Our findings support an active role for torsinA in the retro-translocation of select abnormal proteins targeted for degradation from the lumen of the ER through the cytosolic proteosome pathway, as well as retro-translocation of cholera toxin peptide, CTA1. This retro-translocation function appears to be mediated, at least in part, through association of torsinA with Derlin-1, p97, and VIMP, all of which are implicated in ERAD. However, no indication of an interaction between torsinA and Hrd1 was found. Either downregulation of torsinA or overexpression of torsinAΔE (associated with early onset dystonia) was associated with decreased efficiency of ERAD for a mutant membrane-spanning protein, GFP-CFTRΔF508, thus, potentially sensitizing cells expressing mutant torsinA to ER stress associated with the unfolded protein response33
. As a corollary, elevated expression of human torsinA in cultured cells facilitated degradation of GFP-CFTRΔF508 and an ER-membrane fusion protein, BAP31-RFP. In nematodes, expression of human torsinA reduced ER stress caused by overexpression of wild-type or mutant CFTR. Further, fibroblasts from DYT1 patients showed an increased sensitivity to ER stress-inducing drugs, and a decreased ability to degrade GFP-CFTRΔF508, as compared to control fibroblasts. Our data are consistent with torsinA functioning as a shuttle or chaperone protein transferring substrates to an ERAD channel translocon complex (), with mutant torsinA being deficient in this process and thus predisposing cells to ER stress.
Hypothesized model for torsinA-mediated degradation of the CFTR mutant ΔF508
ERAD is a complex process in which diverse protein substrates may use different retro- translocation channel complexes to exit the ER. For example, CFTR and mutant forms, e.g. ΔF508, as well as the ER membrane protein, CD3delta appear to require Derlin-1 for retro-translocation, while the degradation of TCRalpha is independent of Derlin-129
. Our findings indicate that downregulation of torsinA or the presence of torsinAΔE found in DYT1 patients significantly inhibited elimination of GFP-CFTRΔF508 and BAP31-RFP out of the ER for proteolytic degradation. Likewise, downregulation of torsinA also inhibited the retro-translocation of the CTA1-peptide. However, upregulation of torsinA levels did not appear to affect ERAD of TCRalpha-YFP. Thus, torsinA appears to be involved in the retro-translocation of a subset of ER proteins.
Defective ERAD function and ER stress have a contributing role in neurologic diseases36
. In response to stress, cells temporarily decrease protein synthesis, increase movement of unfolded proteins out of the ER into the cytoplasm via ERAD, and upregulate transcription of some chaperone proteins33
. This response serves to protect cells from a variety of toxic insults which interfere with protein processing. When this response is overwhelmed by acute or long term ER stress, cellular physiology can be compromised, and in extreme cases lead to cell death. Protein components of the ERAD pathway, e.g. p97, as well as torsinA, have been found in inclusion bodies in brain and peripheral nerves from patients with a variety of neurodegenerative diseases, as well as dystonia37,38,39,40
. Additional, complementary evidence indicates that torsinA can reduce ER stress caused by ER protein overload in nematodes and that torsinA deficient mouse embryonic fibroblasts (MEFs) have increased levels of ER stress as compared to wild-type MEFs41
TorsinA has been found to associate with several different proteins, and may have different functions in the NE and ER. Interacting partners in the NE include proteins which span the inner or outer membranes, including LAP1, LULL1, Sun2 and the nesprins, and appear to influence the morphology of the NE11,8,42,12
and positioning of the nucleus during cell migration10
. Within the ER, proteins associated with torsinA include the chaperone protein, calnexin43
and proteins implicated in ERAD, including Derlin-1, p97 and VIMP (this study). Thus, within the ER torsinA appears to participate in decisions concerning the fate of proteins being processed through the secretory pathway.
Ongoing investigations suggest several ways in which the nervous system may become dysfunctional in DYT1 mutant gene carriers. First, reduced ability of mutant torsinA to degrade some abnormal proteins, as compared to wild-type torsinA (this study), may cause increased sensitivity of DYT1 neurons to ER stress. Thus, under stressful conditions, e.g. high body temperature, oxidative stress, trauma or exposure to specific drugs, neurons in DYT1 carriers may experience toxic effects that compromise their function and alter neurotransmission44
. As a corollary, ischemia or resection of nervous tissue in experimental animals results in upregulation of torsinA transcription, possibly as a compensatory mechanism45
. Second, a number of studies have implicated torsinA in processing proteins through the secretory pathway, showing that reduced torsinA, the presence of torsinAΔE or abnormally high levels of wild-type torsinA can interfere with protein processing13,14,15
. This may explain the apparently reduced/altered function of the D2 receptor46
and the dopamine release process47,48
in neurons in the basal ganglia of transgenic mice expressing human torsinAΔE. Third, the normally very high levels of torsinA in the embryonic/perinatal in rodents49,50
supports a role in neuronal development. Studies using torsinA knock-out embryos52
indicate that tangential migration of GABAergic neuroprecursor cells is compromised in the absence of torsinA53
, possibly leading to reduced numbers of inhibitory GABAergic neurons in the adult brain. Thus, dystonia may result from abnormalities at multiple cellular and system levels in the brain with contributions from increased sensitivity to environmental insults, compromised processing of components of neurotransmission, and subtle developmental neuroanatomical anomalies.
This study expands our mechanistic understanding of torsinA in facilitating retro-translocation of abnormal protein substrates, as well as a toxin, from the ER to the cytosol. This function of torsinA helps to explain the increased sensitivity of cells with loss of or mutant torsinA to ER stress, which may be a factor in neuronal dysfunction underlying abnormal movements in DYT1 carriers41
. These data further suggest that increasing levels or function of torsinA to facilitate ERAD degradation of abnormal proteins and thereby alleviate ER stress may have therapeutic benefit. Enhancement of torsinA activity, including increased secretion of a reporter protein and degradation of misfolded proteins, has been achieved using a drug discovered by screening in nematodes54
. Thus, given the low penetrance of DYT1 dystonia (30–40%) it may be possible to prevent onset of dystonic symptoms or alleviate their manifestations by treatment with small molecules that increase torsinA function and/or reduce ER stress during the period of susceptibility in children and adolescents.