Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

A neuroprotective role for the DNA damage checkpoint in tauopathy

A neuroprotective role for the DNA damage checkpoint in tauopathy A number of studies have demonstrated DNA replication and cell cycle activation within postmitotic neurons in tauopathies, including Alzheimer’s disease and related disorders ( Andorfer , 2005 ; Herrup & Yang, 2007 ). A growing literature also demonstrates that DNA damage accompanies neurodegeneration ( Kruman , 2004 ; Kim , 2008 ). In the major DNA damage checkpoint responding to DNA double‐stranded breaks (DSBs), the Ser/Thr PI3 kinase family member ATM activates targets including p53 and Chk2, a transcription factor and Ser/Thr kinase respectively, to mediate either cell cycle arrest or apoptosis ( Lee & McKinnon, 2000 ). Reports of upregulation of p53 and its homolog p73 in Alzheimer’s disease brains ( Kitamura , 1997 ; Wilson , 2004 ), coupled with studies indicating ATM and p53 inhibition in cultured or developing neurons is neuroprotective, has led to arguments that ATM and p53 may be pro‐apoptotic in Alzheimer’s disease ( Kruman , 2004 ; Culmsee & Mattson, 2005 ; Kim , 2008 ). Here, we probe the relationship of the DNA damage checkpoint to tau‐dependent neurodegeneration in mature postmitotic neurons of the genetically tractable fruit fly. To show activation of a DSB checkpoint in experimental tauopathy, we first demonstrated that p53 was upregulated in cortical neurons in tau transgenic mice ( Fig. 1a , arrow; Fig. S1a, Supporting information; Ittner , 2008 ) compared to age‐matched control brains. The phosphorylation at Ser‐139 of the histone variant H2AX (pH2AX) is a specific marker for DSBs in mammalian cells. We found expression of pH2AX in neurons from tau transgenic mice ( Fig. 1c , arrow; Fig. S1b), but not in control tissue. We then investigated whether these changes were mirrored in a Drosophila tauopathy model, in which pan‐neuronal expression of human tau recapitulates key features of human tauopathies ( Khurana , 2006 ). In the fly model, as in tau transgenic mice, there was upregulation of neuronal p53 ( Fig. 1b ; Fig. S2), along with induction of Chk1 (Fig. S3) and the p53 target gene Gadd45 (Fig. S4). In Drosophila , the homologous histone modification to pH2AX is phosphorylation at Ser‐137 of H2Av (pH2Av). We found that antibodies to pH2Av labeled neuronal chromatin in tau‐expressing adult animals ( Fig. 1d , arrow), but not in age‐matched controls. To demonstrate physical evidence of DNA damage, brains from tau‐expressing Drosophila were analyzed with the comet tail assay in which DNA single‐ or DSBs are demonstrated using single‐cell gel electrophoresis. We observed that nuclei of tau‐expressing flies displayed greater than a 2‐fold longer comet tails ( Fig. 1e ). 1 Upregulation of p53 and DNA damage in tauopathy models. (a, c) Increased p53 (a) or pH2AX (c) in neurons of tau transgenic mice (arrows). Scale bars are 10 μm. (b, d) Elevated p53 (b) or pH2AX (d) in neurons of tau transgenic flies (arrows). Scale bars are 3 μm. (e) Comet assay shows an increase in tail moment in tau transgenic fly brains compared to control. * P < 0.01, unpaired t ‐test. Control genotype: elav‐GAL4/+ . Flies are 10 days old. Having established that a DSB response occurs in tauopathy models, we next determined the effect of genetically manipulating the checkpoint. Neurodegeneration in our model can be assessed by TUNEL staining, caspase cleavage, and standard histology ( Khurana , 2006 ). We first determined whether the Drosophila homolog of ATM , dATM , is a modifier of tau‐induced toxicity in the adult fly brain. We found that removing one copy of dATM significantly enhanced tau‐induced neuronal apoptosis ( Fig. 2a ), with two separate dATM loss‐of‐function alleles, indicating a protective role for dATM in tauopathy. 2 Neurodegeneration and cell cycle are promoted by reducing checkpoint function. (a, c–e) Reducing checkpoint function increases neurodegeneration (a–c, TUNEL) and cell cycle re‐entry (d, e, PCNA, arrowheads). (c) No further increases in tau neurotoxicity are observed when two checkpoints are reduced simultaneously, consistent with a nonlinear interaction. * P < 0.01, ANOVA with Student–Neuman–Keuls test. Control genotype: elav‐GAL4/+ . Flies are 10 days old. Fly homologs of Chk2 and p53, dChk2 and Dmp53, also play a homologous role in the DNA damage response. Dominant‐negative transgenic constructs of these modifiers have been created. A dominant‐negative Chk2 transgene and two separate dominant‐negative p53 transgenes led to a marked increase in tau‐induced apoptosis ( Fig. 2b ; Fig. S5a,b). Conversely, increasing expression of p53 rescued toxicity (Fig. S6). Consistent with ATM , Dmp53 and Chk2 acting in the same pathway, combining these individual genetic modifiers demonstrated a nonadditive modifying effect ( Fig. 2c ). Importantly, genetic modifiers did not alter tau expression (Fig. S7). These data support a neuroprotective role for an ATM‐dependent DSB checkpoint in the fly tauopathy model. We reasoned that the DSB checkpoint could be neuroprotective by inhibiting the cell cycle. We have previously shown that abnormal markers of cell cycle activation can be detected in adult neurons within the brain of tau‐expressing flies, including proliferating cell nuclear antigen (PCNA), a marker also upregulated in Alzheimer’s disease and related tauopathies ( Fig. 2 ; Khurana , 2006 ; Herrup & Yang, 2007 ). Furthermore, cell cycle re‐activation is causally linked to tau‐induced neurodegeneration in fly and mouse Alzheimer’s disease models ( Andorfer , 2005 ; Khurana , 2006 ; Kim , 2008 ). We found that decreasing function of ATM, Chk2 or p53 strongly upregulated tau‐induced PCNA ( Fig. 2d,e , arrowheads). These data implicated re‐entry of postmitotic neurons into the cell cycle as a mechanism through which checkpoint inhibition exacerbates neurotoxicity. There is a growing literature on aberrant cell cycle re‐entry, DNA damage and checkpoint activation in Alzheimer’s disease and related tauopathies ( Herrup & Yang, 2007 ; Kim , 2008 ; Myung , 2008 ). The relationship among these three processes, however, has remained unclear. In this article, we provide direct evidence of DNA damage and checkpoint activation in tauopathy models. Our previous work identifying oxidative stress as a critical component of tau‐induced neurodegeneration provides one potential mechanism for tau‐induced DNA damage ( Barlow , 1999 ; Dias‐Santagata , 2007 ). The genetic data presented in this study argue for a neuroprotective role of the DNA damage checkpoint and suggest caution in designing p53 inhibitors as therapeutic agents in Alzheimer’s disease and related tauopathies. A number of recent studies have demonstrated an early protective role for the DNA damage checkpoint in tumorigenesis ( Bartkova , 2005 ). Together with these findings and the known concomitant neurodegeneration and tumorigenesis that occur with ATM loss‐of‐function in the disease ataxia telangiectasia, our data raise the possibility of a generally protective function for the DNA damage checkpoint in diseases of aging. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aging Cell Wiley

A neuroprotective role for the DNA damage checkpoint in tauopathy

Download PDF
3 pages

Loading next page...
 
/lp/wiley/a-neuroprotective-role-for-the-dna-damage-checkpoint-in-tauopathy-1uPnfjKeTP

References (18)

Publisher
Wiley
Copyright
© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland
ISSN
1474-9718
eISSN
1474-9726
DOI
10.1111/j.1474-9726.2011.00778.x
pmid
22181010
Publisher site
See Article on Publisher Site

Abstract

A number of studies have demonstrated DNA replication and cell cycle activation within postmitotic neurons in tauopathies, including Alzheimer’s disease and related disorders ( Andorfer , 2005 ; Herrup & Yang, 2007 ). A growing literature also demonstrates that DNA damage accompanies neurodegeneration ( Kruman , 2004 ; Kim , 2008 ). In the major DNA damage checkpoint responding to DNA double‐stranded breaks (DSBs), the Ser/Thr PI3 kinase family member ATM activates targets including p53 and Chk2, a transcription factor and Ser/Thr kinase respectively, to mediate either cell cycle arrest or apoptosis ( Lee & McKinnon, 2000 ). Reports of upregulation of p53 and its homolog p73 in Alzheimer’s disease brains ( Kitamura , 1997 ; Wilson , 2004 ), coupled with studies indicating ATM and p53 inhibition in cultured or developing neurons is neuroprotective, has led to arguments that ATM and p53 may be pro‐apoptotic in Alzheimer’s disease ( Kruman , 2004 ; Culmsee & Mattson, 2005 ; Kim , 2008 ). Here, we probe the relationship of the DNA damage checkpoint to tau‐dependent neurodegeneration in mature postmitotic neurons of the genetically tractable fruit fly. To show activation of a DSB checkpoint in experimental tauopathy, we first demonstrated that p53 was upregulated in cortical neurons in tau transgenic mice ( Fig. 1a , arrow; Fig. S1a, Supporting information; Ittner , 2008 ) compared to age‐matched control brains. The phosphorylation at Ser‐139 of the histone variant H2AX (pH2AX) is a specific marker for DSBs in mammalian cells. We found expression of pH2AX in neurons from tau transgenic mice ( Fig. 1c , arrow; Fig. S1b), but not in control tissue. We then investigated whether these changes were mirrored in a Drosophila tauopathy model, in which pan‐neuronal expression of human tau recapitulates key features of human tauopathies ( Khurana , 2006 ). In the fly model, as in tau transgenic mice, there was upregulation of neuronal p53 ( Fig. 1b ; Fig. S2), along with induction of Chk1 (Fig. S3) and the p53 target gene Gadd45 (Fig. S4). In Drosophila , the homologous histone modification to pH2AX is phosphorylation at Ser‐137 of H2Av (pH2Av). We found that antibodies to pH2Av labeled neuronal chromatin in tau‐expressing adult animals ( Fig. 1d , arrow), but not in age‐matched controls. To demonstrate physical evidence of DNA damage, brains from tau‐expressing Drosophila were analyzed with the comet tail assay in which DNA single‐ or DSBs are demonstrated using single‐cell gel electrophoresis. We observed that nuclei of tau‐expressing flies displayed greater than a 2‐fold longer comet tails ( Fig. 1e ). 1 Upregulation of p53 and DNA damage in tauopathy models. (a, c) Increased p53 (a) or pH2AX (c) in neurons of tau transgenic mice (arrows). Scale bars are 10 μm. (b, d) Elevated p53 (b) or pH2AX (d) in neurons of tau transgenic flies (arrows). Scale bars are 3 μm. (e) Comet assay shows an increase in tail moment in tau transgenic fly brains compared to control. * P < 0.01, unpaired t ‐test. Control genotype: elav‐GAL4/+ . Flies are 10 days old. Having established that a DSB response occurs in tauopathy models, we next determined the effect of genetically manipulating the checkpoint. Neurodegeneration in our model can be assessed by TUNEL staining, caspase cleavage, and standard histology ( Khurana , 2006 ). We first determined whether the Drosophila homolog of ATM , dATM , is a modifier of tau‐induced toxicity in the adult fly brain. We found that removing one copy of dATM significantly enhanced tau‐induced neuronal apoptosis ( Fig. 2a ), with two separate dATM loss‐of‐function alleles, indicating a protective role for dATM in tauopathy. 2 Neurodegeneration and cell cycle are promoted by reducing checkpoint function. (a, c–e) Reducing checkpoint function increases neurodegeneration (a–c, TUNEL) and cell cycle re‐entry (d, e, PCNA, arrowheads). (c) No further increases in tau neurotoxicity are observed when two checkpoints are reduced simultaneously, consistent with a nonlinear interaction. * P < 0.01, ANOVA with Student–Neuman–Keuls test. Control genotype: elav‐GAL4/+ . Flies are 10 days old. Fly homologs of Chk2 and p53, dChk2 and Dmp53, also play a homologous role in the DNA damage response. Dominant‐negative transgenic constructs of these modifiers have been created. A dominant‐negative Chk2 transgene and two separate dominant‐negative p53 transgenes led to a marked increase in tau‐induced apoptosis ( Fig. 2b ; Fig. S5a,b). Conversely, increasing expression of p53 rescued toxicity (Fig. S6). Consistent with ATM , Dmp53 and Chk2 acting in the same pathway, combining these individual genetic modifiers demonstrated a nonadditive modifying effect ( Fig. 2c ). Importantly, genetic modifiers did not alter tau expression (Fig. S7). These data support a neuroprotective role for an ATM‐dependent DSB checkpoint in the fly tauopathy model. We reasoned that the DSB checkpoint could be neuroprotective by inhibiting the cell cycle. We have previously shown that abnormal markers of cell cycle activation can be detected in adult neurons within the brain of tau‐expressing flies, including proliferating cell nuclear antigen (PCNA), a marker also upregulated in Alzheimer’s disease and related tauopathies ( Fig. 2 ; Khurana , 2006 ; Herrup & Yang, 2007 ). Furthermore, cell cycle re‐activation is causally linked to tau‐induced neurodegeneration in fly and mouse Alzheimer’s disease models ( Andorfer , 2005 ; Khurana , 2006 ; Kim , 2008 ). We found that decreasing function of ATM, Chk2 or p53 strongly upregulated tau‐induced PCNA ( Fig. 2d,e , arrowheads). These data implicated re‐entry of postmitotic neurons into the cell cycle as a mechanism through which checkpoint inhibition exacerbates neurotoxicity. There is a growing literature on aberrant cell cycle re‐entry, DNA damage and checkpoint activation in Alzheimer’s disease and related tauopathies ( Herrup & Yang, 2007 ; Kim , 2008 ; Myung , 2008 ). The relationship among these three processes, however, has remained unclear. In this article, we provide direct evidence of DNA damage and checkpoint activation in tauopathy models. Our previous work identifying oxidative stress as a critical component of tau‐induced neurodegeneration provides one potential mechanism for tau‐induced DNA damage ( Barlow , 1999 ; Dias‐Santagata , 2007 ). The genetic data presented in this study argue for a neuroprotective role of the DNA damage checkpoint and suggest caution in designing p53 inhibitors as therapeutic agents in Alzheimer’s disease and related tauopathies. A number of recent studies have demonstrated an early protective role for the DNA damage checkpoint in tumorigenesis ( Bartkova , 2005 ). Together with these findings and the known concomitant neurodegeneration and tumorigenesis that occur with ATM loss‐of‐function in the disease ataxia telangiectasia, our data raise the possibility of a generally protective function for the DNA damage checkpoint in diseases of aging.

Journal

Aging Cell – Wiley

Published: Apr 1, 2012

There are no references for this article.