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.     

TorsinA and dystonia: from nuclear envelope to synapse

A GAG deletion in the DYT1 gene is responsible for the autosomal dominant movement disorder, early onset primary torsion dystonia, which is characterised by involuntary sustained muscle contractions and abnormal posturing of the limbs. The mutation leads to deletion of a single glutamate residue in the C-terminus of the protein torsinA, a member of the AAA+ ATPase family of proteins with multiple functions. Since no evidence of neurodegeneration has been found in DYT1 patients, the dystonic phenotype is likely to be the result of neuronal functional defect(s), the nature of which is only partially understood. Biochemical, structural and cell biological studies have been performed in order to characterise torsinA. These studies, together with the generation of several animal models, have contributed to identify cellular compartments and pathways, including the cytoskeleton and the nuclear envelope, the secretory pathway and the synaptic vesicle machinery where torsinA function may be crucial. However, the role of torsinA and the correlation between the dysfunction caused by the mutation and the dystonic phenotype remain unclear. This review provides an overview of the findings of the last ten years of research on torsinA, a critical evaluation of the different models proposed and insights towards future avenues of research.     

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.     

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.     

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.     

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.     

Developmental profile of the aberrant dopamine D2 receptor response in striatal cholinergic interneurons in DYT1 dystonia

Background

DYT1 dystonia, a severe form of genetically determined human dystonia, exhibits reduced penetrance among carriers and begins usually during adolescence. The reasons for such age dependence and variability remain unclear.

Methods and Results

We characterized the alterations in D2 dopamine receptor (D2R) signalling in striatal cholinergic interneurons at different ages in mice overexpressing human mutant torsinA (hMT). An abnormal excitatory response to the D2R agonist quinpirole was recorded at postnatal day 14, consisting of a membrane depolarization coupled to an increase in spiking frequency, and persisted unchanged at 3 and 9 months in hMT mice, compared to mice expressing wild-type human torsinA and non-transgenic mice. This response was blocked by the D2R antagonist sulpiride and depended upon G-proteins, as it was prevented by intrapipette GDP-β-S. Patch-clamp recordings from dissociated interneurons revealed a significant increase in the Cav2.2-mediated current fraction at all ages examined. Consistently, chelation of intracellular calcium abolished the paradoxical response to quinpirole. Finally, no gross morphological changes were observed during development.

Conclusions

These results suggest that an imbalanced striatal dopaminergic/cholinergic signaling occurs early in DYT1 dystonia and persists along development, representing a susceptibility factor for symptom generation.     

Intracellular protein degradation and autophagy in isolated pancreatic acini of the rat

Simultaneous investigation of protein degradation and autophagy of isolated exocrine pancreatic cells is carried out here for the first time in a systematic way by a complex biochemical, morphological and morphometrical approach. Protein degradation proceeds with a decreasing rate of 4-1.5 per cent per h over a 4-h period indicating a comparatively low degradation capacity. Cells in freshly isolated acini do not contain autophagic vacuoles but the latter appear within an hour in vitro and their quantity remains close to a steady state during the subsequent 3 h. Both traditional inhibitors of the autophagic-lysosomal pathway, e.g. vinblastine, leupeptin, and lysosomotropic amines together with the recently introduced 3-methyladenine, inhibit degradation to a similar maximal extent, offering the possibility of the estimation of the ratio of lysosomal/non-lysosomal degradation. In pancreatic acinar cells autophagic sequestration is unaffected and protein degradation is inhibited inside secondary lysosomes by leupeptin and lysosomotropic amines, while 3-methyladenine prevents the formation of autophagosomes. Vinblastine seems to act by inhibiting the fusion of autophagosomes with lysosomes and there is no evidence for the stimulation of autophagic sequestration by vinblastine in the present system. The effect of inhibitors of protein breakdown on protein synthesis is variable and does not correlate with their influence on degradation. Amino acids strongly stimulate protein synthesis, but in contrast to what is found in liver cells, they do not seem to affect protein degradation or autophagy significantly, thus indicating major regulatory differences of these processes between pancreatic acinar cells and hepatocytes.     

Strong allelic association between the torsion dystonia gene (DYT1) and loci on chromosome 9q34 in Ashkenazi Jews

The DYT1 gene responsible for early-onset, idiopathic torsion dystonia (ITD) in the Ashkenazi Jewish population, as well as in one large non-Jewish family, has been mapped to chromosome 9q32-34. Using (GT)n and RFLP markers in this region, we have identified obligate recombination events in some of these Jewish families, which further delineate the area containing the DYT1 gene to a 6-cM region bounded by loci AK1 and ASS. In 52 unrelated, affected Ashkenazi Jewish individuals, we have found highly significant linkage disequilibrium between a particular extended haplotype at the ABL-ASS loci and the DYT1 gene. The 4/A12 haplotype for ABL-ASS is present on 69% of the disease-bearing chromosomes among affected Jewish individuals and on only 1% of control Jewish chromosomes (chi 2 = 91.07, P much less than .001). The allelic association between this extended haplotype and DYT1 predicts that these three genes lie within 1-2 cM of each other; on the basis of obligate recombination events, the DYT1 gene is centromeric to ASS. Furthermore, this allelic association supports the idea that a single mutation event is responsible for most hereditary cases of dystonia in the Jewish population. Of the 53 definitely affected typed, 13 appear to be sporadic, with no family history of dystonia. However, the proportion of sporadic cases which potentially carry the A12 haplotype at ASS (8/13 [62%]) is similar to the proportion of familial cases with A12 (28/40 [70%]). This suggests that many sporadic cases are hereditary, that the disease gene frequency is greater than 1/15,000, and that the penetrance is lower than 30%, as previously estimated in this population. Most affected individuals were heterozygous for the ABL-ASS haplotype, a finding supporting autosomal dominant inheritance of the DYT1 gene. The ABL-ASS extended-haplotype status will provide predictive value for carrier status in Jewish individuals. This information can be used for molecular diagnosis, evaluation of subclinical expression of the disease, and elucidation of environmental factors which may modify clinical symptoms.     

Dtorsin, the Drosophila ortholog of the early-onset dystonia TOR1A (DYT1), plays a novel role in dopamine metabolism

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.     

BFRF1 protein is involved in EBV-mediated autophagy manipulation

Viral egress and autophagy are two mechanisms that seem to be strictly connected in Herpesviruses’s biology. Several data suggest that the autophagic machinery facilitates the egress of viral capsids and thus the production of new infectious particles. In the Herpesvirus family, viral nuclear egress is controlled and organized by a well conserved group of proteins named Nuclear Egress Complex (NEC). In the case of EBV, NEC is composed by BFRF1 and BFLF2 proteins, although the alterations of the nuclear host cell architecture are mainly driven by BFRF1, a multifunctional viral protein anchored to the inner nuclear membrane of the host cell. BFRF1 shares a peculiar distribution with several nuclear components and with them it strictly interacts. In this study, we investigated the possible role of BFRF1 in manipulating autophagy, pathway that possibly originates from nucleus, regulating the interplay between autophagy and viral egress.     

Vacuole membrane protein 1, autophagy and much more

Vacuole membrane protein 1 (Vmp1) is a putative transmembrane protein that has been associated with different functions including autophagy, cell adhesion and membrane traffic. Highly similar proteins are present in lower eukaryotes and plants although a homologue is absent in the fungi lineage. We have recently described the first loss-of-function mutation for a Vmp1 homologue in a model system, Dictyostelium discoideum. Our results give a more comprehensive view of the intricate roles played by this new gene. Dictyostelium Vmp1 is an endoplasmic reticulum-resident protein. Cells deficient in Vmp1 display pleiotropic defects in the context of the secretory pathway such as organelle biogenesis, the endocytic pathway and protein secretion. The biogenesis of the contractile vacuole, an organelle necessary to survive under hypoosmotic conditions, is compromised as well as the structure of the endoplasmic reticulum and the Golgi apparatus. Transmission electron microscopy also shows abnormal accumulation of aberrant double-membrane vesicles, suggesting a defect in autophagosome biogenesis or maturation. The expression of a mammalian Vmp1 in the Dictyostelium mutant complements the phenotype suggesting a functional conservation during evolution. We are taking the first steps in understanding the function of this fascinating protein and recent studies have brought us more questions than answers about its basic function and its role in human pathology.

Addendum to: Calvo-Garrido J, Carilla-Latorre S, Lázaro-Diéguez F, Egea G, Escalante R. Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion and development. Mol Biol Cell 2008; Epub ahead of print, June 11, 2008.     

Vacuole membrane protein 1, autophagy and much more

Vacuole membrane protein 1 (Vmp1) is a putative transmembrane protein that has been associated with different functions including autophagy, cell adhesion, and membrane traffic. Highly similar proteins are present in lower eukaryotes and plants although a homologue is absent in the fungi lineage. We have recently described the first loss-of-function mutation for a Vmp1 homologue in a model system, Dictyostelium discoideum. Our results give a more comprehensive view of the intricate roles played by this new gene. Dictyostelium Vmp1 is an endoplasmic reticulum-resident protein. Cells deficient in Vmp1 display pleiotropic defects in the context of the secretory pathway such as organelle biogenesis, the endocytic pathway, and protein secretion. The biogenesis of the contractile vacuole, an organelle necessary to survive under hypoosmotic conditions, is compromised as well as the structure of the endoplasmic reticulum and the Golgi apparatus. Transmission electron microscopy also shows abnormal accumulation of aberrant double-membrane vesicles, suggesting a defect in autophagosome biogenesis or maturation. The expression of a mammalian Vmp1 in the Dictyostelium mutant complements the phenotype suggesting a functional conservation during evolution. We are taking the first steps in understanding the function of this fascinating protein and recent studies have brought us more questions than answers about its basic function and its role in human pathology.     

Ordered bulk degradation via autophagy

During amino acid starvation, cells undergo macroautophagy which is regarded as an unspecific bulk degradation process. Lately, more and more organelle-specific autophagy subtypes such as reticulophagy, mitophagy and ribophagy have been described and it could be shown, depending on the experimental setup, that autophagy specifically can remove certain subcellular components. We used an unbiased quantitative proteomics approach relying on stable isotope labeling by amino acids in cell culture (SILAC) to study global protein dynamics during amino acid starvation-induced autophagy. Looking at proteasomal and lysosomal degradation ample cross-talk between the two degradation pathways became evident. Degradation via autophagy appeared to be ordered and regulated at the protein complex/organelle level. This raises several important questions such as: can macroautophagy itself be specific and what is its role during starvation?     

XBP-1u suppresses autophagy by promoting the degradation of FoxO1 in cancer cells

Autophagy is activated to maintain cellular energy homeostasis in response to nutrient starvation. However, autophagy is not persistently activated, which is poorly understood at a mechanistic level. Here, we report that turnover of FoxO1 is involved in the dynamic autophagic process caused by glutamine starvation. X-box-binding protein-1u (XBP-1u) has a critical role in FoxO1 degradation by recruiting FoxO1 to the 20S proteasome. In addition, the phosphorylation of XBP-1u by extracellular regulated protein kinases1/2 (ERK1/2) on Ser61 and Ser176 was found to be critical for the increased interaction between XBP-1u and FoxO1 upon glutamine starvation. Furthermore, knockdown of XBP-1u caused the sustained level of FoxO1 and the persistent activation of autophagy, leading to a significant decrease in cell viability. Finally, the inverse correlation between XBP-1u and FoxO1 expression agrees well with the expression profiles observed in many human cancer tissues. Thus, our findings link the dynamic process of autophagy to XBP-1u-induced FoxO1 degradation.