Deficiency in classical nonhomologous end-joining–mediated repair of transcribed genes is linked to SCA3 pathogenesis

Abstract

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-expressingDrosophilalarval brains and eyes. Importantly, SCA3 phenotype inDrosophilawas completely amenable to PNKP complementation. Hence, salvaging PNKP’s activity can be a promising therapeutic strategy for SCA3.

Determining the Fate of Neurons in SCA3: ATX3, a Rising Decision Maker in Response to DNA Stresses and Beyond

“…Given the essential role of PNKP in error-free repair of transcribed genes in postmitotic neurons, compromised PNKP activity induced by mutant ATX3 with polyQ expansion might be involved in the pathogenesis of SCA3. In support of that, PNKP overexpression partially rescues SCA3 phenotype in Drosophila model of SCA3 (Chakraborty et al, 2020).…”

“…Additionally, brain extracts from affected cerebellum region in SCA3 patients or affected brainstem (but not unaffected forebrain) in SCA3 mice model, specifically inhibit PNKP activity (Chakraborty et al, 2020). Consistent with the essential role of PNKP-mediated classical NHEJ repair in postmitotic neurons (Chatterjee et al, 2015;Chakraborty et al, 2016), higher level of DNA damage accumulates in affected brain regions of SCA3 patients and mice model (Gao et al, 2019;Chakraborty et al, 2020). Thus, selective PNKP activity impairment, DNA damage accumulation, and neuronal loss might represent potential biomarkers for SCA3 and HD.…”

“…Given the role of ATX3 in promoting PNKP activity, it raises the possibility that ATX3 might be involved in the PNKP-mediated error-free DSB repair of the transcribed genome. Indeed, ATX3 was recently found to be essential for classical NHEJ repair of DSBs in transcribed genes (Chakraborty et al, 2020). ATX3 interacts with classical NHEJ components (such as Ku70, DNA PKcs, 53BP1, PNKP, and Lig IV), nascent transcripts and RNA polymerase II (RNAP II) under physiological conditions.…”

“…Consistently, ATX3 depletion results in a compromised error-free repair of transcribed genes via classical NHEJ. In addition, ATX3 depletion causes a significant reduction in RNAP II level, probably caused by an enhanced ubiquitination of the stalled elongating RNAP II, which adversely impacts classical NHEJ repair and transcription (Chakraborty et al, 2020). Thus, ATX3 plays an important role in classical NHEJ repair of strand breaks in transcribed genes.…”

“…DDR defects are reported to associate with various genetic diseases accompanied by neurodegeneration, such as AT (ataxia telangiectasia), Xeroderma pigmentosum, Trichothiodystrophy, and Cockayne syndrome (Friedberg et al, 1995;Ciccia and Elledge, 2010). Recently, ATX3 has been reported to exert crucial roles in DDR via interacting with various DDR proteins such as polynucleotide kinase 3'-phosphatase (PNKP), mediator of DNA damage checkpoint protein 1 (MDC1), checkpoint kinase 1 (Chk1), Huntingtin (HTT), Ku70, DNA-PKcs, 53BP1 and p97 (Chatterjee et al, 2015;Gao et al, 2015Gao et al, , 2019Pfeiffer et al, 2017;Tu et al, 2017;Singh et al, 2019;Chakraborty et al, 2020). Aberrant polyQ expansion in ATX3 results in accumulation of DNA damage, activation of pro-apoptotic signaling pathway, and neurodegeneration in SCA3.…”