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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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Mutant ATXN3 activates DNA damage-response in vitro and in vivo.(A) Expression of ATXN3-Q84 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine the levels of ATM-S1981, total ATM, γH2AX-S139, total H2AX, Chk2-T68, total Chk2, p53-S15, p53-S20, p53-S46 and total p53; β-actin was used as the loading control in A, C and E. (B) Levels of ATM-S1981, γH2AX-S139, Chk2-T68, p53-S15, p53-S20 and p53-S46 relative to their respective total protein levels in cells expressing ATXN3-Q84. Cells were harvested 0 (Grey), 3 (black), 6 (blue) and 12 (red) days post ATXN3-Q84 expression in Figs. B and D (n = 4, data represent mean ± SD, *** = p < 0.001 for B and F) (C) Expression of ATXN3-Q28 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. (D) Cells expressing ATXN3-Q28 were analyzed as in B and relative levels of ATM-S1981, γH2AX and p53-S15 vs. the respective total protein levels are shown; NS denotes non-significant. (E) Total protein was isolated from the deep cerebellar nuclei (DCN) of SCA3 transgenic mice (24 weeks old) constitutively expressing human mutant ATXN3 (lanes 3 and 4) and age-matched control mice (lanes 1 and 2) and analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. Each lane represents total protein from a pool of DCN tissue from 4–5 wild-type or an equal number of transgenic littermates. (F) Relative levels of ATM-S1981, γH2AX, p53-S15 with respect to total protein in SCA3 transgenic mouse DCN (black bars) vs. age-matched control DCN (grey bars); each bar represents a pool of DCN tissue collected from 4 to 5 littermate mice (either wild-type or transgenic). Data represent mean ± SD; *** = p < 0.001.
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pgen.1004834.g004: Mutant ATXN3 activates DNA damage-response in vitro and in vivo.(A) Expression of ATXN3-Q84 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine the levels of ATM-S1981, total ATM, γH2AX-S139, total H2AX, Chk2-T68, total Chk2, p53-S15, p53-S20, p53-S46 and total p53; β-actin was used as the loading control in A, C and E. (B) Levels of ATM-S1981, γH2AX-S139, Chk2-T68, p53-S15, p53-S20 and p53-S46 relative to their respective total protein levels in cells expressing ATXN3-Q84. Cells were harvested 0 (Grey), 3 (black), 6 (blue) and 12 (red) days post ATXN3-Q84 expression in Figs. B and D (n = 4, data represent mean ± SD, *** = p < 0.001 for B and F) (C) Expression of ATXN3-Q28 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. (D) Cells expressing ATXN3-Q28 were analyzed as in B and relative levels of ATM-S1981, γH2AX and p53-S15 vs. the respective total protein levels are shown; NS denotes non-significant. (E) Total protein was isolated from the deep cerebellar nuclei (DCN) of SCA3 transgenic mice (24 weeks old) constitutively expressing human mutant ATXN3 (lanes 3 and 4) and age-matched control mice (lanes 1 and 2) and analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. Each lane represents total protein from a pool of DCN tissue from 4–5 wild-type or an equal number of transgenic littermates. (F) Relative levels of ATM-S1981, γH2AX, p53-S15 with respect to total protein in SCA3 transgenic mouse DCN (black bars) vs. age-matched control DCN (grey bars); each bar represents a pool of DCN tissue collected from 4 to 5 littermate mice (either wild-type or transgenic). Data represent mean ± SD; *** = p < 0.001.

Mentions: Activated ATM coordinates cell cycle progression with the damage-response checkpoints and DNA repair to preserve genomic integrity, via a well-orchestrated signaling network [23]. To investigate whether mutant ATXN3 activates ATM signaling in SCA3, we expressed ATXN3-Q84 in differentiated SH-SY5Y cells and assessed activation of the ATM pathway. Expression of ATXN3-Q84 strongly activated the ATM pathway, inducing the phosphorylation of ATM and H2AX and ATM’s downstream substrates Chk2 and p53 (Figs. 4A and 4B). By contrast, expression of wild-type ATXN3-Q28 did not activate the ATM pathway (Figs. 4C and 4D), suggesting that mutant ATXN3 strongly activates the DNA damage-response pathway and the polyQ sequence length is important for ATM pathway activation. Likewise, expression of the mutant ATXN3 carrying 72 and 80 poly-glutamines (ATXN3-Q72 and ATXN3-Q80) in SH-SY5Y cells also strongly activated the DNA damage-response ATM pathway (S8 Fig.). Furthermore, to test whether mutant ATXN3 activates p53 and Chk2 via activating ATM, we pre-treated the cells with ATM inhibitor Ku55933 and expressed ATXN3-Q84 and assessed the activation of DNA damage response pathway. Consistent with our hypothesis, ATXN3-Q84 expression failed to stimulate phosphorylation of Chk2 and p53 in the presence of the ATM inhibitor Ku55933 (S9 Fig.), substantiating our interpretation that mutant ATXN3 stimulates the DNA damage response p53 pathway via activating ATM. The dramatic increase in ATM, H2AX, Chk2 and p53 phosphorylation (Figs. 4A and S8) and formation of 53BP1 and γH2AX foci (Fig. 3) in response to mutant ATXN3 expression suggest that mutant ATXN3-induced genomic DNA strand breaks/damage is sufficient to activate the DNA damage- response pathway. Further, analysis of the tissue from the deep cerebellar nuclei (DCN) from SCA3 transgenic mice (CMVMJD135 mice) constitutively expressing mutant ATXN3 showed robust activation of the ATM pathway (increased phosphorylation of ATM, H2AX and p53) (Figs. 4E and 4F), suggesting that mutant ATXN3 strongly activates the DNA damage-response pathway in vivo. To further test whether inactivation of PNKP by mutant ATXN3 stimulates the ATM pathway, we examined PNKP-siRNA-treated differentiated SH-SY5Y cells for ATM pathway activation. Our data showed robust activation of the ATM and p53 pathways in cells transfected with PNKP-siRNA, but not in cells transfected with control-siRNA (S10 Fig.). To rule out the possibility that DNA damage and subsequent activation of the DNA damage response might be due in part to non-specific off-target toxic effects of the PNKP-siRNA, we used micro-RNA-adapted RNA interference (shRNAmir) to achieve more specific knockdown of PNKP in cells, and assessed activation of the DNA damage-response pathway in these cells. Similar to our previous observation described in S7 Fig., depletion of PNKP in SH-SY5Y cells with PNKP-shRNAmir constructs also resulted in increased genomic DNA damage (53BP1 and γH2AX foci formation; shown by arrows; S11 Fig.), and marked activation of the DNA damage-response ATM pathway (S12 Fig.). Moreover, recent studies have indicated that a mutant ATXN3-mediated increase in oxidative stress might be responsible for inducing DNA damage and SCA3 pathology [22]. Since oxidative stress alone can activate the ATM pathway [24], we sought to determine whether mutant ATXN3 activates ATM via an oxidation-dependent mechanism. To test this possibility, we induced ATXN3-Q84 expression in differentiated SH-SY5Y cells pre-treated with the antioxidant N-acetyl cysteine (NAC). However, pre-treating cells with NAC did not block mutant ATXN3-mediated activation of the DNA damage-response pathway (S13A and S13B Figs.). Likewise, expression of ATXN3-Q84 strongly activated the ATM pathway in cells overexpressing the antioxidant enzyme catalase (S13C and S13D Figs.), suggesting that the mutant ATXN3-induced DNA damage-response ATM pathway activation is oxidation-independent.


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)

Mutant ATXN3 activates DNA damage-response in vitro and in vivo.(A) Expression of ATXN3-Q84 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine the levels of ATM-S1981, total ATM, γH2AX-S139, total H2AX, Chk2-T68, total Chk2, p53-S15, p53-S20, p53-S46 and total p53; β-actin was used as the loading control in A, C and E. (B) Levels of ATM-S1981, γH2AX-S139, Chk2-T68, p53-S15, p53-S20 and p53-S46 relative to their respective total protein levels in cells expressing ATXN3-Q84. Cells were harvested 0 (Grey), 3 (black), 6 (blue) and 12 (red) days post ATXN3-Q84 expression in Figs. B and D (n = 4, data represent mean ± SD, *** = p < 0.001 for B and F) (C) Expression of ATXN3-Q28 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. (D) Cells expressing ATXN3-Q28 were analyzed as in B and relative levels of ATM-S1981, γH2AX and p53-S15 vs. the respective total protein levels are shown; NS denotes non-significant. (E) Total protein was isolated from the deep cerebellar nuclei (DCN) of SCA3 transgenic mice (24 weeks old) constitutively expressing human mutant ATXN3 (lanes 3 and 4) and age-matched control mice (lanes 1 and 2) and analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. Each lane represents total protein from a pool of DCN tissue from 4–5 wild-type or an equal number of transgenic littermates. (F) Relative levels of ATM-S1981, γH2AX, p53-S15 with respect to total protein in SCA3 transgenic mouse DCN (black bars) vs. age-matched control DCN (grey bars); each bar represents a pool of DCN tissue collected from 4 to 5 littermate mice (either wild-type or transgenic). Data represent mean ± SD; *** = p < 0.001.
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pgen.1004834.g004: Mutant ATXN3 activates DNA damage-response in vitro and in vivo.(A) Expression of ATXN3-Q84 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine the levels of ATM-S1981, total ATM, γH2AX-S139, total H2AX, Chk2-T68, total Chk2, p53-S15, p53-S20, p53-S46 and total p53; β-actin was used as the loading control in A, C and E. (B) Levels of ATM-S1981, γH2AX-S139, Chk2-T68, p53-S15, p53-S20 and p53-S46 relative to their respective total protein levels in cells expressing ATXN3-Q84. Cells were harvested 0 (Grey), 3 (black), 6 (blue) and 12 (red) days post ATXN3-Q84 expression in Figs. B and D (n = 4, data represent mean ± SD, *** = p < 0.001 for B and F) (C) Expression of ATXN3-Q28 was induced in differentiated SH-SY5Y cells; cells were harvested 0, 3, 6 and 12 days post-induction (lanes 1 to 4) and their lysates analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. (D) Cells expressing ATXN3-Q28 were analyzed as in B and relative levels of ATM-S1981, γH2AX and p53-S15 vs. the respective total protein levels are shown; NS denotes non-significant. (E) Total protein was isolated from the deep cerebellar nuclei (DCN) of SCA3 transgenic mice (24 weeks old) constitutively expressing human mutant ATXN3 (lanes 3 and 4) and age-matched control mice (lanes 1 and 2) and analyzed by Western blotting to determine ATM-S1981, total ATM, γH2AX-S139, total H2AX, p53-S15 and total p53 levels. Each lane represents total protein from a pool of DCN tissue from 4–5 wild-type or an equal number of transgenic littermates. (F) Relative levels of ATM-S1981, γH2AX, p53-S15 with respect to total protein in SCA3 transgenic mouse DCN (black bars) vs. age-matched control DCN (grey bars); each bar represents a pool of DCN tissue collected from 4 to 5 littermate mice (either wild-type or transgenic). Data represent mean ± SD; *** = p < 0.001.
Mentions: Activated ATM coordinates cell cycle progression with the damage-response checkpoints and DNA repair to preserve genomic integrity, via a well-orchestrated signaling network [23]. To investigate whether mutant ATXN3 activates ATM signaling in SCA3, we expressed ATXN3-Q84 in differentiated SH-SY5Y cells and assessed activation of the ATM pathway. Expression of ATXN3-Q84 strongly activated the ATM pathway, inducing the phosphorylation of ATM and H2AX and ATM’s downstream substrates Chk2 and p53 (Figs. 4A and 4B). By contrast, expression of wild-type ATXN3-Q28 did not activate the ATM pathway (Figs. 4C and 4D), suggesting that mutant ATXN3 strongly activates the DNA damage-response pathway and the polyQ sequence length is important for ATM pathway activation. Likewise, expression of the mutant ATXN3 carrying 72 and 80 poly-glutamines (ATXN3-Q72 and ATXN3-Q80) in SH-SY5Y cells also strongly activated the DNA damage-response ATM pathway (S8 Fig.). Furthermore, to test whether mutant ATXN3 activates p53 and Chk2 via activating ATM, we pre-treated the cells with ATM inhibitor Ku55933 and expressed ATXN3-Q84 and assessed the activation of DNA damage response pathway. Consistent with our hypothesis, ATXN3-Q84 expression failed to stimulate phosphorylation of Chk2 and p53 in the presence of the ATM inhibitor Ku55933 (S9 Fig.), substantiating our interpretation that mutant ATXN3 stimulates the DNA damage response p53 pathway via activating ATM. The dramatic increase in ATM, H2AX, Chk2 and p53 phosphorylation (Figs. 4A and S8) and formation of 53BP1 and γH2AX foci (Fig. 3) in response to mutant ATXN3 expression suggest that mutant ATXN3-induced genomic DNA strand breaks/damage is sufficient to activate the DNA damage- response pathway. Further, analysis of the tissue from the deep cerebellar nuclei (DCN) from SCA3 transgenic mice (CMVMJD135 mice) constitutively expressing mutant ATXN3 showed robust activation of the ATM pathway (increased phosphorylation of ATM, H2AX and p53) (Figs. 4E and 4F), suggesting that mutant ATXN3 strongly activates the DNA damage-response pathway in vivo. To further test whether inactivation of PNKP by mutant ATXN3 stimulates the ATM pathway, we examined PNKP-siRNA-treated differentiated SH-SY5Y cells for ATM pathway activation. Our data showed robust activation of the ATM and p53 pathways in cells transfected with PNKP-siRNA, but not in cells transfected with control-siRNA (S10 Fig.). To rule out the possibility that DNA damage and subsequent activation of the DNA damage response might be due in part to non-specific off-target toxic effects of the PNKP-siRNA, we used micro-RNA-adapted RNA interference (shRNAmir) to achieve more specific knockdown of PNKP in cells, and assessed activation of the DNA damage-response pathway in these cells. Similar to our previous observation described in S7 Fig., depletion of PNKP in SH-SY5Y cells with PNKP-shRNAmir constructs also resulted in increased genomic DNA damage (53BP1 and γH2AX foci formation; shown by arrows; S11 Fig.), and marked activation of the DNA damage-response ATM pathway (S12 Fig.). Moreover, recent studies have indicated that a mutant ATXN3-mediated increase in oxidative stress might be responsible for inducing DNA damage and SCA3 pathology [22]. Since oxidative stress alone can activate the ATM pathway [24], we sought to determine whether mutant ATXN3 activates ATM via an oxidation-dependent mechanism. To test this possibility, we induced ATXN3-Q84 expression in differentiated SH-SY5Y cells pre-treated with the antioxidant N-acetyl cysteine (NAC). However, pre-treating cells with NAC did not block mutant ATXN3-mediated activation of the DNA damage-response pathway (S13A and S13B Figs.). Likewise, expression of ATXN3-Q84 strongly activated the ATM pathway in cells overexpressing the antioxidant enzyme catalase (S13C and S13D Figs.), suggesting that the mutant ATXN3-induced DNA damage-response ATM pathway activation is oxidation-independent.

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