We have developed and characterized a highly tractable Drosophila
model of IBMPFD that exhibits VCP mutation-dependent degeneration ( 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 () 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 () and enhancement of degeneration when TBPH or TDP-43 is over-expressed (Figs and ). 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 (). 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 (, 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
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.