Ataxin-2 associates with the endocytosis complex and affects EGF receptor trafficking

Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.     

A computerized database-scan to identify c-MYC targets

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein ataxin-7, a protein of unknown function. In order to analyze the expression pattern of wild type ataxin-7 in detail, the murine SCA7 gene homolog was cloned and the expression pattern in mice analyzed. The SCA7 mouse and human gene exhibit a high degree of identity at both DNA (88.2%) and protein (88.7%) level. The CAG repeat region, known to be polymorphic in man, is conserved in mouse but contained only five repeats in all mouse strains analyzed. The arrestin homology domain and the nuclear localization signal found in human ataxin-7 is also conserved in the murine homolog. Expression of ataxin-7 was detected during mouse embryonic development and in all adult mouse tissues examined by northern and western blots. In brain, immunohistological staining revealed an ataxin-7 expression pattern similar to that in human, with ataxin-7 expression in cerebellum, several brainstem nuclei, cerebral cortex and hippocampus. Our data show high conservation of ataxin-7 both structurally and at the level of expression, suggesting a conserved role for the protein in mice and humans.     

Histone deacetylase-3 interacts with ataxin-7 and is altered in a spinocerebellar ataxia type 7 mouse model

Spinocerebellar ataxia type 7 (SCA7) is caused by a toxic polyglutamine (polyQ) expansion in the N-terminus of the protein ataxin-7. Ataxin-7 has a known function in the histone acetylase complex, Spt/Ada/Gcn5 acetylase (STAGA) chromatin-remodeling complex. We hypothesized that some histone deacetylase (HDAC) family members would impact the posttranslational modification of normal and expanded ataxin-7 and possibly modulate ataxin-7 function or neurotoxicity associated with the polyQ expansion. Interestingly, when we coexpressed each HDAC family member in the presence of ataxin-7 we found that HDAC3 increased the posttranslational modification of normal and expanded ataxin-7. Specifically, HDAC3 stabilized ataxin-7 and increased modification of the protein. Further, HDAC3 physically interacts with ataxin-7. The physical interaction of HDAC3 with normal and polyQ-expanded ataxin-7 affects the toxicity in a polyQ-dependent manner. We detect robust HDAC3 expression in neurons and glia in the cerebellum and an increase in the levels of HDAC3 in SCA7 mice. Consistent with this we found altered lysine acetylation levels and deacetylase activity in the brains of SCA7 transgenic mice. This study implicates HDAC3 and ataxin-7 interaction as a target for therapeutic intervention in SCA7, adding to a growing list of neurodegenerative diseases that may be treated by HDAC inhibitors.     

Oxidative stress-enhanced SUMOylation and aggregation of ataxin-1: Implication of JNK pathway

Although the polyglutamine protein ataxin-1 is modified by SUMO at multiple sites, the functions of such modification or how it is regulated are still unknown. Here we report that SUMO-1 or Ubc9 over-expression stimulated the aggregation of ataxin-1 and that oxidative stress, such as hydrogen peroxide treatment, further enhanced SUMO conjugation and aggregation of ataxin-1. Accordingly, co-treatment with antioxidant N-acetyl-cysteine attenuated the effect of oxidative stress. Ataxin-1, which can activate c-Jun N-terminal kinase (JNK) pathway by itself, strongly associated with apoptosis signal-regulating kinase 1 (ASK1) while not interacting with JNK. Finally, treatment of JNK-specific inhibitor caused a reduction in the oxidant-enhanced SUMOylation and aggregation of ataxin-1. Together these results indicate that SUMO modification of ataxin-1 promotes the aggregation of ataxin-1 and that oxidative stress and JNK pathway play roles in this process.     

Ataxin-3 deubiquitination is coupled to Parkin ubiquitination via E2 ubiquitin-conjugating enzyme

We reported previously that parkin, a Parkinson disease-associated E3 ubiquitin-ligase interacts with ataxin-3, a deubiquitinating enzyme associated with Machado-Joseph disease. Ataxin-3 was found to counteract parkin self-ubiquitination both in vitro and in cells. Moreover, ataxin-3-dependent deubiquitination of parkin required the catalytic cysteine 14 in ataxin-3, although the precise mechanism remained unclear. We report here that ataxin-3 interferes with the attachment of ubiquitin (Ub) onto parkin in real-time during conjugation but is unable to hydrolyze previously assembled parkin-Ub conjugates. The mechanism involves an ataxin-3-dependent stabilization of the complex between parkin and the E2 Ub-conjugating enzyme, which impedes the efficient charging of the E2 with Ub. Moreover, within this complex, the transfer of Ub from the E2 is diverted away from parkin and onto ataxin-3, further explaining how ataxin-3 deubiquitination is coupled to parkin ubiquitination. Taken together, our findings reveal an unexpected convergence upon the E2 Ub-conjugating enzyme in the regulation of an E3/deubiquitinating enzyme pair, with important implications for the function of parkin and ataxin-3, two proteins responsible for closely related neurodegenerative diseases.     

Impact of Ataxin-2 knock out on circadian locomotor behavior and PER immunoreaction in the SCN of mice

In Drosophila melanogaster, Ataxin-2 is a crucial activator of Period and is involved in the control of circadian rhythms. However, in mammals the function of Ataxin-2 is unknown despite its involvement in the inherited neurogenerative disease Spinocerebellar Ataxia type 2 in humans. Therefore, we analyzed locomotor behavior of Atxn2-deficient mice and their WT littermates under entrained- and free-running conditions as well as after experimental jet lag. Furthermore, we compared the PER1 and PER2 immunoreaction (IR) in the SCN. Atxn2^?/? mice showed an unstable rhythmicity of locomotor activity, but the level of PER1 and PER2 IR in the SCN did not differ between genotypes.     

p45, an ATPase subunit of the 19S proteasome, targets the polyglutamine disease protein ataxin-3 to the proteasome

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine tract near the C-terminus of the MJD-1 gene product, ataxin-3. Ataxin-3 is degraded by the proteasome. However, the precise mechanism of ataxin-3 degradation remains to be elucidated. In this study, we show direct links between ataxin-3 and the proteasome. p45, an ATPase subunit of the 19S proteasome, interacts with ataxin-3 in vitro and stimulates the degradation of ataxin-3 in an in vitro reconstituted degradation assay system. The effect of p45 on ataxin-3 degradation is blocked by MG132, a proteasome inhibitor. In N2a or 293 cells, overexpression of p45 strikingly enhances the clearance of both normal and expanded ataxin-3, but not alpha synuclein or SOD1, implying a functional specificity of p45 in this proteolytic process. The N-terminus of ataxin-3, which serves as a recognition site by p45, is necessary for the proteolytic process of ataxin-3. We also show that other three ATPases of the 19S proteasome, MSS1, p48, and p56 have no effect on ataxin-3 degradation. These data provide evidence that p45 plays an important role in regulating ataxin-3 degradation by the proteasome.     

Conserved domains and lack of evidence for polyglutamine length polymorphism in the chicken homolog of the Machado-Joseph disease gene product ataxin-3

Ataxin-3 is a protein of unknown function which is mutated in Machado-Joseph disease by expansion of a genetically unstable CAG repeat encoding polyglutamine. By analysis of chicken ataxin-3 we were able to identify four conserved domains of the protein and detected widespread expression in chicken tissues. In the first such analysis in a non-primate species we found that in contrast to primates, the chicken CAG repeat is short and genetically stable.     

p80 coilin,a coiled body-specific protein,interacts with ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 is associated with an elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system and co-immunoprecipitation experiments, we have found that p80 coilin, coiled body-specific protein, binds to ataxin-1. In further experiments with deletion mutants, we found that the C-terminal regions of ataxin-1 and p80 coilin were essential for this interaction. In HeLa cells that have been co-transfected with ataxin-1 and p80 coilin, the p80 coilin protein co-localizes with ataxin-1 aggregates in the nucleoplasm. However, immunohistochemical analysis and immunofluorescence assays showed that mutant ataxin-1 aggregates do not redistribute p80 coilin's dot-like structures in the Purkinje cells of SCA1 transgenic mice. This feature of the interaction between ataxin-1 and p80 coilin suggests that p80 coilin might be implicated in altering the function of ataxin-1.     

Splice isoforms of the polyglutamine disease protein ataxin-3 exhibit similar enzymatic yet different aggregation properties

Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively). In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5' variants and both of the known 3' ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity.     

Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy

The lysosomal membrane proteins LAMP-1 and LAMP-2 are estimated to contribute to about 50% of all proteins of the lysosome membrane. Surprisingly, mice deficient in either LAMP-1 or LAMP-2 are viable and fertile. However, mice deficient in both LAMP-1 and LAMP-2 have an embryonic lethal phenotype. These results show that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seems to have more specific functions since LAMP-2 single deficiency has more severe consequences than LAMP-1 single deficiency. Mutations in LAMP-2 gene cause a lysosomal glycogen storage disease, Danon disease, in humans. LAMP-2 deficient mice replicate the symptoms found in Danon patients including accumulation of autophagic vacuoles in heart and skeletal muscle. In embryonic fibroblasts, mutual disruption of both LAMPs is associated with an increased accumulation of autophagic vacuoles and unesterified cholesterol, while protein degradation rates are not affected. These results clearly show that the LAMP proteins fulfil functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.