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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 (VCP) gene. VCP (p97 in mouse, TER94 in D. melanogaster, and CDC48 in S. cerevisiae) is a highly conserved AAA+-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 DNA-binding protein (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. Taken 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.
Inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD; MIM167320) is a rare, complex and ultimately lethal autosomal dominant disorder. Affected individuals exhibit variable penetrance of progressive degeneration of muscle, bone and brain caused by mutations in the gene encoding valosin-containing protein (VCP) (Watts et al., 2004) (Kimonis et al., 2008). The molecular chaperone VCP (also known as p97, TER94, and CDC48) is a member of the AAA+ family of proteins (ATPases associated with multiple cellular activities) that segregates ubiquitinated substrates from multimeric protein complexes or structures (Ye, 2006). VCP activity is essential for multiple cellular processes, including ubiquitin-dependent protein degradation, nuclear envelope construction, Golgi and endoplasmic reticulum assembly, and autophagosome maturation (Halawani and Latterich, 2006; Ju et al., 2009; Tresse et al., 2010). The molecular basis of degeneration resulting from VCP mutations is unknown, although ubiquitin-positive pathology is prominent in affected tissues (Guinto et al., 2007; Weihl et al., 2008). TAR DNA-binding protein 43 (TDP-43) has been identified as a major component of the ubiquitin pathology (Neumann et al., 2007; Salajegheh et al., 2009).
TDP-43 is a predominantly nuclear hnRNP that undergoes nucleocytoplasmic shuttling and associates with translation machinery in the cytoplasm (Ayala et al., 2008; Wang et al., 2008; Freibaum et al., 2009). TDP-43 is redistributed to the cytoplasm after neuronal injury where it associates with stress granules (Colombrita et al., 2009; Moisse et al., 2009a). TDP-43 redistribution to the cytoplasm is recognized as a pathological feature of several sporadic and inherited human diseases including IBMPFD, frontotemporal dementia and amyotrophic lateral sclerosis, although the significance of this is unclear (Neumann et al., 2007; Geser et al., 2009; Salajegheh et al., 2009). TDP-43 redistribution has also been observed in vitro in cells expressing mutant VCP, although the role of TDP-43 in mediating disease has not been explored (Gitcho et al., 2009). The recent identification of disease-associated mutations in TDP-43 strongly implicate this protein in disease pathogenesis (Gitcho et al., 2008; Kabashi et al., 2008; Rutherford et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008; Yokoseki et al., 2008), although it is not known whether disease-associated cytoplasmic accumulation of TDP-43 is a mediator of pathology or a physiological response to it.
Here we present the first Drosophila melanogaster model of IBMPFD. Through genetic screening we identified three RNA-binding proteins that suppress degeneration. One of these was TBPH, the fly orthologue of TDP-43. We show in vitro that expression of disease-causing VCP mutants leads to cytotoxicity and coincidental redistribution of TDP-43. To determine the significance of TDP-43 redistribution, we generated transgenic flies expressing wild type (WT) and mutant forms of TDP-43. We demonstrate that VCP and TDP-43 interact genetically, that disease-causing mutations in VCP lead to redistribution of TDP-43 to the cytoplasm in vivo, and that redistribution of TDP-43 is sufficient to induce degeneration in vivo. Thus, our study provides the first evidence that toxic gain-of-function of TDP-43 in the cytoplasm plays a primary role in mediating the pathogenesis initiated by mutations in VCP.
To generate pUAST-dVCP constructs, dVCP cDNA in the pBluescript SK–(pBS sequence variants were generated using Stratagene’s QuikChange SDM Kit (Agilent Technologies, Cedar Creek, CA), changing R to H at amino acid (aa) 152 (R152H), A to E at aa 229 (A229E) and subsequently subcloned into pUAST. To generate DsRed VCP, VCP cDNA was obtained from Origene and sequence variants R95G, R155H, R155C, R191Q and A232E were generated using Stratagene’s QuikChange SDM kit. Wild type and mutant VCP were then subcloned into the BglII/BamH1 cloning site of pDsRed Monomer-C1 vector (Clontech). TBPH cDNA was obtained from Origene and subcloned into the EcoRI/XhoI site of pUAST. WT, NLS- and NES-mutant TDP-43 cDNAs were amplified from the previously described mammalian expression constructs (Winton et al., 2008). Using the Stratagene’s QuikChange SDM kit, TDP-43 M337V (M to V at aa337) was generated from WT TDP-43. All TDP-43 constructs were subcloned into the NotI/XhoI cloning site of pUAST.
All Drosophila stocks were maintained on standard media in 25°C incubators. Double-strand RNAi lines targeting TBPH (ID38377), xl6 (ID31202, ID31203) and Hrb27C (ID16040, 16041) were obtained from the Vienna Drosophila RNAi Center. Flies transgenic for UAS-dVCP (WT or mutant), UAS-TBPH and UAS-TDP-43 (WT or mutant) were generated by injecting the constructs described above into embryos of w1118 using standard techniques.
Deficiency (Df) lines for all four chromosomes obtained from the Bloomington stock center were used to identify dominant modifiers of mutant dVCP in a genetic screen. For the primary screen, balanced virgin female dVCP R152H (recombined with Gmr GAL4) flies were crossed with Df/Balancer males from 270 deficiency lines and progeny were examined for changes in eye phenotype (including color, ommatidia structure and bristle formation). At least ten progeny were examined and scored on a twenty-point scale. Eyes were examined for the presence of: supernumerary inter-ommatidial bristles (IOBs), IOBs with abnormal orientation, necrotic patches, a decrease in size, retinal collapse, fusion of ommatidia, disorganization of ommatidial array and loss of pigmentation. Points were added if: there was complete loss of IOBs (+1), more than 3 small or 1 large necrotic patch (+1), retinal collapse extended to the midline of the eye (+1) or beyond (+2), loss of ommatidial structure in less than 50% (+1) or more than 50% (+2) of the eye, and if pigmentation loss resulted in change of eye color from red to orange (+1) or pale orange/white (+2). Gmr, GAL4, UAS dVCP R152H / Balancer served as an internal control. In a secondary screen to filter nonspecific modifiers of cell death, deficiencies defined as hits (either enhancing or suppressing the dVCP mutant phenotype) were then crossed with flies expressing the pro-apoptotic gene Reaper (recombined with Gmr GAL4). Any hits that similarly affected dVCP R152H and Reaper in the secondary screen were excluded from further study due to the possibility of non–specific anti-apoptotic effects. As regions of interest were identified from the primary and secondary screens, additional Df lines were obtained that overlapped with interacting deficiencies to verify and refine the position of potential modifiers. For the final step of gene identification, individual RNAi lines corresponding to the genes within the candidate intervals were obtained from the Vienna Drosophila RNAi Center. Gmr GAL4, UAS dVCP R152H females were crossed with males from the RNAi lines and the progeny eyes were evaluated for changes. A modifier was defined as an RNAi line that replicated the enhancement or suppression of the corresponding deficiency.
HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamate. HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Primary cortical neurons were cultured from postnatal day zero C57Bl/6J pups. Briefly, pups were decapitated into Hanks medium without Ca2+ and Mg2+, and cortices were dissected in Neurobasal-A medium supplemented with 10 mM HEPES. After dissection, cortices were trypsinized for 25 minutes at 37°C and dissociated. Neurons were plated in Neurobasal-A with B27 supplement at a density of 2.5 × 105 cells per well in 4-well chamber CC2 slides (Nalge-Nunc). After 4 days in vitro (DIV), cells were transfected with 1ug plasmid DNA using Lipofectamine 2000 (Invitrogen) as suggested by the manufacturer.
To examine soluble and insoluble fractions of TDP-43 in cells, HEK293T cells were harvested at the indicated times and RIPA-soluble and insoluble fractions were prepared as previously described (Winton et al., 2008). To examine proteins expressed in flies, 3 fly heads (or thoraces) of the appropriate genotype were lysed in 20 μL of RIPA. For immunoblots (IB), 20 μg of cell lysates or lysate from 3 fly head equivalents were resolved on 10% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad), and IB was performed as described (Pandey et al., 2007). Mouse monoclonal anti-VCP antibody (Affinity BioReagent) was used at 1:10000; rabbit polyclonal anti-TDP-43 antibody raised against recombinant TDP-43 (Protein Tech Group) at 1:1000; mouse monoclonal anti-tubulin (Sigma) at 1:10000; and rabbit polyclonal anti-actin (Santa Cruz) at 1:3000. Primary antibodies were detected with horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch) and proteins were visualized using Immobilon Western Chemiluminescent AP Substrate (Millipore).
Total RNA was isolated from 10 animals of the appropriate genotype with TRIzol reagent (Invitrogen) and cDNA was generated using the iScript cDNA Synthesis kit (BioRad) following the manufacturer’s protocol. The concentration of each primer probe set was individually optimized. Quantitative real-time PCR reactions were carried out in a total reaction volume of 25 μl of iQ Supermix (BioRad) using BioRad iCycler iQ5. Transcript levels were normalized to dGAPDH2. The primer/probe set for genes purchased from Applied Biosystems were as follows: Hrb27C--Dm01803323_g1, xl6--Dm01803314_m1, TBPH--Dm01820181_g1 and GAPDH2 ctl.-- Dm01843776_S1.
To assess toxicity in HEK293T cells, cells were harvested at the indicated times with versene, rinsed with PBS, and resuspended in PBS + 1% FBS. TOPRO-3 (Molecular Probes) in 1 mM DMSO was diluted 1:50000 in PBS and 50 μL was added to 450μL of the cell suspension. After incubation for 1-2 min, cells were analyzed by FACS. Transfected cells were identified by DsRed fluorescence and dead cells were identified by TOPRO-3 fluorescence. Cell viability was calculated and expressed as previously described (Taylor et al., 2003). To assess toxicity in primary cortical neurons, we transfected these cells on day 4 in vitro with DsRed-conjugated WT or mutant VCP. Twenty-four or 48 hours post-transfection, neurons were immunostained for MAP2 and stained with DAPI to visualize nuclear morphology as described above. A blinded investigator scored VCP-expressing neurons for the presence or absence of toxicity. Only neurons with both condensed nuclei and loss of MAP2 staining were scored positive for cytotoxicity. More than100 neurons from at least 3 trials were analyzed and results were compared by using the Student t-test with a significance threshold of p<0.05).
HEK293T cells were transfected with firefly luciferase reporter constructs (pLTR, pSP10 or pSP10D) combined with 100ng pRL-CMV and 0.5ug VCP WT, VCP R155H, VCP A232E or empty vector control (pcDNA 3.1) by using Fugene (Roche) according to the manufacturer’s protocol. Forty-eight hours after transfection, cell lysates were analyzed using the Stop and Glo dual reporter system (Promega) in a 96-well format with a Vector3 luminometer, as directed by the Promega protocol. Firefly luciferase activity was then normalized to the Renilla luciferase activity to control for transfection efficiency. Data was then normalized to activity in cells transfected with empty vector control, which was given a value of 1.
For immunofluorescence analysis in cell culture, HEK293T and HeLa cells were washed twice with PBS, fixed with 4% PFA for 10 minutes, washed twice with PBS, then permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and blocked with 0.2% Triton/5% goat serum in PBS for 30 minutes. Cells were incubated with primary antibody (rabbit polyclonal anti-TDP-43 antibody at 1:200 and mouse monoclonal anti-MAP2 at 1:500, diluted in PBS containing 0.2% Triton/5% goat serum) for 1.5 hour and washed twice with PBS (15 minutes each). After the final wash, the cells were incubated with secondary antibody (diluted in PBS containing 0.2% Triton/5% goat serum) for 1 hour, washed three times with PBS (5 minutes each), and mounted with Vectashield + DAPI (Vector Laboratories Inc). Digital imaging was performed with a Leica DMIRE2 fluorescent microscope using IP-LAB or Slidebook 5.1 software. To quantify fluorescence, regions of interest were drawn around the nuclei or cytoplasm of transfected (DsRed-positive) cells or untransfected neighboring cells and the intensity of FITC fluorescence emission from >100 cells was measured in at least three experiments. For immunofluorescence analysis in Drosophila salivary glands, at least 5 third instar wandering larvae expressing dVCP (WT or mutant) and TDP-43 (WT or mutant) under control of the driver fkh-GAL4 were collected. Salivary glands from these larvae were dissected in PBS and fixed in 4% PFA and heptane at room temperature. After 20 minutes, the PFA was removed and 100% methanol added. After vigorous shaking for 1 minute the heptane was removed and the samples washed in methanol 3 times. After 3 washes in PBST (PBS/0.1% Tween-20) and 3 washes in PBSBT (PBS/0.1% Tween-20/1% BSA) the samples were blocked for 2 hours in PBSBT at room temperature. Samples were incubated with primary antibody (rabbit polyclonal anti-TDP-43 antibody at 1:300, diluted in PBSBT) overnight and washed 4 times with PBSBT (30 minutes each). After the final wash, the cells were incubated with secondary antibody (diluted in PBSBT) for 2 hours, washed three times with PBSBT (10 minutes each), and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Digital imaging was performed with a Leica DMIRE2 fluorescent microscope using Slidebook software. All cells from the glands were evaluated for the absence of nuclear TDP-43 staining. Using Slidebook software, DAPI was pseudo-colored in red.
For immunohistochemistry of fly eyes, heads of the appropriate genotype were collected and fixed in 4% buffered paraformaldehyde in PBS for 2 hours at room temperature. Samples were serially dehydrated in ethanol (1 h each in 50%, 70%, 80%, 90%, 95%, and twice in 100%). Samples were then either: (1) embedded in JB-4 according to the manufacturer’s protocol (JB-4 Plus Embedding Kit, Polysciences, Inc.) and sectioned at 1 micron for staining with Richardson’s or toluidine blue stains, or (2) embedded into paraffin, sectioned at 5 microns, and placed on glass slides. Immunohistochemistry was performed using (ProteinTech) rabbit anti-TDP43 at 1:200, Vector Rabbit HRP ImmPress detection, and Vector AEC substrate.
To develop an in vivo model of VCP-mediated degeneration, we used Drosophila melanogaster, in which the gene TER94 encodes the highly conserved orthologue of VCP. The Drosophila VCP orthologue, hereafter referred to as dVCP, is 92% similar and 83% identical to human VCP at the amino acid level. Conservation is even higher (95% similar, 84% identical) across the 250–amino acid N-terminus, which hosts most known disease-causing mutations, and all amino acid residues altered in disease are perfectly conserved. We introduced R152H and A229E mutations into dVCP to create homologs of the R155H and A232E mutations, which cause the most common and most severe forms of IBMPFD, respectively, and generated transgenic flies. When these mutants were expressed in the fly eye or brain using the UAS/GAL4 system (Brand and Perrimon, 1993) we observed mutation-dependent degenerative phenotypes despite equivalent levels of dVCP expression (Fig 1, Supplemental Fig 1). In the eye, expression of WT dVCP caused a very modest phenotypic change, whereas matched expression of the R152H and A229E mutants caused severe external rough eye phenotypes with necrotic patches and histologically evident marked vacuolar degeneration. The more severe phenotype seen in flies expressing the A229E mutant is consistent with the more severe disease in patients with the A232E mutation.
When expressed broadly in the central nervous system, mutant dVCP reduced viability by diminishing eclosion rates (Supplemental Fig. 1A), and surviving male flies had a significantly reduced lifespan indicative of neurodegeneration (Supplemental Fig. 1B-C). Thus, this fly model recapitulates the degeneration associated with disease-causing mutations in VCP in tissue known to be affected (brain) and in a non-essential tissue (eye), which allows screening to identify modifying genes.
VCP participates in a range of cellular activities and thus in diverse biological pathways. The specific pathways underlying pathogenesis of IBMPFD, however, are unknown. As an unbiased approach to identify molecules and pathways involved in mutant VCP–mediated degeneration, we performed a dominant-modifier screen, using the moderate dVCP mutant phenotype of dVCP R152H to maximize the likelihood of visualizing enhancement or suppression of the mutant dVCP phenotype. To carry out the screen, we obtained a deficiency (Df) collection representing all four chromosomes from the Bloomington Drosophila Stock Center in order to scan the maximum proportion of genome (~80%) with the smallest number of lines. From the primary screen, seventy-four deficiencies were identified as dominant modifiers (enhancers or suppressors) of the dVCP R152H eye phenotype. After the secondary screen, validation studies and individual gene interrogation by double-strand RNAi lines, we identified three related genes: TBPH (CG10327), xl6 (CG10203) and Hrb27C (CG10377) that dominantly suppress the degenerative phenotype (Figure 2A-B). RNAi-mediated knockdown of these genes in the eye did not result in a phenotypic change independent of mutant dVCP expression (Supplementary Figure 2A-D). However, in flies expressing dVCP R152H, knockdown of these genes suppressed mutation-dependent degeneration (Figure 2B). Suppression of degeneration in VCP-mutant flies was corroborated by seeing a significant reduction in the blinded phenotypic severity score (Figure 2C) and by using additional RNAi lines and classical alleles (Supplementary Figure 2E-F). We also generated transgenic lines over-expressing TBPH, which resulted in a degenerative phenotype evident externally and histologically when targeted to the eye. When exogenous TBPH was co-expressed with dVCP R152H, degeneration associated with mutant VCP was enhanced, confirming the genetic interaction (Fig 2D). Hrb27C, xl6 and TBPH correspond to the human genes DAZAP1, 9G8 and TDP-43, respectively. These are all RNA recognition motif (RRM)-containing RNA-binding proteins that shuttle between the nucleus and cytoplasm (Huang and Steitz, 2001; Lin and Yen, 2006; Ayala et al., 2008). Furthermore, all three have been shown to regulate multiple aspects of RNA metabolism, including transcription, export, splicing and translation (Elvira et al., 2006b; Swartz et al., 2007; Yang et al., 2009). We focused on TDP-43 for further assessment, because cytoplasmic deposition of this protein is a prominent feature of IBMPFD and other degenerative diseases (Geser et al., 2009).
In the brain, TDP-43 is found predominantly in the nuclei of neurons and some glial cells (Fig. 3A), although dynamic studies in vitro have shown TDP-43 to shuttle between the nucleus and cytoplasm (Ayala et al., 2008). In IBMPFD, affected brain regions show gross abnormalities in TDP-43 localization, including clearance of TDP-43 from many nuclei and accumulation in the cytoplasm; some neurons show dense nuclear inclusions of TDP-43 (Fig. 3B). To examine the subcellular localization of TDP-43 in vitro, we first investigated whether VCP mutation-dependent toxicity could be recapitulated in primary mouse cortical neurons transfected with FLAG-conjugated WT VCP or mutant VCP (R155H and A232E). At 24 or 48 hours post-transfection, a blinded examiner scored VCP-positive neurons for changes in MAP2 staining and nuclear morphology (changes in DAPI staining) to assess cytotoxicity (Figure 3C). Quantitative analysis of the data revealed significant mutation-dependent toxicity in primary neurons 48 hours post-transfection (Figure 3D). This time-dependent cytotoxicity caused by mutant VCP was also demonstrated by fluorescence-activated cell sorting for living vs. dead HEK293T cells. Exogenous expression of mutant but not WT VCP caused cell death despite equivalent levels of VCP expression (Supplemental Fig. 3A-B).
To examine TDP-43 subcellular localization, we fixed and immunostained primary mouse cortical neurons twenty-four hours post-transfection, before occurrence of the changes in MAP2 staining or nuclear morphology. In neurons expressing FLAG vector alone or FLAG-conjugated WT VCP, TDP-43 was consistently nuclear. By contrast, in cells transfected with mutant VCP, we observed significant clearance of endogenous TDP-43 from nuclei and accumulation in the cytoplasm (Fig. 3E-G). Redistribution of endogenous TDP-43 to the cytoplasm was also observed in HEK293T cells expressing mutant but not wild type VCP (Supplemental Fig. 3C-D). Exogenous expression of TDP-43 increased the amount of cytoplasmic redistribution (Supplemental Fig. 3E-F). The nuclear depletion of TDP-43 in primary neurons and HEK293T cells was corroborated by an associated loss of TDP-43’s known nuclear function as a transcriptional repressor of testis-specific SP-10 gene (Acharya et al., 2006). TDP-43 has been shown to interact with the SP-10 insulator, which prevents luciferase expression by repressing the interaction between the CMV enhancer and SP-10 core promoter in the pSP-10 construct (Fig. 3H). Mutation of the two TDP-43 binding sites (ACACAC to GGGTTG) compromises the enhancer-blocking ability of the SP-10 insulator, allowing luciferase expression (Fig. 3H). HEK293T cells were transfected with pSP10 or pSP10-mutant luciferase reporter constructs and luciferase expression was quantified forty-eight hours post-transfection as an indicator of TDP-43 nuclear function. When TDP-43 binding to SP10 was disrupted by use of the pSP10-mutant or by siRNA knockdown of TDP-43, the normal repression of luciferase expression was attenuated. When mutant VCP, but not WT VCP, was co-transfected with pSP10, the repression was released, allowing a significant increase in luciferase expression. Cells co-transfected with VCP constructs and mutant pSP10 did not show greater luciferase activity than cells transfected with pSP10-mutant alone. Similar results were found using the HIV-1 luciferase reporter plasmid (pLTR) (Supplemental Fig. 3G), a construct previously shown to rely on TDP-43 as a transcriptional repressor (Ou et al., 1995). Thus, mutation-dependent VCP toxicity in primary neurons and HEK293T cells was coincident with TDP-43 redistribution from the nucleus to the cytoplasm, as was also recently observed in immortalized SH-SY5Y and U20S cells (Gitcho et al., 2009; Ju et al., 2009). TDP-43 redistribution is also a conspicuous feature of numerous TDP-43 proteinopathies. However, these observations do not tell us whether TDP-43 redistribution represents an adaptive response to neuronal injury or, alternatively whether change in subcellular localization mediates disease. Furthermore, if TDP-43 redistribution mediates disease, it leaves unclear whether the mechanism involves loss of nuclear function or toxic gain of cytoplasmic function.
To further investigate the potential role of TDP-43 mislocalization in mutant–VCP-associated degeneration in vivo, we generated multiple transgenic flies expressing human WT or mutant TDP-43, using the UAS/GAL4 system. To target TDP-43 specifically to the nucleus or the cytoplasm, we used TDP-43 with previously characterized mutations that disrupt the nuclear export sequence (NES) or the nuclear localization sequence (NLS)(Winton et al., 2008). Five independent transgenic lines expressing WT TDP-43 in the fly eye showed a modest degenerative phenotype (Figure 4 and Supplemental Fig 4A-B and 6A). A normal eye phenotype was observed in 5 independent transgenic lines expressing NES-mutant TDP-43 (Fig 4 and Supplemental Fig 4C-D and 6A). By contrast, expression of NLS-mutant TDP-43 in the fly eye in 6 independent lines resulted in a strong degenerative phenotype and high-molecular-weight smears of TDP-43 in immunoblots (Supplemental Fig. 5A-B and 6). Immunostaining of NLS-mutant TDP-43 confirmed predominantly cytoplasmic localization, while WT and NES-mutant TDP-43 remained predominantly nuclear (Fig. 4). To confirm that the degenerative phenotype was caused by cytoplasmic localization of TDP-43 rather than another effect of the NLS mutation, we also generated lines with truncated TDP-43 (80-414). Deletion of the first seventy-nine amino acids, which disrupts the NLS, led to cytoplasmic localization of TDP-43 and a degenerative phenotype indistinguishable from that observed with NLS-mutant TDP-43 (Supplemental Fig. 5C and data not shown). These findings indicate that accumulation of TDP-43 in the cytoplasm is sufficient to cause degeneration. Together with the observation that knockdown of TBPH in flies causes no phenotypic change alone but suppresses the mutant dVCP phenotype, these results indicate that a toxic gain of cytoplasmic TDP-43 function underlies pathogenesis. However, these observations do not preclude the possibility that a loss of TDP-43 nuclear function contributes to toxicity.
Missense mutations in TDP-43 have recently been identified in association with dominantly inherited and sporadic ALS. To assess the effect of mutant TDP-43 in vivo, we generated transgenic lines expressing TDP-43 with the ALS-causing mutation M337V (Sreedharan et al., 2008). In 6 independent lines evaluated, we observed degenerative phenotypes ranging from modest to severe (Fig 5A and Supplemental Fig 6A). The severity of the phenotype correlated with the amount of abnormal TDP-43 species, including a C-terminal 25-kDa fragment and high molecular weight bands (Fig 5 and Supplemental Fig 6B). This is similar to the pathogenic biochemical signature seen in patient tissue with TDP-43 proteinopathy (Neumann et al., 2007). Immunohistochemistry for TDP-43 in these eyes showed a marked cytoplasmic distribution of TDP-43, consistent with a recent report that this mutation increases cytoplasmic TDP-43 inclusions in vitro (Nonaka et al., 2009).
To assess the relationship between dVCP and TDP-43 in vivo, we performed an epistasis study by co-expressing dVCP (WT or mutant) with TDP-43 (WT or mutant) and analyzing changes to external eye phenotype by light microscopy (Fig 6A) and in subcellular localization of TDP-43 by immunohistochemistry (Fig 6B). Crosses with most of our NLS-mutant TDP-43 lines resulted in lethality; we therefore used line #5 that exhibits a very mild phenotype (all lines are shown in Supplemental Fig. 5A). Due to the severe degradative loss of internal eye tissue in some genotypes, immunohistochemistry of TDP-43 in the eye (Fig 6B) was corroborated with quantitative immunofluorescence analysis of TDP-43’s subcellular distribution in larval salivary glands (Fig 6C-D).
This epistasis study indicated no interaction between WT dVCP and WT or NES-mutant TDP-43 (Fig. 6A, C). However the combination of WT dVCP with a weak NLS-mutant resulted in mild enhancement of the pathologic phenotype. The combination of WT dVCP with a strong NLS-mutant TDP-43 resulted in lethality. Notably, the subcellular distribution of WT and mutant TDP-43 was not altered by co-expression with WT dVCP (Fig. 6B-D). By contrast, the degenerative phenotype of mutant dVCP-R152H was strongly enhanced by co-expression of WT TDP-43 (Fig. 6A, C), and this result was associated with significant clearance of WT TDP-43 from nuclei and accumulation in cytoplasm (Fig. 6B-D). Importantly, the dVCP-R152H phenotype was not enhanced by co-expression with NES-mutant TDP-43 (Fig. 6A, C), and the subcellular distribution of NES-mutant TDP-43 remained nuclear (Fig. 6B-D). Unsurprisingly, the dVCP-R152H phenotype was enhanced by NLS-mutant TDP-43 (Fig. 6A, C), and NLS-mutant TDP-43 was found predominantly in the cytoplasm (Fig. 6B-D). When the ALS-associated mutation TDP-43 M337V was co-expressed with WT dVCP, the phenotype was enhanced, and lethality resulted when the M337V mutant was co-expressed with mutant dVCP (Fig. 6A, C). Thus, genetic interaction between dVCP and WT TDP-43 was dependent on a disease-causing mutation in dVCP and was associated with redistribution of TDP-43 from the nucleus to the cytoplasm. TDP-43 restricted to the nucleus failed to interact with mutant VCP. The interaction of VCP with a known ALS-causing TDP-43 mutant further underscores the importance of cytoplasmic TDP-43 in VCP mutation-dependent degeneration.
We have developed and characterized a highly tractable Drosophila model of IBMPFD that exhibits VCP mutation-dependent degeneration (Fig.1 and Supplemental Fig. 1). Using this model we identified multiple, related RNA-binding proteins that genetically modified degeneration and one of these was TBPH, the Drosophila orthologue of TDP-43. We further demonstrated that VCP and TDP-43 interacted genetically, that disease-causing mutations in VCP led to redistribution of TDP-43 to the cytoplasm in vitro and in vivo, and that this redistribution was sufficient to cause degeneration in vivo. We also determined that a pathogenic mutation in TDP-43 enhanced the genetic interaction with VCP. Taken together, our results show that toxic gain-of-function of TDP-43 in the cytoplasm contributes to degeneration initiated by mutations in VCP.
TDP-43 pathology is a prominent pathological feature in a broad array of sporadic and inherited human diseases including ALS, FTD-TDP, Perry Syndrome and IBMPFD (Neumann et al., 2007; Geser et al., 2009; Salajegheh et al., 2009). In these diseases, TDP-43 is found to be redistributed from the nucleus to the cytoplasm in affected neurons, although the significance of this has been unclear. In brain and muscle of IBMPFD patients, TDP-43 redistributes to the cytoplasm where it co-localizes with ubiquitin-immunopositivity, and is also present in lenticular nuclear inclusions (Guinto et al., 2007). Expression of mutant VCP in SH-SY5Y cells (Gitcho et al., 2009), U20S cells (Ju et al., 2009) and in primary neurons (Fig 3) resulted in redistribution of TDP-43 to the cytoplasm, although no nuclear inclusions were observed. TDP-43 also redistributes to the cytoplasm in response to neuronal injury where it co-localizes with stress granules (Colombrita et al., 2009; Moisse et al., 2009a). Therefore, it was unclear whether the redistribution of TDP-43 in the setting of mutant VCP-related disease was a mediator of pathogenesis or an indicator of cytotoxic stress caused by disease. The results presented here clarify this issue, indicating that TDP-43 is a mediator of toxicity initiated by disease-causing mutations in VCP. This is illustrated by suppression of degeneration in the IBMPFD model when endogenous TBPH is depleted (Fig 2B, C) and enhancement of degeneration when TBPH or TDP-43 is over-expressed (Figs (Figs2D2D and and6).6). While our results indicate that accumulation of TDP-43 in the cytoplasm contributes to cytotoxicity, they do not exclude the possibility that depletion of nuclear TDP-43 also contributes to cytotoxicity.
The mechanism by which mutations in VCP influence the subcellular distribution of TDP-43 is unknown, but we outline several possibilities here (Figure 7). First, the nuclear depletion and cytoplasmic accumulation of TDP-43 might reflect a defect in the well-established role of VCP in ubiquitin-dependent segregation of substrates from multiprotein complexes. It is known that TDP-43 is present in the cytoplasm at low levels normally, where it is found in ribonucleoprotein complexes implicated in translation regulation (Freibaum et al.; Elvira et al., 2006a; Wang et al., 2008; Moisse et al., 2009b). Perhaps VCP activity is necessary for removal of TDP-43 from ribonucleoprotein complexes to permit recycling and impairment of this activity by disease-causing mutations leads to progressive accumulation of TDP-43 in the cytoplasm. A second possibility is that VCP directly participates in nuclear import of TDP-43. VCP was previously shown to regulate the nuclear import of the TSAd protein (T cell-specific adaptor protein) in T cell signal transduction (Marti and King, 2005). This aspect of VCP function may involve the adaptor Npl4 (nuclear protein localization-4), originally discovered in a yeast screen for mutants deficient in nuclear protein import (DeHoratius and Silver, 1996) (Fabre and Hurt, 1997). Perhaps VCP regulates nucleocytoplasmic shuttling of additional proteins, including TDP-43, and that disease mutations impair this activity. A third possibility relates to the recently discovered role of VCP in autophagy, and the finding that disease-causing mutations in VCP impair autophagy (Ju and Weihl; Tresse et al.). Since autophagy may be important for turnover of cytoplasmic TDP-43 (Wang et al.), accumulation of TDP-43 in the cytoplasm may simply reflect a defect in this degradation pathway. These three possibilities are not mutually exclusive and it is also possible that redistribution of TDP-43 in IBMPFD reflects defects in VCP functions that are presently unknown.
The present study shows that degeneration initiated by mutations in VCP is mediated in part through toxic gain-of-function of TDP-43 in the cytoplasm. The basis for toxicity associated with excess cytoplasmic TDP-43 is unclear, but this observation is consistent with the recent report showing that (1) cytoplasmic mislocalization of TDP-43 is toxic to neurons and (2) mutations in TDP-43 that cause familial ALS promote cytoplasmic mislocalization (Barmada et al., 2010). There is some evidence to suggest that TDP-43, or a fragment of TDP-43, is intrinsically prone to aggregation resulting in the formation of a toxic species (Johnson et al., 2009; Zhang et al., 2009). Indeed, we have observed a correlation between TDP-43 toxicity in vivo and the presence of TDP-43 cleavage products or high molecular weight species of TDP-43 (Figure 5, Supplemental Figures 4-6), although this study does not address whether there is a cause and effect relationship between these abnormal species and toxicity. Whether or not TDP-43 aggregation promotes toxicity, we are particularly intrigued by the possibility that excess cytoplasmic TDP-43 perturbs some aspect of cytoplasmic RNA metabolism. The notion that a defect in RNA metabolism contributes to IBMPFD pathogenesis is supported in the present study by the identification of xl6 (the Drosophila ortholog of human SR protein 9G8) and Hrb27C (the fly ortholog of the human hnRNP DAZAP1) as dominant modifiers of mutant-VCP toxicity in vivo. This notion is further supported by the high frequency with which inherited neurodegenerative diseases are caused by mutations that impair RNA metabolism, either through mutations in RNA-binding proteins or through mutations in RNA that impair the function of RNA binding proteins (La Spada and Taylor; Cooper et al., 2009). The extent to which perturbation in RNA metabolism contributes to TDP-43 proteinopathies in general, and IBMPFD in particular, will be fascinating to learn as the field moves forward.
We are indebted to the Cell and Tissue Imaging Core of the Hartwell Center at St. Jude Children’s Research Hospital for assistance with electron microscopy. This work was funded in part by grant AG10124 to JQT, grant AG17586 to VMYL and grants from the Association of Frontotemporal Dementias, the Dana Foundation, the Packard Foundation for ALS Research at Johns Hopkins University, the Comprehensive Neuroscience Center at the University of Pennsylvania, and the American-Syrian-Lebanese Associated Charities to JPT. GPR and JBG were supported by T32 AG000255.