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Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Gao R, Liu Y, Silva-Fernandes A, Fang X, Paulucci-Holthauzen A, Chatterjee A, Zhang HL, Matsuura T, Choudhary S, Ashizawa T, Koeppen AH, Maciel P, Hazra TK, Sarkar PS - PLoS Genet. (2015)

Bottom Line: We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3.Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death.Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

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Proposed mechanism where interaction of PNKP with mutant expanded polyQ-containing ATXN3 abrogates PNKP’s 3’-phosphatase activity.This atypical interaction perturbs the efficacy of DNA repair, leading to the persistent accumulation of DNA damage/strand breaks in SCA3. The wild-type ATXN3 binds with and stimulates PNKP’s enzymatic activity to repair damaged DNA, contributing to genomic DNA sequence integrity and neuronal survival. By contrast, mutant ATXN3 interacts with and decreases PNKP’s 3’-phosphatase activity, leading to the persistent accumulation of DNA damage in the post-mitotic neurons and resulting in chronic activation of the DNA damage-response ATM→p53 signaling pathway in SCA3. This triggers apoptosis by increasing expression of such p53 target genes as BAX, PUMA and NOXA in SCA3. In parallel, activated ATM phosphorylates c-Abl kinase, which in turn phosphorylates PKCδ, facilitating nuclear translocation of PKCδ and thus further amplifying the pro-apoptotic output in SCA3 ultimately leading to neuronal death in SCA3.
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pgen.1004834.g008: Proposed mechanism where interaction of PNKP with mutant expanded polyQ-containing ATXN3 abrogates PNKP’s 3’-phosphatase activity.This atypical interaction perturbs the efficacy of DNA repair, leading to the persistent accumulation of DNA damage/strand breaks in SCA3. The wild-type ATXN3 binds with and stimulates PNKP’s enzymatic activity to repair damaged DNA, contributing to genomic DNA sequence integrity and neuronal survival. By contrast, mutant ATXN3 interacts with and decreases PNKP’s 3’-phosphatase activity, leading to the persistent accumulation of DNA damage in the post-mitotic neurons and resulting in chronic activation of the DNA damage-response ATM→p53 signaling pathway in SCA3. This triggers apoptosis by increasing expression of such p53 target genes as BAX, PUMA and NOXA in SCA3. In parallel, activated ATM phosphorylates c-Abl kinase, which in turn phosphorylates PKCδ, facilitating nuclear translocation of PKCδ and thus further amplifying the pro-apoptotic output in SCA3 ultimately leading to neuronal death in SCA3.

Mentions: We next assessed whether blocking PKCδ phosphorylation by inhibiting c-Abl kinase activity ameliorates mutant ATXN3-mediated caspase-3 activation. Pre-treating cells with STI-571 significantly inhibited mutant ATXN3-Q84-mediated caspase-3 activation (S19A Fig.). Likewise, pre-treating cells with STI-571 also ameliorated PNKP-siRNA-mediated caspase-3 activation (S19B Fig.). Collectively, these data, together with the data presented in the accompanying manuscript by Chatterjee et al, suggest that the wild-type ATXN3 interacts with and stimulates PNKP’s 3’-phosphatase activity, and this interaction possibly modulates the efficacy of DNA repair, and helps maintain genomic integrity and neuronal survival. In contrast, mutant ATXN3 interacts with PNKP and abrogates its 3’-phosphatase activity, resulting in increased accumulation of DNA damage that chronically activates ATM→p53 and ATM→c-Abl→PKCδ pro-apoptotic signaling to trigger neuronal dysfunction and apoptosis in SCA3 (illustrated in Fig. 8).


Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Gao R, Liu Y, Silva-Fernandes A, Fang X, Paulucci-Holthauzen A, Chatterjee A, Zhang HL, Matsuura T, Choudhary S, Ashizawa T, Koeppen AH, Maciel P, Hazra TK, Sarkar PS - PLoS Genet. (2015)

Proposed mechanism where interaction of PNKP with mutant expanded polyQ-containing ATXN3 abrogates PNKP’s 3’-phosphatase activity.This atypical interaction perturbs the efficacy of DNA repair, leading to the persistent accumulation of DNA damage/strand breaks in SCA3. The wild-type ATXN3 binds with and stimulates PNKP’s enzymatic activity to repair damaged DNA, contributing to genomic DNA sequence integrity and neuronal survival. By contrast, mutant ATXN3 interacts with and decreases PNKP’s 3’-phosphatase activity, leading to the persistent accumulation of DNA damage in the post-mitotic neurons and resulting in chronic activation of the DNA damage-response ATM→p53 signaling pathway in SCA3. This triggers apoptosis by increasing expression of such p53 target genes as BAX, PUMA and NOXA in SCA3. In parallel, activated ATM phosphorylates c-Abl kinase, which in turn phosphorylates PKCδ, facilitating nuclear translocation of PKCδ and thus further amplifying the pro-apoptotic output in SCA3 ultimately leading to neuronal death in SCA3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295939&req=5

pgen.1004834.g008: Proposed mechanism where interaction of PNKP with mutant expanded polyQ-containing ATXN3 abrogates PNKP’s 3’-phosphatase activity.This atypical interaction perturbs the efficacy of DNA repair, leading to the persistent accumulation of DNA damage/strand breaks in SCA3. The wild-type ATXN3 binds with and stimulates PNKP’s enzymatic activity to repair damaged DNA, contributing to genomic DNA sequence integrity and neuronal survival. By contrast, mutant ATXN3 interacts with and decreases PNKP’s 3’-phosphatase activity, leading to the persistent accumulation of DNA damage in the post-mitotic neurons and resulting in chronic activation of the DNA damage-response ATM→p53 signaling pathway in SCA3. This triggers apoptosis by increasing expression of such p53 target genes as BAX, PUMA and NOXA in SCA3. In parallel, activated ATM phosphorylates c-Abl kinase, which in turn phosphorylates PKCδ, facilitating nuclear translocation of PKCδ and thus further amplifying the pro-apoptotic output in SCA3 ultimately leading to neuronal death in SCA3.
Mentions: We next assessed whether blocking PKCδ phosphorylation by inhibiting c-Abl kinase activity ameliorates mutant ATXN3-mediated caspase-3 activation. Pre-treating cells with STI-571 significantly inhibited mutant ATXN3-Q84-mediated caspase-3 activation (S19A Fig.). Likewise, pre-treating cells with STI-571 also ameliorated PNKP-siRNA-mediated caspase-3 activation (S19B Fig.). Collectively, these data, together with the data presented in the accompanying manuscript by Chatterjee et al, suggest that the wild-type ATXN3 interacts with and stimulates PNKP’s 3’-phosphatase activity, and this interaction possibly modulates the efficacy of DNA repair, and helps maintain genomic integrity and neuronal survival. In contrast, mutant ATXN3 interacts with PNKP and abrogates its 3’-phosphatase activity, resulting in increased accumulation of DNA damage that chronically activates ATM→p53 and ATM→c-Abl→PKCδ pro-apoptotic signaling to trigger neuronal dysfunction and apoptosis in SCA3 (illustrated in Fig. 8).

Bottom Line: We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3.Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death.Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Show MeSH
Related in: MedlinePlus