RESUMO
Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative spinocerebellar ataxia caused by a polyglutamine-coding CAG repeat expansion in the ATXN3 gene. While the CAG length correlates negatively with the age at onset, it accounts for approximately 50% of its variability only. Despite larger efforts in identifying contributing genetic factors, candidate genes with a robust and plausible impact on the molecular pathogenesis of MJD are scarce. Therefore, we analysed missense single nucleotide polymorphism variants in the PRKN gene encoding the Parkinson's disease-associated E3 ubiquitin ligase parkin, which is a well-described interaction partner of the MJD protein ataxin-3, a deubiquitinase. By performing a correlation analysis in the to-date largest MJD cohort of more than 900 individuals, we identified the V380L variant as a relevant factor, decreasing the age at onset by 3 years in homozygous carriers. Functional analysis in an MJD cell model demonstrated that parkin V380L did not modulate soluble or aggregate levels of ataxin-3 but reduced the interaction of the two proteins. Moreover, the presence of parkin V380L interfered with the execution of mitophagy-the autophagic removal of surplus or damaged mitochondria-thereby compromising cell viability. In summary, we identified the V380L variant in parkin as a genetic modifier of MJD, with negative repercussions on its molecular pathogenesis and disease age at onset.
Assuntos
Doença de Machado-Joseph , Mitofagia , Ubiquitina-Proteína Ligases , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/patologia , Humanos , Ubiquitina-Proteína Ligases/genética , Mitofagia/genética , Mitofagia/fisiologia , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Polimorfismo de Nucleotídeo Único , Ataxina-3/genética , Idade de Início , Proteínas RepressorasRESUMO
Ataxin-3 is a deubiquitinating enzyme and the affected protein in the neurodegenerative disorder Machado-Joseph disease (MJD). The ATXN3 gene is alternatively spliced, resulting in protein isoforms that differ in the number of ubiquitin-interacting motifs. Additionally, nonsynonymous SNPs in ATXN3 cause amino acid changes in ataxin-3, and one of these polymorphisms introduces a premature stop codon in one isoform. Here, we examined the effects of different ataxin-3 isoforms and of the premature stop codon on ataxin-3's physiological function and on main disease mechanisms. At the physiological level, we show that alternative splicing and the premature stop codon alter ataxin-3 stability and that ataxin-3 isoforms differ in their enzymatic deubiquitination activity, subcellular distribution, and interaction with other proteins. At the pathological level, we found that the expansion of the polyglutamine repeat leads to a stabilization of ataxin-3 and that ataxin-3 isoforms differ in their aggregation properties. Interestingly, we observed a functional interaction between normal and polyglutamine-expanded ATXN3 allelic variants. We found that interactions between different ATXN3 allelic variants modify the physiological and pathophysiological properties of ataxin-3. Our findings indicate that alternative splicing and interactions between different ataxin-3 isoforms affect not only major aspects of ataxin-3 function but also MJD pathogenesis. Our results stress the importance of considering isoforms of disease-causing proteins and their interplay with the normal allelic variant as disease modifiers in MJD and autosomal-dominantly inherited diseases in general.
Assuntos
Processamento Alternativo , Ataxina-3/genética , Ataxina-3/metabolismo , Doença de Machado-Joseph/genética , Agregação Patológica de Proteínas/genética , Ataxina-3/análise , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Polimorfismo de Nucleotídeo Único , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Mapas de Interação de Proteínas , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Ubiquitina/metabolismoRESUMO
BACKGROUND & AIMS: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited neurodegenerative disorder and the most common form of SCA worldwide. It is caused by the expansion of a polyglutamine (polyQ) tract in the ataxin-3 protein. Nuclear localization of the affected protein is a key event in the pathology of SCA3 via affecting nuclear organization, transcriptional dysfunction, and seeding aggregations, finally causing neurodegeneration and cell death. So far, there is no effective therapy to prevent or slow the progression of SCA3. METHODS: In this study, we explored the effect of divalproex sodium as an HDACi in SCA3 cell models and explored how divalproex sodium interferes with pathogenetic processes causing SCA3. RESULTS: We found that divalproex sodium rescues the hypoacetylation levels of histone H3 and attenuates cellular cytotoxicity induced by expanded ataxin-3 partly via preventing nuclear transport of ataxin-3 (particularly heat shock-dependent). CONCLUSION: Our study provides novel insights into the mechanisms of action of divalproex sodium as a possible treatment for SCA3, beyond the known regulation of transcription.