar degeneration disrupt localization of TDP-43 and induce cell death

Michael A Gitcho et al. J Biol Chem. .

Abstract

Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.

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Figures

FIGURE 1.
FIGURE 1.
Model of pathogenic mutations and domains in valosin-containing protein. CDC48 (magenta), located within the N terminus (residues 22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1 (23-25, 28). There are two AAA-ATPase domains (AAA; blue) at residues 240-283 and 516-569, which are joined by two linker regions (L1 and L2; red).
FIGURE 2.
FIGURE 2.
Neuropathology of the brain of a 47-year-old man with frontotemporal lobar degeneration and VCP mutation R155H. A, low power micrograph of the superficial laminae of the frontal lobe showing two neurons (arrows) containing intranuclear aggregates of VCP. B, high power micrograph showing a VCP-immunoreactive intranuclear inclusion. C, a TDP-43-immunoreactive intranuclear aggregate. D, a ubiquitin-immunoreactive neuronal intranuclear inclusion. E, a TDP-43 NCI. F, a ubiquitin-immunoreactive NCI. A and B, VCP immunohistochemistry (IHC); C and E, TDP-43 immunohistochemistry; D and F, ubiquitin immunohistochemistry. Scale bar, 20 μm (A) and 5 μm (B-F).
FIGURE 3.
FIGURE 3.
Mutations in VCP decrease cell viability and proteasome activity and increase the amount of soluble ubiquitin in neuroblastoma, SHSY-5Y, cells. A, trypan blue exclusion assay measured cell viability over a 3-day time course after transfection in three separate experiments. The graph shows viability of WT compared with mutant VCP transiently transfected SHSY-5Y cells (n = 3; *, p < 0.05; **, p < 0.001). B, proteasome chymotrypsin cleavage activity in retinoic acid induced differentiated SHSY-5Y cells transiently transfected with VCP constructs assessed 24 h post-transfection (n = 6; *, p < 0.05; **, p < 0.001). C, SHSY-5Y cells transiently transfected with monomeric dsRED-VCP constructs; arrows indicate colocalization of TDP-43/VCP (yellow) and visualization by confocal microscopy (VCP dsRED fusion constructs (red), anti-FK2 (blue), and anti-TDP-43 (green)). D (top), immunoblotting of low salt and urea-soluble cell fractions reveals a distinct ubiquitin-positive band of ∼37 kDa. Soluble low salt (L) and soluble urea (U) fractions are shown. 5Y, control SHSY-5Y cells not transfected. Top, blot probed with anti-ubiquitin antibodies. D (bottom), fractions were probed with anti-VCP and anti-TDP-43 antibodies. A single band of ∼43 kDa of TDP-43 was identified; no other bands and no cleavage products were detected in the cellular protein fractions. VCP was detected using anti-VCP antibodies, and two bands were identified: full-length VCP of ∼97 kDa and VCP-dsRED fusion protein of ∼150 kDa.
FIGURE 4.
FIGURE 4.
Mutations in VCP increase markers of ER stress. A, Western blot analysis of VCP constructs in SHSY-5Y cells under different conditions: untreated SHSY-5Y cells (5Y), retinoic acid (RA)-induced differentiation, and cells in the presence of phosphatidylinositol 3-kinase inhibitor, LY294002 (LY). B, there was an increase, but not statistically significant, in GRP78 levels in mutants compared with controls. Values represent relative optical density values of GRP78 in untreated cells in arbitrary units normalized to β-actin (ACTB) in three separate experiments. C, GRP78 mRNA normalized to GAPDH was significantly increased in mutants, as determined by quantitative RT-PCR (qRT-PCR). D, quantitative RT-PCR of calreticulin mRNA levels were significantly reduced in mutants compared with WT control when normalized to GAPDH 24 h after transfection and 24 h after retinoic acid-induced differentiation in SHSY-5Y cells. Data are expressed as mean ± S.D.; Student's t test; *, p < 0.05; **, p < 0.001.
FIGURE 5.
FIGURE 5.
Apoptotic markers are elevated in mutant cell lines compared with controls. A, caspase-4 and/or -9 activity was significantly increased in all mutants. B, caspase-3 activity was also elevated in all mutants compared with WT control cells. Transiently transfected overexpressed WT VCP was compared with mutant VCP in SHSY-5Y cells (n = 10; *, p < 0.05; **, p < 0.001).
FIGURE 6.
FIGURE 6.
VCP colocalizes with the ER-Golgi network. Overexpressed dsRED-VCP fusion protein (red), GOLGB1 (anti-giantin, trans-Golgi) (green), and protein-disulfide isomerase (PDI; ER marker, secondary; blue) were visualized by confocal microscopy.
FIGURE 7.
FIGURE 7.
Mutant VCP alters endogenous TDP-43 localization 24 h post-transfection. At 24 h post-transfection, dsRED-VCP fusion protein (red), TDP-43 (green), and TOPRO-3 (nuclear marker; blue) were visualized by confocal microscopy (arrows indicate TDP-43 and VCP colocalization and accumulation (merge/yellow)).
FIGURE 8.
FIGURE 8.
Mutant VCP localizes to the nucleus from cytoplasm 24 h post-transfection. Wild-type VCP shows no change in cytosolic distribution. R95G and R155H showed a similar spatial pattern of protein localization; the graphs show the intensity distribution profile of dsRED-VCP (red line), TDP-43 (green line), and TOPRO-3, nuclear marker (blue line) of the merged image. Intensity profile of R155H (A) and control, nontransfected cell (SHSY-5Y) profile are represented in the graph (B) and show predominantly nuclear and subtle TDP-43 cytoplasmic staining.
FIGURE 9.
FIGURE 9.
Mutant VCP translocates to the nucleus from the cytoplasm. VCP translocates to the nucleus and TDP-43 appears more abundant in the cytoplasm of R155C-, R191Q-, and A232E-transfected cells. The graphs show the intensity distribution profile of dsRED-VCP (red line), TDP-43 (green line), and TOPRO-3 (nuclear marker; blue line) in the analyzed cells.
FIGURE 10.
FIGURE 10.
Mutant VCP alters endogenous TDP-43 localization 48 h post-transfection. dsRED-VCP fusion protein (red), TDP-43 (green), and TOPRO-3 (nuclear marker; blue) were visualized by confocal microscopy At 48 h post-transfection. The arrows indicate TDP-43 and VCP colocalization in the cytosol (merge/yellow).
FIGURE 11.
FIGURE 11.
Mutant VCP induces TDP-43 distribution to the cytosol 48 h post-transfection. Wild-type VCP-expressing cells show no change in nuclear distribution of TDP-43. In contrast, VCP R95G and R155H mutants display a relative increase in cytosolic TDP-43. The graphs show the intensity distribution profile of dsRED-VCP (red line), TDP-43 (green line), and TOPRO-3 (nuclear marker; blue line) of the merged image.
FIGURE 12.
FIGURE 12.
Mutant VCP induces TDP-43 distribution from the nucleus to the cytosol 48 h post-transfection. R155C, R191Q, and A232E mutants display a relative increase in cytosolic TDP-43. The graphs show the intensity distribution profile of dsRED-VCP (red line), TDP-43 (green line), and TOPRO-3 (nuclear marker; blue line) of the merged image.
FIGURE 13.
FIGURE 13.
Overexpression of dsRED-VCP E305Q/E578Q (dominant negative) leads to nuclear fragmentation consistent with neuronal cell death at 24 h post-transfection. There is little to no TDP-43 present either in the nucleus or cytoplasm. dsRED-VCP E305Q/E578Q (dominant negative) (red), TDP-43 (green) and TOPRO-3 (nuclear marker; blue) and visualized by confocal microscopy.
FIGURE 14.
FIGURE 14.
TDP-43 complexes with VCP in co-immunoprecipitation assays in both SHSY-5Y cells and human brain. A, TDP-43 immunoprecipitation (IP) in VCP-transfected SHSY-5Y cells probed with anti-VCP (top) and 5% loading control (bottom). B, VCP immunoprecipitation in high salt (HS) fraction of human brain probed with anti-TDP-43. Samples were immunoprecipitated with VCP: normal adult control cases (NL-1 and NL-2); Alzheimer disease (AD); familial FTLD-U with GRN A9D mutation (GRN); sporadic (FTLD-U); and FTLD-U with the VCP R155H mutation (VCP) (top) and 2% loading control (bottom). C, TDP-43 immunoprecipitation in HS of human brain probed with anti-VCP: age-matched control (NL-1); familial FTLD-U with GRN A9D mutation (GRN); sporadic (FTLD-U); and FTLD-U with VCP R155H mutation (VCP).
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