Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinAΔE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinAΔE when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinAΔE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinAΔE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinAΔE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinAΔE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.
A specific mutation (ΔE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3α co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3α. This association with human nesprin-3 appeared to be stronger for torsinAΔE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3 was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.
Nesprin; Dystonia; Cell migration; Nuclear polarization; DYT1; Vimentin; Actin
TorsinA is an AAA+ ATPase located within the lumen of the endoplasmic reticulum and nuclear envelope, with a mutant form causing early onset torsion dystonia (DYT1). Here we report a new function for torsinA in endoplasmic reticulum-associated degradation (ERAD). Retro-translocation and proteosomal degradation of a mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508) was inhibited by downregulation of torsinA or overexpression of mutant torsinA, and facilitated by increased torsinA. Retro-translocation of cholera toxin was also decreased by downregulation of torsinA. TorsinA associates with proteins implicated in ERAD, including Derlin-1, VIMP, and p97. Further, torsinA reduces endoplasmic reticulum stress in nematodes overexpressing CFTRΔF508, and fibroblasts from DYT1 dystonia patients are more sensitive than controls to endoplasmic reticulum stress and less able to degrade mutant CFTR. Therefore, compromised ERAD function in the cells of DYT1 patients may increase sensitivity to endoplasmic reticulum stress with consequent alterations in neuronal function contributing to the disease state.
dystonia; movement disorder; secretory pathway; retro-translocation; protein degradation; proteosome; cystic fibrosis; cholera toxin
DYT1 early-onset generalized torsion dystonia (DYT1 dystonia) is an inherited movement disorder caused by mutations in one allele of DYT1 (TOR1A), coding for torsinA. The most common mutation is a trinucleotide deletion (ΔGAG), which causes a deletion of a glutamic acid residue (ΔE) in the C-terminal region of torsinA. Although recent studies using cultured cells suggest that torsinA contributes to protein processing in the secretory pathway, endocytosis, and the stability of synaptic proteins, the nature of how this mutation affects synaptic transmission remains unclear. We previously reported that theta-burst-induced long-term potentiation (LTP) in the CA1 region of the hippocampal slice is not altered in Dyt1 ΔGAG heterozygous knock-in (KI) mice. Here, we examined short-term synaptic plasticity and synaptic transmission in the hippocampal slices. Field recordings in the hippocampal Schaffer collaterals (SC) pathway revealed significantly enhanced paired pulse ratios (PPRs) in Dyt1 ΔGAG heterozygous KI mice, suggesting an impaired synaptic vesicle release. Whole-cell recordings from the CA1 neurons showed that Dyt1 ΔGAG heterozygous KI mice exhibited normal miniature excitatory post-synaptic currents (mEPSC), suggesting that action-potential independent spontaneous pre-synaptic release was normal. On the other hand, there was a significant decrease in the frequency, but not amplitude or kinetics, of spontaneous excitatory post-synaptic currents (sEPSC) in Dyt1 ΔGAG heterozygous KI mice, suggesting that the action-potential dependent pre-synaptic release was impaired. Moreover, hippocampal torsinA was significantly reduced in Dyt1 ΔGAG heterozygous KI mice. Although the hippocampal slice model may not represent the neurons directly associated with dystonic symptoms, impaired release of neurotransmitters caused by partial dysfunction of torsinA in other brain regions may contribute to the pathophysiology of DYT1 dystonia.
Early-onset torsion dystonia is the most severe heritable form of dystonia, a human movement disorder that typically starts during a developmental window in early adolescence. Deletion in the DYT1 gene, encoding the torsinA protein, is responsible for this dominantly inherited disorder, which is non-degenerative and exhibits reduced penetrance among carriers. Here, we explore the hypothesis that deficits in torsinA function result in an increased vulnerability to stress associated with protein folding and processing in the endoplasmic reticulum (ER), where torsinA is located. Using an in vivo quantitative readout for the ER stress response, we evaluated the consequences of torsinA mutations in transgenic nematodes expressing variants of human torsinA. This analysis revealed that, normally, torsinA serves a protective function to maintain a homeostatic threshold against ER stress. Furthermore, we show that the buffering capacity of torsinA is greatly diminished by the DYT1-associated deletion or mutations that prevent its translocation to the ER, block ATPase activity, or increase the levels of torsinA in the nuclear envelope versus ER. Combinations of transgenic Caenorhabditis elegans designed to mimic clinically relevant genetic modifiers of disease susceptibility also exhibit a direct functional correlation to changes in the ER stress response. Furthermore, using mouse embryonic fibroblasts (MEFs) from torsinA knockout mice, we demonstrated that loss of endogenous torsinA results in enhanced sensitivity to ER stress. This study extends our understanding of molecular mechanisms underlying dystonia, and establishes a new functional paradigm to evaluate therapeutic strategies to compensate for reduced torsinA activity in the ER as a means to restore homeostatic balance and neuronal function.
DYT1 dystonia is a dominantly inherited, disabling neurological disorder with low penetrance that is caused by the deletion of a glutamic acid (ΔE) in the protein torsinA. We previously showed that torsinA(wt) is degraded through macroautophagy while torsinA(ΔE) is targeted to the ubiquitin proteasome pathway (UPP). The different catabolism of torsinA(wt) and (ΔE) potentially modulates torsinA(wt):torsinA(ΔE) stoichiometry. Therefore, gaining a mechanistic understanding on how the protein quality control machinery clears torsinA(ΔE) in neurons may uncover important regulatory steps in disease pathogenesis. Here, we asked whether FBG1, a ubiquitin ligase known to degrade neuronal glycoproteins, is implicated in the degradation of torsinA(ΔE) by the UPP. In a first set of studies completed in cultured cells, we show that FBG1 interacts with and influences the steady-state levels of torsinA(wt) and (ΔE). Interestingly, FBG1 achieves this effect promoting the degradation of torsinA not only through the UPP, but also by macroautophagy. To determine the potential clinical significance of these findings, we asked if eliminating expression of Fbg1 triggers a motor phenotype in torsinA(ΔE) knock in mice, a model of non-manifesting DYT1 mutation carriers. We detected differences in spontaneous locomotion between aged torsinA(ΔE) knock in-Fbg1 knock out and control mice. Furthermore, neuronal levels of torsinA were unaltered in Fbg1 null mice, indicating that redundant systems likely compensate in vivo for the absence of this ubiquitin ligase. In summary, our studies support a non-essential role for FBG1 on the degradation of torsinA and uncover a novel link of FBG1 to the autophagy pathway.
TorsinA; DYT1 dystonia; FBG1; ubiquitin proteasome pathway; autophagy; protein degradation
TorsinA is a member of the AAA+ ATPase family of proteins and, notably, is the only known ATPase localized to the ER lumen. It has been suggested to act as a molecular chaperone, while a mutant form associated with early-onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss of function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both wild-type (WT) and mutant torsinA fusion proteins in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase (CS) aggregation. We also assessed their ability to hold proteins in an intermediate state for refolding. As measured by light scattering and SDS-PAGE, both WT and mutant torsinA effectively, and similarly, suppressed protein aggregation compared to controls. This function was not further enhanced by the presence of ATP. Further, we found that while neither form of torsinA could protect CS from heat-induced inactivation, they were both able to reactivate luciferase when ATP and rabbit reticulocyte lysate were added. This suggests that torsinA holds luciferase in an intermediate state, which can then be refolded in the presence of other chaperones. These data provide conclusive evidence that torsinA acts as a molecular chaperone in vitro and suggests that early-onset torsion dystonia is likely not a consequence of a loss in torsinA chaperone activity but might be an outcome of insufficient torsinA localization at the ER to manage protein folding or trafficking.
AAA+ protein; Endoplasmic reticulum; Chaperone; Dystonia; TorsinA
Early-onset generalized dystonia (DYT1) is a debilitating neurological disorder characterized by involuntary movements and sustained muscle spasms. DYT1 dystonia has been associated with two mutations in torsinA that result in the deletion of a single glutamate residue (torsinA ΔE) and six amino-acid residues (torsinA Δ323-8). We recently revealed that torsinA, a peripheral membrane protein, which resides predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), is a long-lived protein whose turnover is mediated by basal autophagy. Dystonia-associated torsinA ΔE and torsinA Δ323-8 mutant proteins show enhanced retention in the NE and accelerated degradation by both the proteasome and autophagy. Our results raise the possibility that the monomeric form of torsinA mutant proteins is cleared by proteasome-mediated ER-associated degradation (ERAD), whereas the oligomeric and aggregated forms of torsinA mutant proteins are cleared by ER stress-induced autophagy. Our findings provide new insights into the pathogenic mechanism of torsinA ΔE and torsinA Δ323-8 mutations in dystonia and emphasize the need for a mechanistic understanding of the role of autophagy in protein quality control in the ER and NE compartments.
Dystonia; autophagy; torsinA; endoplasmic reticulum-associated degradation; endoplasmic reticulum; nuclear envelope; protein misfolding; protein quality control
A glutamic acid deletion (ΔE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the ΔE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of “substrate trap” EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.
Early onset torsion dystonia is characterized by involuntary movements and distorted postures and is usually caused by a 3-bp (GAG) deletion in the DYT1 (TOR1A) gene. DYT1 codes for torsinA, a member of the AAA+ family of proteins, implicated in membrane recycling and chaperone functions. A close relative, torsinB may be involved in similar cellular functions. We investigated torsinA and torsinB message and protein levels in the developing mouse brain. TorsinA expression was highest during prenatal and early postnatal development (until postnatal day 14; P14), whereas torsinB expression was highest during late postnatal periods (from P14 onwards) and in the adult. In addition, significant regional variation in the expression of the two torsins was seen within the developing brain. Thus, torsinA expression was highest in the cerebral cortex from embryonic day 15 (E15)–E17 and in the striatum from E17–P7, while torsinB was highest in the cerebral cortex between P7–P14 and in the striatum from P7–P30. TorsinA was also highly expressed in the thalamus from P0–P7 and in the cerebellum from P7–P14. Although functional significance of the patterns of torsinA and B expression in the developing brain remains to be established, our findings provide a basis for investigating the role of torsins in specific processes such as neurogenesis, neuronal migration, axon/dendrite development, and synaptogenesis.
Torsion dystonia; Brain development
An in-frame 3 bp deletion in the torsinA gene resulting in the loss of a glutamate residue at position 302 or 303 (torsinA ΔE) is the major cause for early-onset torsion dystonia (DYT1). In addition, an 18 bp deletion in the torsinA gene resulting in the loss of residues 323–328 (torsinA Δ323–8) has also been associated with dystonia. Here we report that torsinA ΔE and torsinA Δ323–8 mutations cause neuronal cell-type-specific mislocalization of torsinA protein to the nuclear envelope without affecting torsinA oligomerization. Furthermore, both dystonia-associated mutations destabilize torsinA protein in dopaminergic cells. We find that wild-type torsinA protein is degraded primarily through the macroautophagy–lysosome pathway. In contrast, torsinA ΔE and torsinA Δ323–8 mutant proteins are degraded by both the proteasome and macroautophagy–lysosome pathways. Our findings suggest that torsinA mutation-induced premature degradation may contribute to the pathogenesis of dystonia via a loss-of-function mechanism and underscore the importance of both the proteasome and macroautophagy in the clearance of dystonia-associated torsinA mutant proteins.
An in-frame 3-bp deletion in the torsinA gene resulting in the loss of a glutamate residue at position 302 or 303 (torsinA ΔE) is the major cause for early-onset torsion dystonia (DYT1). In addition, an 18-bp deletion in the torsinA gene resulting in the loss of residues 323–328 (torsinA Δ323-8) has also been associated with dystonia. Here we report that torsinA ΔE and torsinA Δ323-8 mutations cause neuronal cell type-specific mislocalization of torsinA protein to the nuclear envelope without affecting torsinA oligomerization. Furthermore, both dystonia-associated mutations destabilize torsinA protein in dopaminergic cells. We find that wild-type torsinA protein is degraded primarily through the macroautophagy-lysosome pathway. In contrast, torsinA ΔE and torsinA Δ323-8 mutant proteins are degraded by both the proteasome and macroautophagy-lysosome pathways. Our findings suggest that torsinA mutation-induced premature degradation may contribute to the pathogenesis of dystonia via a loss-of-function mechanism, and underscore the importance of both the proteasome and macroautophagy in the clearance of dystonia-associated torsinA mutant proteins.
DYT1 is the most common inherited dystonia, a neurological syndrome that causes disabling involuntary muscle contractions. This autosomal dominant disease is caused by a glutamic acid deletion near the carboxy-terminus in the protein torsinA. Cell and animal based studies have shown how the DYT1 mutation causes mutant torsinA to redistribute from the endoplasmic reticulum to the nuclear envelope, acting through a dominant negative effect over the wild type protein. As a result, the wild type:mutant torsinA expression ratio would be important for disease pathogenesis, and events that influence it, such as a differential degradation process for each protein, might modulate DYT1 pathobiology. The DYT1 mutation also triggers the formation of abnormal intermolecular disulfide bonds in torsinA, although the significance of this finding is unclear. How the protein quality control machinery handles torsinA, and whether this process is affected by its abnormal oligomerization remain unknown. Here, we first explored how the disease-linked mutation influences the catabolic process of torsinA, demonstrating that the differences in subcellular localization between both forms of torsinA lead to divergences in their degradation pathways and, whereas torsinA is normally recycled through autophagy, the proteasome is also required for the efficient clearance of the mutated form. Subsequently, we determined that the abnormal disulfide bond-dependent oligomerization of mutant torsinA is not a result of its redistribution to the nuclear envelope, but a direct consequence of the mutation. Finally, we established that the presence of disulfide links in mutant torsinA oligomers interfere with their degradation by the proteasome, thus relying on autophagy as the main pathway for clearance. In conclusion, the abnormal subcellular localization and oligomerization of DYT1-linked torsinA influences its catabolic process, opening the door to the modulation of the wildtype:mutant torsinA ratio through pharmacological manipulation of protein degradation pathways.
Dystonia; autophagy; proteasome
DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been documented. The loss of LTD and SD on one hand, and the increase in LTP on the other, demonstrate that a ‘loss of inhibition’ characterizes the impairment of synaptic plasticity in this model of DYT1 dystonia. More importantly, our results indicate that an unbalanced cholinergic transmission plays a pivotal role in these alterations, providing a clue to understand the ability of anticholinergic agents to restore motor deficits in dystonia.
dystonia; synaptic plasticity; striatum; acetylcholine; electrophysiology
Early-onset torsion dystonia is a severe, life-long disease that leads to loss of motor control and involuntary muscle contractions. While the molecular etiology of the disease is not fully understood, a mutation in an AAA+ ATPase, torsinA, has been linked to disease onset. Previous work on torsinA has shown that it localizes to the endoplasmic reticulum, where there is evidence that it plays roles in protein trafficking, and potentially also protein folding. Given the high level of evolutionary conservation among proteins involved in these processes, the ability of human such proteins to function effectively in yeast, as well as the previous successes achieved in examining other proteins involved in complex human diseases in yeast, we hypothesized that Saccharomyces cerevisiae might represent a useful model system for studying torsinA function and the effects of its mutants. Since torsinA is proposed to function in protein homeostasis, we tested cells for their ability to respond to various stressors, using a fluorescent reporter to measure the unfolded protein response, as well as their rate of protein secretion. TorsinA did not impact these processes, even after co-expression of its recently identified interacting partner, printor. In light of these findings, we propose that yeast may lack an additional cofactor necessary for torsinA function or proteins required for essential post-translational modifications of torsinA. Alternatively, torsinA may not function in endoplasmic reticulum protein homeostasis. The strains and assays we describe may provide useful tools for identifying and investigating these possibilities and are freely available.
DYT1, the most common inherited dystonia, is caused by a common dominant mutation in the TOR1A gene that leads to a glutamic acid deletion in the protein torsinA. Wild type torsinA locates preferentially in the endoplasmic reticulum while the disease-linked mutant accumulates in the nuclear envelope. As a result, it has been proposed that DYT1 pathogenesis could result either from transcriptional dysregulation caused by abnormal interactions of mutant torsinA with nuclear envelope proteins, or from a loss of torsinA function in the endoplasmic reticulum that would impair specific neurobiological pathways. Aiming to determine whether one or both of these potential mechanisms are implicated in DYT1 pathogenesis, we completed unbiased transcriptional and proteomic profiling in well-characterized neural cell lines that inducibly express wild type or mutant torsinA. These experiments demonstrated that the accumulation of mutant torsinA in the nuclear envelope is not sufficient to cause transcriptional dysregulation. However, we detected expression changes at the protein level that, together with other reports, suggest a potential implication of torsinA on energy metabolism and regulation of the redox state. Furthermore, several proteins identified in this study have been previously linked to other forms of dystonia. In conclusion, our results argue against the hypothesis of transcriptional dysregulation in DYT1 dystonia, suggesting potential alternative pathogenic pathways.
Early onset torsion dystonia (DYT1), the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein, torsinA. We previously examined the effect of the human mutant torsinA on striatal dopaminergic function in a conventional transgenic mouse model of DYT1 dystonia (hMT1), in which human mutant torsinA is expressed under the cytomegalovirus promotor. Systemic administration of amphetamine did not increase dopamine (DA) release as efficiently in these mice as compared with wild-type transgenic and non-transgenic mice. We, now, studied the contribution of the DA transporter (DAT) to amphetamine-induced DA release in hMT1 transgenic mice using in vivo no-net flux microdialysis. This method applies different concentrations of DA through the microdialysis probe and measures DA concentration at the output of the probe following an equilibrium period. The slope (extraction fraction) is the measure of the DAT activity in vivo. The slope for hMT1 transgenic mice was 0.58 ± 0.07 and for non-transgenic animals, 0.87 ± 0.06 (p < 0.05). We further investigated the efficacy of nomifensine (a specific DAT inhibitor) in inhibiting amphetamine-induced DA release. Local application of nomifensine 80 min before the systemic application of amphetamine inhibited DA release in both transgenic mice and their non-transgenic littermates. The efficiency of the inhibition appeared to be different, with mean values of 48% for hMT1 transgenic mice versus 84% for non-transgenic littermates. Moreover, we have evaluated basal and amphetamine-induced locomotion in hMT1 transgenic mice compared with their non-transgenic littermates, using an O-maze behavioral chamber. Basal levels of locomotion in the hMT1 transgenic mice showed that they moved much less than their non-transgenic littermates (0.9 ± 0.3 m for transgenic mice vs. 2.4 ± 0.7 m for non-transgenic littermates, p < 0.05). This relative reduction in locomotion was also observed following amphetamine administration (48.5 ± 6.7 m for transgenics vs. 73.7 ± 9.8 m for non-transgenics, p < 0.05). These results support the finding that there are altered dynamics of DA release and reuptake in hMT1 transgenic mice in vivo, with DAT activity is reduced in the presence of mutant torsinA, which is consistent with behavioral consequences such as reduced locomotion and (previously described) abnormal motor phenotypes such as increased hind-base width and impaired performance on the raised-beam task. These data implies that altered DAT function may contribute to impaired DA neurotransmission and clinical symptoms in human DYT1 dystonia.
dopamine; DYT1 dystonia; locomotion; mouse; no-net flux microdialysis; striatum
A specific mutation (ΔE302/303) in the torsinA gene underlies most cases of dominantly inherited early-onset torsion dystonia. This mutation causes the protein to aggregate and form intracellular inclusion bodies in cultured cells and animal models. Co-expression of the wildtype and mutant proteins resulted in the redistribution of the wildtype protein from the endoplasmic reticulum to inclusion bodies in cultured HEK293 cells, and this was associated with increased interaction between the two proteins. Expression of ΔE302/303 but not wildtype torsinA in primary postnatal midbrain neurons resulted in the formation of intracellular inclusion bodies, predominantly in dopaminergic neurons. Tyrosine hydroxylase was sequestered in these inclusions and this process was mediated by increased protein-protein interaction between mutant torsinA and tyrosine hydroxylase. Analysis in an inducible neuroblastoma cell culture model demonstrated altered tyrosine hydroxylase activity in the presence of the mutant but not wildtype torsinA protein. Our results suggest that the interaction of tyrosine hydroxylase and mutant torsinA may contribute to the phenotype and reported dopaminergic dysfunction in torsinA-mediated dystonia.
Dystonia; dopamine; catecholaminergic neurons; inclusion body
Molecular mechanisms underlying neurodegenerative diseases converge at the interface of pathways impacting cellular stress, protein homeostasis and aging. Targeting the intrinsic capacities of neuroprotective proteins to restore neuronal function and/or attenuate degeneration represents a potential means toward therapeutic intervention. The product of the human DYT1 gene, torsinA, is a member of the functionally diverse AAA+ family of proteins and exhibits robust molecular-chaperone-like activity, both in vitro and in vivo. Although mutations in DYT1 are associated with a rare form of heritable generalized dystonia, the native function of torsinA seems to be cytoprotective in maintaining the cellular threshold to endoplasmic reticulum (ER) stress. Here we explore the potential for torsinA to serve as a buffer to attenuate the cellular consequences of misfolded-protein stress as it pertains to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). The selective vulnerability of motor neurons to degeneration in ALS mouse models harboring mutations in superoxide dismutase (SOD1) has been found to correlate with regional-specific ER stress in brains. Using Caenorhabditis elegans as a system to model ER stress, we generated transgenic nematodes overexpressing either wild-type or mutant human SOD1 to evaluate their relative impact on ER stress induction in vivo. These studies revealed a mutant-SOD1-specific increase in ER stress that was further exacerbated by changes in temperature, all of which was robustly attenuated by co-expression of torsinA. Moreover, through complementary behavioral analysis, torsinA was able to restore normal neuronal function in mutant G85R SOD1 animals. Furthermore, torsinA targeted mutant SOD1 for degradation via the proteasome, representing mechanistic insight on the activity that torsinA has on aggregate-prone proteins. These results expand our understanding of proteostatic mechanisms influencing neuronal dysfunction in ALS, while simultaneously highlighting the potential for torsinA as a novel target for therapeutic development.
ALS; ER stress; Chaperone; Neurotransmission; TorsinA
Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.
Most cases of early onset DYT1 dystonia in humans are caused by a GAG deletion in the TOR1A gene leading to loss of a glutamic acid (ΔE) in the torsinA protein, which underlies a movement disorder associated with neuronal dysfunction without apparent neurodegeneration. Mutation/deletion of the gene (Dst) encoding dystonin in mice results in a dystonic movement disorder termed dystonia musculorum, which resembles aspects of dystonia in humans. While torsinA and dystonin proteins do not share modular domain architecture, they participate in a similar function by modulating a structural link between the nuclear envelope and the cytoskeleton in neuronal cells. We suggest that through a shared interaction with the nuclear envelope protein nesprin-3α, torsinA and the neuronal dystonin-a2 isoform comprise a bridge complex between the outer nuclear membrane and the cytoskeleton, which is critical for some aspects of neuronal development and function. Elucidation of the overlapping roles of torsinA and dystonin-a2 in nuclear/endoplasmic reticulum dynamics should provide insights into the cellular mechanisms underlying the dystonic phenotype.
DYT1 dystonia, a common and severe primary dystonia, is caused by a 3-bp deletion in TOR1A which encodes torsinA, a protein found in the endoplasmic reticulum. Several cellular functions are altered by the mutant protein, but at a systems level the link between these and the symptoms of the disease is unclear. The most effective known therapy for DYT1 dystonia is use of anticholinergic drugs. Previous studies have revealed that in mice, transgenic expression of human mutant torsinA under a non-selective promoter leads to abnormal function of striatal cholinergic neurons. To investigate what pathological role torsinA plays in cholinergic neurons, we created a mouse model in which the Dyt1 gene, the mouse homolog of TOR1A, is selectively deleted in cholinergic neurons (ChKO animals). These animals do not have overt dystonia, but do have subtle motor abnormalities. There is no change in the number or size of striatal cholinergic cells or striatal acetylcholine content, uptake, synthesis, or release in ChKO mice. There are, however, striking functional abnormalities of striatal cholinergic cells, with paradoxical excitation in response to D2 receptor activation and loss of muscarinic M2/M4 receptor inhibitory function. These effects are specific for cholinergic interneurons, as recordings from nigral dopaminergic neurons revealed normal responses. Amphetamine stimulated dopamine release was also unaltered. These results demonstrate a cell-autonomous effect of Dyt1 deletion on striatal cholinergic function. Therapies directed at modifying the function of cholinergic neurons may prove useful in the treatment of the human disorder.
DYT1 early-onset generalized torsion dystonia is an inherited movement disorder caused by mutations in DYT1 coding for torsinA with ∼30% penetrance. Most of the DYT1 dystonia patients exhibit symptoms during childhood and adolescence. On the other hand, DYT1 mutation carriers without symptoms during these periods mostly do not exhibit symptoms later in their life. Little is known about what controls the timing of the onset, a critical issue for DYT1 mutation carriers. DYT11 myoclonus-dystonia is caused by mutations in SGCE coding for ε-sarcoglycan. Two dystonia patients from a single family with double mutations in DYT1 and SGCE exhibited more severe symptoms. A recent study suggested that torsinA contributes to the quality control of ε-sarcoglycan. Here, we derived mice carrying mutations in both Dyt1 and Sgce and found that these double mutant mice showed earlier onset of motor deficits in beam-walking test. A novel monoclonal antibody against mouse ε-sarcoglycan was developed by using Sgce knock-out mice to avoid the immune tolerance. Western blot analysis suggested that functional deficits of torsinA and ε-sarcoglycan may independently cause motor deficits. Examining additional mutations in other dystonia genes may be beneficial to predict the onset in DYT1 mutation carriers.
antibody; dystonia; ε-sarcoglycan; myoclonus-dystonia; torsinA
Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1.
cerebellum; conditional knock-out mouse; dystonia; DYT1; Purkinje cell; torsinA
DYT1 early-onset generalized dystonia is a neurological movement disorder characterized by involuntary muscle contractions. It is caused by a trinucleotide deletion of a GAG (ΔGAG) in the DYT1 (TOR1A) gene encoding torsinA; the mouse homolog of this gene is Dyt1 (Tor1a). Although structural and functional alterations in the cerebellum have been reported in DYT1 dystonia, neuronal morphology has not been examined in vivo.
In this study, we examined the morphology of the cerebellum in Dyt1 ΔGAG knock-in (KI) mice. Golgi staining of the cerebellum revealed a reduction in the length of primary dendrites and a decrease in the number of spines on the distal dendrites of Purkinje cells. To determine if this phenomenon was cell autonomous and mediated by a loss of torsinA function in Purkinje cells, we created a knockout of the Dyt1 gene only in Purkinje cells of mice. We found the Purkinje-cell specific Dyt1 conditional knockout (Dyt1 pKO) mice have similar alterations in Purkinje cell morphology, with shortened primary dendrites and decreased spines on the distal dendrites.
These results suggest that the torsinA is important for the proper development of the cerebellum and a loss of this function in the Purkinje cells results in an alteration in dendritic structure.