RESUMO
Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.
Assuntos
Ataxina-3/genética , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Doença de Machado-Joseph/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Ataxina-3/metabolismo , Encéfalo/patologia , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Drosophila , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Peptídeos/genética , RNA Interferente Pequeno/metabolismoRESUMO
We previously reported that the loss of activity of an essential DNA repair enzyme, polynucleotide kinase 3'-phosphatase (PNKP), resulted in accumulation of double strand breaks (DSB) in patient's brain genome in Huntington's disease (HD) and Spinocerebellar ataxia type 3 (SCA3). Here we document that PNKP interacts with the nuclear isoform of phosphofructokinase fructose-2,6-bisphosphatase 3 (PFKFB3), which converts fructose-6-phosphate (F6P) into fructose-2,6-bisphosphate (F2,6BP), a potent allosteric modulator of glycolysis. Depletion of PFKFB3 markedly abrogates PNKP activity, thereby affecting PNKP mediated transcription-coupled non-homologous end joining (TC-NHEJ). Both PFKFB3 and F2,6BP levels are significantly lower in the nuclear extracts of HD and SCA3 patients' brains. Exogenous F2,6BP restored PNKP activity in the brain nuclear extracts of those samples. Moreover, delivery of F2,6BP into HD mouse striata-derived neuronal cells restored PNKP activity, transcribed genome integrity and cellular viability. We thus postulate that F2,6BP serves in vivo as a cofactor for proper functionality of PNKP and thereby of brain health. Our results thus provide a compelling rationale for exploring therapeutic use of F2,6BP and related compounds for treating polyQ diseases.