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. 2010 Jun 2;30(22):7729-39.
doi: 10.1523/JNEUROSCI.5894-09.2010.

TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97

Gillian P Ritson  1 , Sara K Custer, Brian D Freibaum, Jake B Guinto, Dyanna Geffel, Jennifer Moore, Waixing Tang, Matthew J Winton, Manuela Neumann, John Q Trojanowski, Virginia M-Y Lee, Mark S Forman, J Paul Taylor
Affiliations

TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97

Gillian P Ritson et al. J Neurosci. .

Abstract

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP7) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster, and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA(+) (ATPases associated with multiple cellular activities) ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. To elucidate the pathogenic mechanism, we developed and characterized a Drosophila model of IBMPFD (mutant-VCP-related degeneration). Based on genetic screening of this model, we identified three RNA-binding proteins that dominantly suppressed degeneration; one of these was TBPH, the Drosophila homolog of TAR (trans-activating response region) DNA-binding protein 43 (TDP-43). Here we demonstrate that VCP and TDP-43 interact genetically and that disease-causing mutations in VCP lead to redistribution of TDP-43 to the cytoplasm in vitro and in vivo, replicating the major pathology observed in IBMPFD and other TDP-43 proteinopathies. We also demonstrate that TDP-43 redistribution from the nucleus to the cytoplasm is sufficient to induce cytotoxicity. Furthermore, we determined that a pathogenic mutation in TDP-43 promotes redistribution to the cytoplasm and enhances the genetic interaction with VCP. Together, our results show that degeneration associated with VCP mutations is mediated in part by toxic gain of function of TDP-43 in the cytoplasm. We suggest that these findings are likely relevant to the pathogenic mechanism of a broad array of TDP-43 proteinopathies, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

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Figures

Figure 1.
Figure 1.
Exogenous expression of disease-related VCP mutants enhances toxicity in vivo. A, Top row, Stereomicrographs of 1-d-old adult fly eyes in which expression of WT or mutant dVCP is driven by GMR-GAL4. The eyes of control flies (GMR-GAL4/+) and flies expressing WT dVCP have a highly organized ommatidial array. The eyes of flies expressing mutant dVCP (R152H) show loss of individual ommatidia, partial collapse, and small necrotic patches. The eyes of flies expressing mutant dVCP (A229E) are severely degenerated with large necrotic patches. Bottom row, Corresponding Richardson's stained frontal sections. B, Western blot showing expression levels of WT and mutant forms of dVCP in the eyes of 1-d-old flies. Actin served as a loading control. C, Quantitative analysis of eye phenotypes. Flies from each genotype were randomly selected for objective scoring according to the criteria described in Materials and Methods. Values are the mean results; error bars represent SEM.
Figure 2.
Figure 2.
A dominant modifier screen identifies TBPH as a modifier of dVCP toxicity. A, The positions of the two deficiency lines from the Bloomington Deficiency Kit Df(2L)Dwee1-W05 and Df(2R)BSC136 and the genes within them in the Drosophila genome. The size of each deletion region is noted. Genes in red were identified by genetic screening to suppress the degenerative phenotype of mutant dVCP. B, Stereomicrographs of eyes of 1-d-old adult flies in which dVCP R152H and suppressors identified in a dominant modifier screen were expressed by using the driver GMR-GAL4. Top row, dVCP R152H alone and coexpressed with two deficiency lines (Df(2R)BSC136 and Df(2L)Dwee1–W05) or the individual RNAi lines corresponding to the genes within the regions defined by the deficiency lines, which all suppress the degenerative phenotype associated with mutant dVCP. Bottom row, Corresponding toluidine blue-stained frontal sections. C, Quantitative analysis of eye phenotypes. Flies from each genotype were randomly selected for objective scoring according to the criteria described in Materials and Methods. Quantitation of the phenotype shows a significant decrease in severity score when the deficiency and RNAi lines are coexpressed with mutant dVCP compared to mutant dVCP alone. Comparisons were made using Student's t test. Data show mean phenotype severity score (error bars represent SD; *p < 0.05). D, Top row, Stereomicrographs of eyes of 1-d-old flies expressing WT TBPH alone and with dVCP R152H. Overexpression of WT TBPH causes a profound degenerative phenotype and enhances the degenerative phenotype associated with mutant dVCP. Bottom row, Corresponding toluidine blue-stained frontal sections.
Figure 3.
Figure 3.
Exogenous expression of disease-related VCP mutants in vitro causes cytotoxicity and redistribution of TDP-43. A, B, Localization of TDP-43 in a patient with IBMPFD was first confirmed by immunohistochemistry. In an unaffected region (occipital cortex) (A), TDP-43 is predominantly nuclear, whereas in the affected frontal cortex (B), TDP-43 is largely redistributed to the cytoplasm and some nuclei show dense inclusions (arrowheads). C, Mouse primary cortical neurons were transfected on day 4 in vitro with DsRed-conjugated WT or mutant VCP, then immunostained 24 or 48 h later for MAP2 (green). DAPI staining (blue) shows nuclear morphology. Arrows indicate neurons expressing mutant VCP that have lost MAP2 staining and have condensed nuclei. Scale bar, 20 μm. D, A blinded investigator scored VCP-expressing neurons for cytotoxicity (condensed nuclei plus altered MAP2 staining). Values are the mean results from three trials; error bars represent SEM; *p < 0.05, Student's t test. E, Mouse primary cortical neurons transfected with DsRed-conjugated WT or mutant VCP were fixed and immunostained with anti-TDP-43 and DAPI 24 h after transfection. Neurons expressing mutant VCP showed reduced nuclear TDP-43 and increased cytoplasmic TDP-43. Representative examples are shown (arrows). Scale bar, 20 μm. F, G, A blinded examiner scored >100 VCP-expressing neurons from three separate cultures for the presence or absence of TDP-43 in the cytoplasm (F) and, independently, in the nucleus (G). Expression of mutant VCP (R155H or A232E) was associated with significantly less nuclear TDP-43 and significantly more cytoplasmic TDP-43 (values are the means from three trials; error bars represent SEM; *p < 0.05, Student's t test). H, The firefly luciferase reporter constructs used to detect TDP-43 transcriptional repression activity. I, Luciferase values relative to control values (values are the means from three trials; error bars represent SEM; *p < 0.05, Student's t test). The SP-10 insulator inserted between the CMV enhancer and the core promoter (CP) repressed luciferase expression (Luc). Knockdown of TDP-43 by siRNA or mutation of the TDP-43 binding sites (ACACAC to GGGTTG) released the enhancer-blocking effect of the insulator. Cotransfection of VCP R155H and A232E, but not of WT VCP, with pSP-10 also significantly compromised the enhancer-blocking ability. Cells cotransfected with VCP constructs and mutant pSP-10 did not show greater luciferase activity than cells transfected with pSP-10-mutant alone.
Figure 4.
Figure 4.
Mislocalization of exogenous TDP-43 to the cytoplasm causes degeneration in vivo. Stereomicrographs of the eyes of 1-d-old adult flies in which LacZ (control), WT, or mutant TDP-43 was expressed by using the driver GMR-GAL4. Shown beside each panel is immunohistochemical analysis of a longitudinal section with anti-TDP-43. WT and NES-mutant TDP-43 are predominantly nuclear, and eye morphology is normal (see also supplemental Figs. 5, 6, available at www.jneurosci.org as supplemental material). NLS-mutant TDP-43 is predominantly cytoplasmic and causes a severe rough eye phenotype (see also supplemental Fig. 7, available at www.jneurosci.org as supplemental material).
Figure 5.
Figure 5.
Exogenous expression of ALS-causing mutant M337V TDP-43 results in degeneration in vivo. A, Stereomicrographs of the eyes of 1-d-old adult flies in which expression of TDP-43 M337V is driven by GMR-GAL4. All seven of the independent transgenic lines generated are shown. The eyes of some lines exhibit a mild rough eye phenotype. Immunohistochemistry with anti-TDP-43 reveals cytoplasmic TDP-43 in M337V#6. B, Western blot analysis of head homogenates from TDP-43 M337V flies showing TDP-43 expression in all transgenic lines generated. All lines with a degenerative phenotype show both monomeric TDP-43 and a ∼25 kDa fragment of TDP-43. Staining for tubulin served as a loading control.
Figure 6.
Figure 6.
Mutation-dependent interaction of dVCP with TDP-43. A, Stereomicrographs of the eyes of 1-d-old adult flies coexpressing WT or mutant (R152H) dVCP and WT or mutant (NES-mutant, NLS-mutant or ALS-causing mutant M337V) TDP-43. Coexpression with LacZ served as a negative control. B, Immunohistochemical detection of the subcellular distribution of TDP-43 in longitudinal eye sections, with nuclei highlighted. C, Immunohistochemical detection of the subcellular distribution of TDP-43 in whole-mount salivary glands from Drosophila larvae. Scale bar, 10 μm. D, Quantitation of nuclear depletion of TDP-43 in salivary glands (values are the means from at least five larvae of each genotype; error bars represent SEM; *p < 0.05, Student's t test). WT TDP-43 coexpressed with WT dVCP did not modify the phenotype and remained predominantly nuclear. By contrast, WT TDP-43 enhanced the rough eye phenotype in flies expressing mutant dVCP and was significantly redistributed to the cytoplasm. NES-mutant TDP-43 did not modify the phenotype of WT or mutant dVCP and remained predominantly nuclear. The phenotype associated with mutant dVCP (but not WT dVCP) was strongly enhanced in a weakly degenerative NLS-mutant TDP-43 line, and the TDP-43 remained predominantly cytoplasmic. In the strongly degenerative NLS-mutant TDP-43 line, coexpression of either WT or mutant dVCP caused lethality. The ALS-causing mutant M337V enhanced the dVCP WT phenotype and resulted in lethality when coexpressed with mutant dVCP.
Figure 7.
Figure 7.
VCP's role in the nucleocytoplasmic balance of TDP-43 The mechanism whereby VCP mutations lead to cytoplasmic accumulation of TDP-43 is unknown. We speculate that the possibilities include, but are not limited to, dysfunction in the following: (1) VCP's segregase activity for extracting TDP-43 from RNP complexes in the cytoplasm, (2) a role for VCP in nuclear import of TDP-43 (analogous to the role of VCP in nuclear import of TSAd), and (3) degradation of TDP-43 by VCP, a known component of the autophagic pathway.
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