Parp1 hyperactivity couples DNA breaks to aberrant neuronal calcium signalling and lethal seizures
2021; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.15252/embr.202051851
ISSN1469-3178
AutoresEmilia Komulainen, Jack Badman, Stéphanie Rey, Stuart L. Rulten, Limei Ju, Kate Fennell, Ilona Kalasová, Kristýna Ilievová, Peter J. McKinnon, Hana Hanzlíková, Kevin Staras, Keith W. Caldecott,
Tópico(s)Plant Molecular Biology Research
ResumoArticle1 May 2021Open Access Transparent process Parp1 hyperactivity couples DNA breaks to aberrant neuronal calcium signalling and lethal seizures Emilia Komulainen Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Jack Badman Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Stephanie Rey Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Stuart Rulten Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Limei Ju Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Kate Fennell Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Ilona Kalasova orcid.org/0000-0001-8235-1805 Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Kristyna Ilievova Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Peter J McKinnon orcid.org/0000-0002-0485-3778 Department of Genetics, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Hana Hanzlikova orcid.org/0000-0001-7235-7269 Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Kevin Staras Corresponding Author [email protected] orcid.org/0000-0003-4141-339X Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Keith W Caldecott Corresponding Author [email protected] orcid.org/0000-0003-4255-9016 Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Emilia Komulainen Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Jack Badman Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Stephanie Rey Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Stuart Rulten Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Limei Ju Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Kate Fennell Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Ilona Kalasova orcid.org/0000-0001-8235-1805 Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Kristyna Ilievova Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Peter J McKinnon orcid.org/0000-0002-0485-3778 Department of Genetics, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Hana Hanzlikova orcid.org/0000-0001-7235-7269 Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Kevin Staras Corresponding Author [email protected] orcid.org/0000-0003-4141-339X Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Keith W Caldecott Corresponding Author [email protected] orcid.org/0000-0003-4255-9016 Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Author Information Emilia Komulainen1, Jack Badman1,2, Stephanie Rey2, Stuart Rulten1, Limei Ju1, Kate Fennell2, Ilona Kalasova3, Kristyna Ilievova3, Peter J McKinnon4, Hana Hanzlikova1,3, Kevin Staras *,2 and Keith W Caldecott *,1,3 1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK 2Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton, UK 3Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic 4Department of Genetics, St Jude Children's Research Hospital, Memphis, TN, USA *Corresponding author. Tel: +44 01273 678478; E-mail: [email protected] *Corresponding author. Tel: +44 01273 877519; E-mail: [email protected] EMBO Rep (2021)22:e51851https://doi.org/10.15252/embr.202051851 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Defects in DNA single-strand break repair (SSBR) are linked with neurological dysfunction but the underlying mechanisms remain poorly understood. Here, we show that hyperactivity of the DNA strand break sensor protein Parp1 in mice in which the central SSBR protein Xrcc1 is conditionally deleted (Xrcc1Nes-Cre) results in lethal seizures and shortened lifespan. Using electrophysiological recording and synaptic imaging approaches, we demonstrate that aberrant Parp1 activation triggers seizure-like activity in Xrcc1-defective hippocampus ex vivo and deregulated presynaptic calcium signalling in isolated hippocampal neurons in vitro. Moreover, we show that these defects are prevented by Parp1 inhibition or deletion and, in the case of Parp1 deletion, that the lifespan of Xrcc1Nes-Cre mice is greatly extended. This is the first demonstration that lethal seizures can be triggered by aberrant Parp1 activity at unrepaired SSBs, highlighting PARP inhibition as a possible therapeutic approach in hereditary neurological disease. SYNOPSIS Excessive PARP1 activity at unrepaired DNA single-strand breaks in Xrcc1-defective brain causes aberrant synaptic calcium signaling, seizures, and shortened lifespan. These effects are prevented by Parp1 inhibition, revealing possibilities for the treatment of human neurological disease. Parp1 hyperactivity triggers aberrant synaptic activity in Xrcc1-mutated neurons Parp1 hyperactivity causes seizures and shortened life-span Aberrant synaptic and seizure-like activity are prevented by Parp1 inhibition or deletion Introduction DNA single-strand breaks (SSBs) are the commonest DNA lesions arising in cells and are rapidly detected by poly(ADP-ribose) polymerase-1 (PARP1) and/or poly(ADP-ribose) polymerase-2 (PARP2), enzymes that are activated at DNA breaks and modify themselves and other proteins with mono-ADP-ribose and/or poly-ADP-ribose (Benjamin & Gill, 1980; Chaudhuri & Nussenzweig, 2017; Hanzlikova et al, 2017; Azarm & Smith, 2020). Poly(ADP-ribose) triggers recruitment of the DNA single-strand break repair (SSBR) scaffold protein XRCC1 and its protein partners to facilitate the repair of SSBs (Breslin et al, 2015; Hanzlikova et al, 2017; Caldecott, 2019). If not repaired rapidly, SSBs can result in replication fork stalling and/or collapse and can block the progression of RNA polymerases during gene transcription (Hsiang et al, 1989; Ryan et al, 1991; Zhou & Doetsch, 1993; Kuzminov, 2001; Kathe et al, 2004). Notably, mutations in proteins involved in SSBR in humans are associated with cerebellar ataxia, neurodevelopmental defects and episodic seizures (Caldecott, 2008; Yoon & Caldecott, 2018). To date, all identified SSBR-defective human diseases are mutated in either XRCC1 or one of its protein partners (Caldecott, 2019). Recently, we demonstrated using an Xrcc1-defective mouse model (Xrcc1Nes-Cre) that SSBR-defective cerebellum possesses elevated steady-state levels of poly(ADP-ribose) resulting from the hyperactivation of Parp1, leading to the loss of cerebellar interneurons and cerebellar ataxia (Lee et al, 2009; Hoch et al, 2017). PARP1 hyperactivity can trigger cellular dysfunction and/or cytotoxicity by several mechanisms including excessive depletion of NAD+/ATP and/or by generating excessive amounts of poly(ADP-ribose) (Zhang et al, 1994; Andrabi et al, 2006; Yu et al, 2006; Andrabi et al, 2014). However, the extent to which Parp1 hyperactivation might account for the spectrum of neurological pathology induced by unrepaired endogenous SSBs is unknown. Here, we have addressed this question. We show that aberrant Parp1 activity extends beyond the cerebellum in Xrcc1Nes-Cre mice and is evident across the brain, resulting in deregulated neuronal presynaptic calcium signalling, lethal seizures and shortened lifespan. Importantly, we demonstrate that Parp1 inhibition or deletion prevents the Ca2+ signalling defects and elevated seizure-like activity in Xrcc1Nes-Cre hippocampus and that Parp1 deletion prolongs the lifespan of Xrcc1Nes-Cre mice. These findings highlight the potential of Parp1 as a target in the therapeutic treatment of XRCC1-defective disease. Results Parp1 is hyperactive throughout Xrcc1Nes-Cre brain We reported previously that Parp1 is hyperactive in the cerebellum of Xrcc1Nes-Cre mice, resulting in cerebellar ataxia (Hoch et al, 2017). Here, to extend this, we measured the steady-state level of pan-ADP-ribose signal across Xrcc1-defective brain by immunohistochemistry. We detected increased levels of ADP-ribose throughout Xrcc1Nes-Cre brain, including the cortex, with particularly strong immunostaining in the cerebellum and hippocampus (Fig 1A). In contrast, we did not detect elevated levels of Atm protein, an unrelated DNA repair-associated antigen, ruling out that the elevated anti-ADP-ribose signal was a non-specific artefact (Fig EV1). Indeed, we confirmed that the elevated ADP-ribose signal was the product of endogenous Parp1 activity, because Parp1 deletion in Xrcc1Nes-Cre mice reduced this signal to levels below those in wild-type brain (Fig 1A; Parp1−/−/Xrcc1Nes-Cre). Consistent with this, the elevated ADP-ribose signal in Xrcc1Nes-Cre brain was also detected if we employed antibody specific for poly(ADP-ribose), which is the primary ADP-ribosylation product of Parp1 activity (Fig EV1). Figure 1. Hyperactivity of Parp1 in Xrcc1Nes-Cre brain Sagittal sections obtained from mice (p15) of the indicated genotypes were immunostained for ADP-ribose using the pan-ADP-ribose detection reagent MABE1016. Representative images showing levels of ADP-ribose in the hippocampal regions CA1, CA3 and dentate gyrus (DG), and in the cerebral cortex. Red dotted boxes highlight the elevated ADP-ribose staining in Xrcc1Nes-Cre cerebellum (left dotted box) and hippocampus (right doted box). Scale bars: 5 mm, 50 μm. WT (n = 4 mice), Xrcc1Nes-Cre (n = 4), Parp1+/- /Xrcc1Nes-Cre (n = 4), Parp1−/−/Xrcc1Nes-Cre (n = 3) and Parp1−/− (n = 3). Summary histograms show mean ± SEM. Pairwise comparisons between WT versus Xrcc1Nes-Cre mice and WT versus Parp1−/−/Xrcc1Nes-Cre mice were conducted by Kruskal–Wallis ANOVA with Dunn's post hoc test, and statistically significant differences (*P < 0.05) are shown. Protein extracts from wild-type (WT), Xrcc1Nes-Cre and Parp1−/− forebrain tissue, containing hippocampus and cortex, were incubated with 1 mM NAD+ for 45 min in the presence or absence of PARP inhibitor as indicated, and ADP-ribosylation detected by Western blotting using the poly(ADP-ribose)-specific detection reagent MABE1031. Representative images from two or more independent experiments are shown. A Western blot showing the level of Parp1 and Xrcc1 in the forebrain tissue extracts is also shown, Inset. Protein extracts from wild-type (WT) and Xrcc1Nes-Cre forebrain or cerebellum were incubated for 0, 5, 10, 15, 30 and 45 min in the presence of NAD+ as above. NAD+ levels in wild-type (WT), Xrcc1Nes-Cre and Parp+/-/Xrcc1Nes-Cre forebrain tissue. Data are the scatterplots of individual measurements from at least three mice per genotype, with error bars representing the mean ± SEM. Statistically significant differences with Xrcc1Nes-cre are indicated (Kruskal–Wallis ANOVA with Dunn's post hoc test *P < 0.05). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Levels of poly(ADP-ribose) and Atm in Wild-type and Xrcc1Nes-cre brain Sagittal sections obtained from mice (p15) of the indicated genotypes were immunostained using anti-poly(ADP-ribose) antibody (Trevigen; 4336) or for Atm protein using the antibody EPR17059 (Abcam; ab199726). Representative images of the hippocampal regions CA1, CA3 and dentate gyrus (DG), and in the cerebral cortex, are shown as indicated. Scale bar 50 µm. Download figure Download PowerPoint To examine whether we could recapitulate the increased ADP-ribosylation in Xrcc1Nes-Cre brain biochemically, we incubated tissue extracts from wild-type and Xrcc1Nes-Cre forebrain, containing cortex and hippocampus, with NAD+ to stimulate ADP-ribosylation in vitro. Indeed, wild-type but not Parp1−/− forebrain extracts rapidly accumulated ADP-ribosylated proteins when incubated with NAD+ (Fig 1B). More importantly, tissue extracts prepared from Xrcc1Nes-Cre forebrain accumulated ADP-ribosylated proteins more rapidly and to a greater extent than did wild-type extracts, and similar results were observed if we employed tissue extracts from cerebellum (Fig 1B and C). Consistent with ongoing Parp1 hyperactivity, the steady-state level of NAD+ in Xrcc1Nes-Cre forebrain was half that present in wild-type forebrain and was increased by deletion of even a single Parp1 allele (Fig 1D). Collectively, these data implicate widespread Parp1 hyperactivation in Xrcc1-defective brain, presumably as a result of the underlying defect in SSB repair (Lee et al, 2009). In agreement with this, we did not detect elevated ADP-ribose in brain from Ku70−/− mice in which the primary pathway for DNA double-strand break (DSB) repair in brain is defective, confirming that the elevated ADP-ribose in Xrcc1Nes-cre brain was the result of unrepaired SSBs, not DSBs (Fig 1A). Parp1 hyperactivation triggers juvenile seizures and mortality in Xrcc1Nes-Cre mice Next, we generated Kaplan–Meier survival curves to determine the influence of Xrcc1 on lifespan. As expected (Lee et al, 2009), Xrcc1Nes-Cre animals exhibited greatly reduced longevity when compared to wild-type mice, with the cohort employed in these experiments having a median lifespan of ~ 3–4 weeks (Fig 2A). Given the widespread Parp1 hyperactivity across the brain in Xrcc1Nes-Cre mice, we examined the impact of Parp1 deletion on lifespan. Remarkably, the median lifespan of Parp1+/-/Xrcc1Nes-Cre and Parp−/−/Xrcc1Nes-Cre littermates in which one or both Parp1 alleles were additionally deleted was increased ~ 25-fold and ~ 7-fold, to 79 and 23 weeks, respectively (Fig 2A). This result demonstrates that aberrant Parp1 activity in Xrcc1-defective brain is a major contributor to organismal death. It is noteworthy that deletion of one Parp1 allele prolonged the lifespan of Xrcc1Nes-Cre mice to a greater extent than deletion of both Parp1 alleles. This suggests that whilst the loss of a single Parp1 allele is sufficient to suppress Parp1-induced toxicity, the loss of the second allele eradicates an additional role for Parp1 in Xrcc1-defective brain that is important for survival. Figure 2. Parp1 hyperactivation triggers juvenile seizures and mortality in the absence of Xrcc1 Kaplan–Meier curves for survival of mice of the indicated genotypes. The table shows number of individuals in each group, median survival (weeks) and P-values from pairwise curve comparisons; Und: undetermined (log-rank Mantel–Cox tests). Video monitoring and recording of generalized running/bouncing seizures in mice of the indicated genotypes from P15-P19. The point of death of Xrcc1Nes-Cre mice by fatal seizure is indicated (cross). Note no seizures from mice of other genotypes were detected. WT (n = 9 mice), Xrcc1Nes-cre (n = 3 mice), Parp1+/−/Xrcc1Nes-Cre (n = 3 mice) and Parp1−/−/Xrcc1Nes-Cre (n = 3). Download figure Download PowerPoint Next, to establish the cause of Parp1-dependent death in Xrcc1Nes-Cre mice, we conducted infrared video imaging for a four-day period starting at P15. These experiments revealed that Xrcc1Nes-Cre mice experienced sporadic seizures, culminating ultimately in a lethal seizure from which animals did not recover (Fig 2B). In contrast, we did not observe any seizures in wild-type mice over the same time period. The cause of death induced by the seizures is unclear but, similar to sudden unexpected death during epilepsy (SUDEP) in humans, it is likely to result from the disruption of normal cardiac or respiratory function (Surges et al, 2009). Importantly, we also did not detect seizures in Xrcc1Nes-Cre mice in which one or both alleles of Parp1 were deleted over the time course of the experiment, consistent with the increased lifespan of these mice (Fig 2B). To our knowledge, this is the first demonstration that seizures can be triggered by aberrant Parp1 activity. Seizure-like activity in Xrcc1Nes-Cre brain slices is corrected by Parp1 deletion The induction of seizures by Parp1 in Xrcc1Nes-Cre mice is consistent with the strikingly high level of poly(ADP-ribose) in the hippocampus of these animals, because defects in this region of the brain are often associated with seizure activity (Gunn & Baram, 2017). To examine directly whether elevated seizure-like activity is present in Xrcc1Nes-Cre hippocampus, we carried out targeted extracellular electrophysiological recording experiments in brain slices prepared from wild-type mice, Xrcc1Nes-Cre mice and Xrcc1Nes-Cre mice in which Parp1 was additionally deleted. When the brain slices were washed into an epileptogenic solution, the mean cumulative number of seizure-like events in the CA3 region of Xrcc1Nes-Cre hippocampus was ~ 2.5-fold greater than in wild-type hippocampus (Fig 3A–C). Moreover, strikingly, this elevated seizure-like activity was reduced or prevented if one or both alleles of Parp1 were deleted, respectively (Fig 3A–C), confirming Parp1 as the cause of the elevated seizure activity in Xrcc1−/− hippocampus. Figure 3. Suppression of seizure-like activity in Xrcc1Nes-Cre brain slices by Parp1 deletion Plots of enveloped spike activity waveforms based on targeted extracellular recordings from hippocampal CA3 region perfused into epileptogenic buffer for the indicated genotypes. Upward deflections indicate seizure-like episodes (SLEs). Scale bars, 5% of maximum amplitude. Heatplots of cumulative SLEs: WT (n = 11 slices from three mice), Xrcc1Nes-Cre (n = 12 slices from four mice), Parp1+/−/Xrcc1Nes-Cre (n = 12 slices from three mice) and Parp1−/−/Xrcc1Nes-Cre (n = 10 slices from three mice). Each horizontal bar corresponds to one slice with the colour code indicating cumulative SLEs. Summary of mean ± SEM cumulative SLE counts. The number of replicates was the same as in (B). At the recording endpoint, the total SLE count in Xrcc1Nes-Cre is significantly higher than both WT and Parp1−/− Xrcc1Nes-Cre (Kruskal–Wallis ANOVA, P < 0.0017, * indicates significance with Dunn's post hoc comparisons). Download figure Download PowerPoint To extend these analyses, we employed a high-density multi-electrode array (HD-MEA) platform (Fig 4A). This approach allowed us to assay the spatial organization of network activity across hippocampal and cortical structures simultaneously and with high temporal resolution. In particular, we recorded the onset and threshold of seizure-like events in the cortex, CA1 and CA3 regions of brain slices perfused into an epileptogenic solution, and plotted the cumulative activity in each. In agreement with the results described above, we found that seizure-like events were significantly higher in Xrcc1Nes-Cre cortex and hippocampus when compared to wild type, with the strongest effects observed in the hippocampal CA3 region (Fig 4B–D). Figure 4. Suppression of seizure-like activity in Xrcc1Nes-Cre brain slices by PARP1 inhibitor Brightfield image (left) of an acute brain slice positioned on HD-MEA with targeted regions indicated. (Right) progression of a typical seizure event in a slice perfused with epileptogenic buffer; colour code indicates voltage changes in microvolts. Representative traces from four channels in the CA3 region of hippocampus recorded from 10 to 15 min in epileptogenic buffer, showing seizure-like activity in Xrcc1Nes-Cre. Vertical scale bar indicates 300 μV. Mean cumulative activity plots in CA1 and CA3 regions of hippocampus and cortex over 15 min of recording in epileptogenic buffer. WT (n = 8 slices from four mice), Xrcc1Nes-Cre (n = 9 slices from four mice). Summary histograms of the mean (± SEM) cumulative seizure-like activity at 15 min, from the data in panel C. Statistical significance was assessed by ANOVA with post hoc pairwise comparisons (*P < 0.05). Summary histograms (mean ± SEM) of cumulative seizure-like activity at 5-, 10- and 15-min timepoints in cortex, CA1 and CA3 regions of wild-type and Xrcc1Nes-Cre brain slices from mice treated or not for 5–8 days ad libitum with PARP1 inhibitor (ABT-888) prior to analysis. WT (n = 8 slices from four mice), Xrcc1Nes-Cre (n = 10 from five mice), WT + PARPi (n = 6 from three mice) and Xrcc1Nes-Cre + PARPi (n = 9 from three mice). Pairwise comparisons at the 15-min timepoint between WT versus Xrcc1Nes-Cre mice and WT versus Xrcc1Nes-Cre mice + PARPi were conducted by Kruskal–Wallis with Dunn's post hoc tests, and statistically significant differences (*P < 0.05) are shown. Download figure Download PowerPoint Given the ability of Parp1 deletion to rescue normal levels of seizure-like activity in Xrcc1Nes-Cre brain, we extended these experiments to examine the impact of PARP1 inhibitor. To do this, we administered PARP1 inhibitor (ABT-888; veliparib) ad libitum in the drinking water from day 10 until their analysis at days 14–17. Strikingly, whereas this application of PARP1 inhibitor had little effect on the seizure-like activity of brain sections prepared from wild-type mice, it ablated the elevated seizure-like activity in Xrcc1Nes-Cre brain slices, in all three regions of the brain tested (Fig 4E). Together, these data confirm that Parp1 hyperactivity in Xrcc1Nes-Cre brain triggers increased seizure-like activity that can be prevented by Parp1 deletion or pharmacological inhibition. Aberrant Parp1 activity deregulates presynaptic calcium signalling in Xrcc1Nes-Cre neurons It is currently unclear how aberrant Parp1 activity at unrepaired SSBs might trigger seizures. However, it is known that Parp1 activity can affect the expression of many genes that might influence seizure activity, including those affecting Ca2+ homeostasis (Stoyas et al, 2019). Consequently, we examined whether the seizure-like activity in Xrcc1Nes-Cre mice reflects a defect in Ca2+ signalling at the level of single synapses in isolated hippocampal neurons. Similar to whole brain sections, we detected elevated endogenous levels of ADP-ribosylation in isolated Xrcc1Nes-Cre neurons, although as observed previously in other cultured cell types (Hanzlikova et al, 2018) the detection of endogenous poly(ADP-ribose) required incubation for 1 h with an inhibitor of poly(ADP-ribose) glycohydrolase (PARGi), the enzyme primarily responsible for poly(ADP-ribose) catabolism (Fig 5A and B). To confirm that the elevated poly(ADP-ribose) detected here was nascent polymer resulting from hyperactive Parp1, rather than pre-existing poly(ADP-ribose), we co-incubated the neurons with an inhibitor of PARP1 (PARPi). Indeed, the presence of PARPi ablated the appearance of poly(ADP-ribose) in the Xrcc1Nes-Cre neurons (Fig 5A and B). This result demonstrates that Parp1 hyperactivation occurs continuously in Xrcc1Nes-Cre hippocampal neurons, presumably as a result of the elevated steady-state level of unrepaired SSBs. Figure 5. Parp1 hyperactivation in isolated Xrcc1Nes-Cre hippocampal neurons Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes-Cre mouse pups, immunostained for ADP-ribose (red), NeuN to identify neurons (green) and counterstained with DAPI (blue). Cells were pretreated with PARP inhibitor (10 µM) or vehicle for 2 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Scale bar 10 µm. Histogram of mean (± SEM) relative pan-ADP-ribose fluorescence in NeuN-positive hippocampal neurons pretreated with PARP inhibitor (5 µM) or vehicle for 5 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Neurons were cultured from WT (n = 6 mice, > 180 cells per condition), Xrcc1Nes-Cre (n = 6, > 180), Parp1+/-/Xrcc1Nes-Cre (n = 3, > 90) and Parp1−/−/Xrcc1Nes-Cre (n = 3, > 90). * indicates significant differences from WT (Kruskal–Wallis ANOVA, P = 0.0013 and Dunn's post hoc tests). Download figure Download PowerPoint Next, to measure presynaptic calcium signalling, we transduced dissociated hippocampal cultures from different genotypes with SyGCaMP6f, a presynaptically targeted optical Ca2+ reporter (Fig 6A and B) (Dreosti et al, 2009). We then carried out time-lapse imaging at DIV15–17 to assess Ca2+ dynamics in response to electrical stimulation. We found that with repeated presentations of 10 Hz stimulus trains, SyGCaMP6f-positive puncta in wild-type mouse cultures showed characteristic transient increases in fluorescence consistent with activity-evoked Ca2+ influx at the presynaptic terminal (Fig 6C). Strikingly, however, the amplitude of these responses was ~ 2-fold higher in Xrcc1Nes-Cre neurons, indicating that Xrcc1 loss results in excessive activity-evoked synaptic Ca2+ influx (Fig 6C and D). Moreover, this defect was partially or fully suppressed by deletion of one or both alleles of Parp1, respectively, suggesting that the excessive activity-evoked synaptic Ca2+ influx was a result of Parp1 hyperactivity (Fig 6C, E and G). To confirm this, we incubated cultures with PARP1 inhibitor (PARPi) continuously for 9–11 days prior to recording. Strikingly, this treatment fully suppressed the aberrant Ca2+ response in Xrcc1Nes-Cre neurons (Fig 6C, F and G). To our knowledge, this is the first demonstration that aberrant Parp1 activity deregulates synaptic Ca2+ signalling, providing a compelling explanation for the elevated seizures and, consequently, shortened lifespan in Xrcc1Nes-Cre mice. Figure 6. Aberrant presynaptic calcium signalling in Xrcc1−/− hippocampal neurons is rescued by Parp1 deletion or inhibition A. Representative image of DIV15 dissociated cultured neurons used. Scale bar: 100 µm. B. (Top) Cartoon schematic illustrates SyGCaMP6f targeting and action. (Bottom) Image shows typical punctate SyGCaMP6f expression at DIV15. Scale bar, 20 µm. C. Representative images of fluorescence responses in synaptic terminals expressing SyGCaMP6f to a train of 10 stimuli at 10 Hz in dissociated hippocampal neurons derived from wild-type and mutant mice. Scale bar: 5 µm. D, E. Mean SyGCaMP6f responses to three rounds of 10 action potentials stimulation from mice of the following genotypes: WT (n = 1,946 synapses, nine coverslips, three animals), Xrcc1Nes-Cre (n = 3,313, 12, 4), Parp1+/−/Xrcc1Nes-Cre (n = 2,272, 9, 3) and Parp1−/−/Xrcc1Nes-Cre (n = 2,122, 11, 3). F. Response to chronic treatment with the PARP inhibitor, KU 0058948 Hydrochloride (1 µM) for 9–11 days prior to imaging for Xrcc1+/+ (n = 1,264 synapses, eight coverslips, three animals) and Xrcc1Nes-Cre (n = 2,231, 10, 4) mice. G. Summary histogram of the mean (± SEM) of the integrated fluorescence responses for all individual synapses for each condition. Synapse number is the same as in (D–F). Responses are significantly higher in Xrcc1Nes-Cre synapses versus both WT and Parp1−/−/Xrcc1Nes-Cre (one-way ANOVA, *P < 0.0033 and pairwise Student's t-tests). Data information: (D–F) Data are from three or more independent experiments per genotype/treatment type, combined into one experimental data set and plotted on three separate graphs for clarity. Dashed lines in panels E and F are the WT and/or Xrcc1Nes-Cre curves transposed from panel D for comparative purposes. Download figure Download PowerPoint Discussion DNA single-strand breaks (SSBs) are the commonest DNA lesions arising in cells and can block the progression of DNA and RNA polymerases (Hsiang et al, 1989; Zhou & Doetsch, 1993, 1994; Tsao et al, 1993; Kathe et al, 2004; Caldecott, 2008; Neil et al, 2012). The collision of DNA polymerases with SSBs can also result in DNA replication fork collapse and the formation of DSBs (Ryan et a
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