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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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Expression of mutant ATXN3 in cells induces DNA strand breaks.(A) Expression of GFP-ATXN3-Q84 and GFP-ATXN3-Q28 was induced in SH-SY5Y cells; 48 hours after induction, the cells were harvested and their lysates analyzed by Western blotting with anti-ATXN3 monoclonal antibody to detect endogenous ATXN3 and exogenous GFP-ATXN3-Q28 and GFP-ATXN3-Q84 levels (shown by arrows). Lane 1, control SH-SY5Y cells; lane 2, SH-SY5Y cells expressing GFP-ATXN3-Q28; lane 3, SH-SY5Y cells expressing ATXN3-Q84; β-actin was used as loading control. (B) Confocal images showing expression of GFP-tagged ATXN3-Q28 and ATXN3-Q84 in SH-SY5Y cells. Nuclei were stained with DAPI in B, C and E. (C) Expression of either GFP-ATXN3-Q28 or GFP-ATXN3-Q84 was induced and the cells analyzed by immunostaining with anti-p-53BP1-S1778 antibody (red) to assess DNA strand breaks; 53BP1 foci are shown by arrows. (D) Relative number of 53BP1 foci in the SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represent mean ± SD, *** = p < 0.001). (E) SH-SY5Y cells expressing either ATXN3-Q84 or ATXN3-Q28 analyzed by immunostaining with anti-γH2AX-S139 antibody (red); γH2AX foci are shown by arrows. (F) Relative number of γH2AX foci in SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represents mean ± SD, *** = p < 0.001).
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pgen.1004834.g003: Expression of mutant ATXN3 in cells induces DNA strand breaks.(A) Expression of GFP-ATXN3-Q84 and GFP-ATXN3-Q28 was induced in SH-SY5Y cells; 48 hours after induction, the cells were harvested and their lysates analyzed by Western blotting with anti-ATXN3 monoclonal antibody to detect endogenous ATXN3 and exogenous GFP-ATXN3-Q28 and GFP-ATXN3-Q84 levels (shown by arrows). Lane 1, control SH-SY5Y cells; lane 2, SH-SY5Y cells expressing GFP-ATXN3-Q28; lane 3, SH-SY5Y cells expressing ATXN3-Q84; β-actin was used as loading control. (B) Confocal images showing expression of GFP-tagged ATXN3-Q28 and ATXN3-Q84 in SH-SY5Y cells. Nuclei were stained with DAPI in B, C and E. (C) Expression of either GFP-ATXN3-Q28 or GFP-ATXN3-Q84 was induced and the cells analyzed by immunostaining with anti-p-53BP1-S1778 antibody (red) to assess DNA strand breaks; 53BP1 foci are shown by arrows. (D) Relative number of 53BP1 foci in the SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represent mean ± SD, *** = p < 0.001). (E) SH-SY5Y cells expressing either ATXN3-Q84 or ATXN3-Q28 analyzed by immunostaining with anti-γH2AX-S139 antibody (red); γH2AX foci are shown by arrows. (F) Relative number of γH2AX foci in SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represents mean ± SD, *** = p < 0.001).

Mentions: Our studies described in the accompanying manuscript by Chatterjee et al suggest that PNKP is a native ATXN3-interacting protein, and that ATXN3 modulates PNKP activity and DNA repair (Chatterjee et al, Figs. 1–3). Immunoprecipitation of PNKP from the nuclear extract from human neuroblastoma SH-SY5Y cells and subsequent mass spectrometric analysis showed the presence of ATXN3 in the immunoprecipitated (IP) pellet; conversely, immunoprecipitation of ATXN3 and Western blot analysis revealed the presence of PNKP in the ATXN3 IP (Chatterjee et al, Figs. 1, S1, 2A and 2B). Further, GST pull-down from the nuclear extract, followed by Western blot analysis, indicated that both wild-type and mutant ATXN3 directly interact with PNKP in vitro, (Chatterjee et al; Fig. 2D). The wild-type ATXN3 protein stimulated, and in contrast, the mutant ATXN3 dramatically diminished, the 3’ phosphatase activity of PNKP in vitro (Chatterjee et al; Figs. 3A and 3B). The interaction between these two proteins was further validated in SH-SY5Y cells co-transfected with the plasmids pCherry-PNKP and pGFPC-ATXN3–28, expressing cherry-tagged PNKP and GFP-tagged ATXN3-Q28, respectively, and imaged by confocal microscopy. Analysis of the transfected cells showed significant co-localization of the red fluorescence of PNKP with the green fluorescence of ATXN3-Q28 (Fig. 1A). Similarly, cells co-transfected with pCherry-PNKP and pGFP-ATXN3-Q84 (a plasmid expressing mutant ATXN3-Q84 encoding 84 glutamines) showed marked co-localization of PNKP and ATXN3-Q84 (Fig. 1B). However, co-transfection of plasmid pCherry-PNKP and pAcGFPC1 (an empty control vector expressing GFP) did not show any detectable reconstitution of yellow/orange fluorescence (S1 Fig.), suggesting specificity of these interactions. Together, these data support our previous interpretation that both wild-type and mutant ATXN3 interact with PNKP in the cell (Chatterjee et al).


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)

Expression of mutant ATXN3 in cells induces DNA strand breaks.(A) Expression of GFP-ATXN3-Q84 and GFP-ATXN3-Q28 was induced in SH-SY5Y cells; 48 hours after induction, the cells were harvested and their lysates analyzed by Western blotting with anti-ATXN3 monoclonal antibody to detect endogenous ATXN3 and exogenous GFP-ATXN3-Q28 and GFP-ATXN3-Q84 levels (shown by arrows). Lane 1, control SH-SY5Y cells; lane 2, SH-SY5Y cells expressing GFP-ATXN3-Q28; lane 3, SH-SY5Y cells expressing ATXN3-Q84; β-actin was used as loading control. (B) Confocal images showing expression of GFP-tagged ATXN3-Q28 and ATXN3-Q84 in SH-SY5Y cells. Nuclei were stained with DAPI in B, C and E. (C) Expression of either GFP-ATXN3-Q28 or GFP-ATXN3-Q84 was induced and the cells analyzed by immunostaining with anti-p-53BP1-S1778 antibody (red) to assess DNA strand breaks; 53BP1 foci are shown by arrows. (D) Relative number of 53BP1 foci in the SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represent mean ± SD, *** = p < 0.001). (E) SH-SY5Y cells expressing either ATXN3-Q84 or ATXN3-Q28 analyzed by immunostaining with anti-γH2AX-S139 antibody (red); γH2AX foci are shown by arrows. (F) Relative number of γH2AX foci in SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represents mean ± SD, *** = p < 0.001).
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Related In: Results  -  Collection

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pgen.1004834.g003: Expression of mutant ATXN3 in cells induces DNA strand breaks.(A) Expression of GFP-ATXN3-Q84 and GFP-ATXN3-Q28 was induced in SH-SY5Y cells; 48 hours after induction, the cells were harvested and their lysates analyzed by Western blotting with anti-ATXN3 monoclonal antibody to detect endogenous ATXN3 and exogenous GFP-ATXN3-Q28 and GFP-ATXN3-Q84 levels (shown by arrows). Lane 1, control SH-SY5Y cells; lane 2, SH-SY5Y cells expressing GFP-ATXN3-Q28; lane 3, SH-SY5Y cells expressing ATXN3-Q84; β-actin was used as loading control. (B) Confocal images showing expression of GFP-tagged ATXN3-Q28 and ATXN3-Q84 in SH-SY5Y cells. Nuclei were stained with DAPI in B, C and E. (C) Expression of either GFP-ATXN3-Q28 or GFP-ATXN3-Q84 was induced and the cells analyzed by immunostaining with anti-p-53BP1-S1778 antibody (red) to assess DNA strand breaks; 53BP1 foci are shown by arrows. (D) Relative number of 53BP1 foci in the SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represent mean ± SD, *** = p < 0.001). (E) SH-SY5Y cells expressing either ATXN3-Q84 or ATXN3-Q28 analyzed by immunostaining with anti-γH2AX-S139 antibody (red); γH2AX foci are shown by arrows. (F) Relative number of γH2AX foci in SH-SY5Y cells expressing ATXN3-Q28 or ATXN3-Q84 (n = 100, data represents mean ± SD, *** = p < 0.001).
Mentions: Our studies described in the accompanying manuscript by Chatterjee et al suggest that PNKP is a native ATXN3-interacting protein, and that ATXN3 modulates PNKP activity and DNA repair (Chatterjee et al, Figs. 1–3). Immunoprecipitation of PNKP from the nuclear extract from human neuroblastoma SH-SY5Y cells and subsequent mass spectrometric analysis showed the presence of ATXN3 in the immunoprecipitated (IP) pellet; conversely, immunoprecipitation of ATXN3 and Western blot analysis revealed the presence of PNKP in the ATXN3 IP (Chatterjee et al, Figs. 1, S1, 2A and 2B). Further, GST pull-down from the nuclear extract, followed by Western blot analysis, indicated that both wild-type and mutant ATXN3 directly interact with PNKP in vitro, (Chatterjee et al; Fig. 2D). The wild-type ATXN3 protein stimulated, and in contrast, the mutant ATXN3 dramatically diminished, the 3’ phosphatase activity of PNKP in vitro (Chatterjee et al; Figs. 3A and 3B). The interaction between these two proteins was further validated in SH-SY5Y cells co-transfected with the plasmids pCherry-PNKP and pGFPC-ATXN3–28, expressing cherry-tagged PNKP and GFP-tagged ATXN3-Q28, respectively, and imaged by confocal microscopy. Analysis of the transfected cells showed significant co-localization of the red fluorescence of PNKP with the green fluorescence of ATXN3-Q28 (Fig. 1A). Similarly, cells co-transfected with pCherry-PNKP and pGFP-ATXN3-Q84 (a plasmid expressing mutant ATXN3-Q84 encoding 84 glutamines) showed marked co-localization of PNKP and ATXN3-Q84 (Fig. 1B). However, co-transfection of plasmid pCherry-PNKP and pAcGFPC1 (an empty control vector expressing GFP) did not show any detectable reconstitution of yellow/orange fluorescence (S1 Fig.), suggesting specificity of these interactions. Together, these data support our previous interpretation that both wild-type and mutant ATXN3 interact with PNKP in the cell (Chatterjee et al).

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