TorsinA protein degradation and autophagy in DYT1 dystonia

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 DeltaE) and six amino-acid residues (torsinA Delta323-8). 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 DeltaE and torsinA Delta323-8 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 DeltaE and torsinA Delta323-8 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.     

TorsinA protein degradation and autophagy in DYT1 dystonia

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 �”E) and six amino-acid residues (torsinA �”323-8). 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 �”E and torsinA �”323-8 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 �”E and torsinA �”323-8 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.

Addendum to: Giles LM, Chen J, Li L, Chin L-S. Dystonia-associated torsinA mutations cause premature degradation of torsinA protein and cell-type-specific mislocalization to the nuclear envelope. Hum Mol Genet 2008; 17:2712-22; PMID: 18552369; DOI: 10.1093/hmg/ddn173.     

Pre-Synaptic Release Deficits in a DYT1 Dystonia Mouse Model

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.     

DYT1 Knock-In Mice Are Not Sensitized against Mitochondrial Complex-II Inhibition

DYT1 is caused by a partly penetrant dominant mutation in TOR1A that leads to a glutamic acid deletion (ΔE) in torsinA. Identifying environmental factors that modulate disease pathogenesis and penetrance could help design therapeutic strategies for dystonia. Several cell-based studies suggest that expression of torsinA(ΔE) increases the susceptibility of neuronal cells to challenges to their oxidative/energy metabolism. Based on those reports, we hypothesized that mice expressing torsinA(ΔE) would be more susceptible than control littermates to the effects of oxidative stress and ATP deficits caused by disruption of the mitochondrial respiratory chain in neurons. To test this hypothesis, we administered 20 or 50 mg/kg/day of the irreversible complex-II inhibitor 3-nitropropionic acid (3-NP) intraperitoneally for 15 consecutive days to young heterozygote DYT1 knock-in (KI) mice and wild type littermates. Repeated phenotypic assessments were performed at baseline, during and after the injections. Animals were then sacrificed and their brains processed for protein analysis. The administration of 20 mg/kg 3-NP led to increased levels of torsinA in the striatum, the main target of 3-NP, but did not cause motor dysfunction in DYT1 KI or control mice. The administration of 50 mg/kg/day of 3-NP caused the death of ～40% of wild type animals. Interestingly, DYT1 KI animals showed significantly reduced mortality. Surviving animals exhibited abnormal motor behavior during and right after the injection period, but recovered by 4 weeks postinjection independent of genotype. In contrast to the findings reported in cultured cells, these studies suggest the DYT1 mutation does not sensitize central neurons against the toxic effects of oxidative stress and energy deficits.     

Dopamine release is impaired in a mouse model of DYT1 dystonia

Early onset torsion dystonia, the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein torsinA. This form of dystonia is referred to as DYT1. We have used a transgenic mouse model of DYT1 dystonia [human mutant-type (hMT)1 mice] to examine the effect of the mutant human torsinA protein on striatal dopaminergic function. Analysis of striatal tissue dopamine (DA) and metabolites using HPLC revealed no difference between hMT1 mice and their non-transgenic littermates. Pre-synaptic DA transporters were studied using in vitro autoradiography with [(3)H]mazindol, a ligand for the membrane DA transporter, and [(3)H]dihydrotetrabenazine, a ligand for the vesicular monoamine transporter. No difference in the density of striatal DA transporter or vesicular monoamine transporter binding sites was observed. Post-synaptic receptors were studied using [(3)H]SCH-23390, a ligand for D(1) class receptors, [(3)H]YM-09151-2 and a ligand for D(2) class receptors. There were again no differences in the density of striatal binding sites for these ligands. Using in vivo microdialysis in awake animals, we studied basal as well as amphetamine-stimulated striatal extracellular DA levels. Basal extracellular DA levels were similar, but the response to amphetamine was markedly attenuated in the hMT1 mice compared with their non-transgenic littermates (253 +/- 71% vs. 561 +/- 132%, p < 0.05, two-way anova). These observations suggest that the mutation in the torsinA protein responsible for DYT1 dystonia may interfere with transport or release of DA, but does not alter pre-synaptic transporters or post-synaptic DA receptors. The defect in DA release as observed may contribute to the abnormalities in motor learning as previously documented in this transgenic mouse model, and may contribute to the clinical symptoms of the human disorder.     

Genetic background modulates the phenotype of a mouse model of DYT1 dystonia

DYT1 dystonia is a debilitating neurological disease characterized by involuntary twisting movements. The disease is caused by an in-frame deletion (GAG, "ΔE") mutation in the TOR1A gene that encodes the torsinA protein. Intriguingly, only 30% of mutation carriers exhibit motor symptoms despite the fact that functional brain imaging studies show abnormal brain metabolism in all carriers. Because genetic modifiers may be a determinant of this reduced penetrance, we examined the genetic contribution of three different inbred strains of mice on the DYT1 mutation in animals that are homozygous (Tor1a(ΔE/ΔE)) or heterozygous (Tor1a(ΔE/+); disease state) for the disease-causing ΔE mutation. We find that the DBA/2J, C57BL/6J, and CD1-ICR contribution of genes significantly alter lifespan in Tor1a(ΔE/ΔE) mice, which die during the first few days of life on the 129S6/SvEvTac (129) background. The C57BL/6J (B6) strain significantly decreases life expectancy of Tor1a(ΔE/ΔE) animals but, like 129S6/SvEvTac Tor1a(ΔE/+) mice, congenic C57BL/6J Tor1a(ΔE/+) mice do not exhibit any motor abnormalities. In contrast, the DBA/2J (D2) strain significantly increases life expectancy. This effect was not present in congenic DBA/2J Tor1a(ΔE/ΔE) mice, indicating that the extended lifespan of F2 129/D2 mice was due to a combination of homozygous and heterozygous allelic effects. Our observations suggest that genetic modifiers may alter the penetrance of the ΔE mutation, and that mapping these modifiers may provide fresh insight into the torsinA molecular pathway.     

Intragenic Cis and Trans modification of genetic susceptibility in DYT1 torsion dystonia

A GAG deletion in the DYT1 gene is a major cause of early-onset dystonia, but clinical disease expression occurs in only 30% of mutation carriers. To gain insight into genetic factors that may influence penetrance, we evaluated three DYT1 single-nucleotide polymorphisms, including D216H, a coding-sequence variation that moderates the effects of the DYT1 GAG deletion in cellular models. We tested DYT1 GAG-deletion carriers with (n=119) and without (n=113) clinical signs of dystonia and control individuals (n=197) and found the frequency of the 216H allele to be increased in GAG-deletion carriers without dystonia and to be decreased in carriers with dystonia, compared with the control individuals. Analysis of haplotypes demonstrated a highly protective effect of the H allele in trans with the GAG deletion; there was also suggestive evidence that the D216 allele in cis is required for the disease to be penetrant. Our findings establish, for the first time, a clinically relevant gene modifier of DYT1.     

拟南芥转录因子DYT1的原核表达与多克隆抗体制备

以拟南芥雄性不育突变体ms157为材料,对其转录因子DYT1的原核表达进行了分析及多克隆抗体的制备.结果表明:构建的原核表达载体转化入BL21菌株后,经IPTG诱导,表达了分子量约为50 kD的重组蛋白.SDS-PAGE检测结果表明,该重组表达蛋白以可融性形式表达.用该蛋白作为抗原注射新西兰兔后,成功获取了多克隆抗体.Western blotting证实其具良好的免疫原性,ELISA结果表明融合蛋白的效价大于1:5 120.     

The TOR1A (DYT1) gene family and its role in early onset torsion dystonia

Most cases of early onset torsion dystonia are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A gene (alias DYT1, DQ2), resulting in loss of a glutamic acid in the carboxy terminal of the encoded protein, torsin A. TOR1A and its homologue TOR1B (alias DQ1) are located adjacent to each other on human chromosome 9q34. Both genes comprise five similar exons; each gene spans a 10-kb region. Mutational analysis of most of the coding region and splice junctions of TOR1A and TOR1B did not reveal additional mutations in typical early onset cases lacking the GAG deletion (N = 17), in dystonic individuals with apparent homozygosity in the 9q34 chromosomal region (N = 5), or in a representative Ashkenazic Jewish individual with late onset dystonia, who shared a common haplotype in the 9q34 region with other late onset individuals in this ethnic group. A database search revealed a family of nine related genes (50-70% similarity) and their orthologues in species including human, mouse, rat, pig, zebrafish, fruitfly, and nematode. At least four of these genes occur in the human genome. Proteins encoded by this gene family share functional domains with the AAA/HSP/Clp-ATPase superfamily of chaperone-like proteins, but appear to represent a distinct evolutionary branch.     

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by the DYT1 Dystonia Mutation

TorsinA (TorA) is an AAA+ ATPase in the endoplasmic reticulum (ER) lumen that is mutated in early onset DYT1 dystonia. TorA is an essential protein in mice and is thought to function in the nuclear envelope (NE) despite localizing throughout the ER. Here, we report that transient interaction of TorA with the ER membrane protein LULL1 targets TorA to the NE. FRAP and Blue Native PAGE indicate that TorA is a stable, slowly diffusing oligomer in either the absence or presence of LULL1. Increasing LULL1 expression redistributes both wild-type and disease-mutant TorA to the NE, while decreasing LULL1 with shRNAs eliminates intrinsic enrichment of disease-mutant TorA in the NE. When concentrated in the NE, TorA displaces the nuclear membrane proteins Sun2, nesprin-2G, and nesprin-3 while leaving nuclear pores and Sun1 unchanged. Wild-type TorA also induces changes in NE membrane structure. Because SUN proteins interact with nesprins to connect nucleus and cytoskeleton, these effects suggest a new role for TorA in modulating complexes that traverse the NE. Importantly, once concentrated in the NE, disease-mutant TorA displaces Sun2 with reduced efficiency and does not change NE membrane structure. Together, our data suggest that LULL1 regulates the distribution and activity of TorA within the ER and NE lumen and reveal functional defects in the mutant protein responsible for DYT1 dystonia.