The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis (original) (raw)

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1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia

*To whom correspondence should be addressed at: Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, 2 Technology Place, Macquarie University, NSW, 2109, Australia. Tel: 61 2 9850 2772; Fax 61 2 9850 2701; Department of Biochemistry and Genetics, LaTrobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. Tel: 61 3 9479 2354; Fax: 61 3 9479 1266; Email: m.farg@latrobe.edu.au (M.A.F.); julie.atkin@mq.edu.au (J.D.A.)

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2Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, 2 Technology Place, Macquarie University, NSW 2109, Australia

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1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia

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3Department of Neurology, School of Medicine, Keio University, Tokyo 160-8582, Japan

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1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia

2Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, 2 Technology Place, Macquarie University, NSW 2109, Australia

*To whom correspondence should be addressed at: Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, 2 Technology Place, Macquarie University, NSW, 2109, Australia. Tel: 61 2 9850 2772; Fax 61 2 9850 2701; Department of Biochemistry and Genetics, LaTrobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. Tel: 61 3 9479 2354; Fax: 61 3 9479 1266; Email: m.farg@latrobe.edu.au (M.A.F.); julie.atkin@mq.edu.au (J.D.A.)

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Received:

08 January 2017

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29 April 2017

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Manal A. Farg, Anna Konopka, Kai Ying Soo, Daisuke Ito, Julie D. Atkin, The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis, Human Molecular Genetics, Volume 26, Issue 15, 1 August 2017, Pages 2882–2896, https://doi.org/10.1093/hmg/ddx170
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Abstract

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease affecting motor neurons. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9orf72 are the major cause of familial ALS and frontotemporal dementia (FTD) worldwide. The C9orf72 repeat expansion undergoes repeat-associated non-ATG (RAN) translation to produce five dipeptide repeat proteins (DRPs), including poly(GR) and poly(PR). Whilst it remains unclear how mutations in C9orf72 lead to neurodegeneration in ALS/FTD, dysfunction to the nucleolus and R loop formation are implicated as pathogenic mechanisms. These events can damage DNA and hence genome integrity. Cells activate the DNA damage response (DDR) with the aim of repairing this damage. However, if the damage cannot be repaired, apoptosis is triggered. In lumbar motor neurons from C9orf72-positive ALS patients, we demonstrate significant up-regulation of markers of the DDR compared to controls: phosphorylated histone 2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated (p-ATM), cleaved poly (ADP-Ribose) polymerase 1 (PARP-1) and tumour suppressor p53-binding protein (53BP1). Similarly, significant up-regulation of γ-H2AX and p-ATM was detected in neuronal cells expressing poly(GR)100 and poly(PR)100 compared to controls, revealing that DNA damage is triggered by the DRPs. Nucleophosmin (NPM1) is a histone chaperone induced during the DDR, which interacts with APE1 to enhance DNA repair. We also demonstrate that more NPM1 precipitates with APE1 in C9orf72 patients compared to controls. Furthermore, overexpression of NPM1 inhibits apoptosis in cells expressing poly(GR)100 and poly(PR)100. This study therefore demonstrates that DNA damage is activated by the C9orf72 repeat expansion in ALS.

Introduction

Hexanucleotide repeat expansions (GGGGCC) in a non-coding region of C9orf72 are the most common genetic abnormality in amyotrophic lateral sclerosis (ALS) and the related disorder frontotemporal dementia (FTD) (1,2). The exact mechanisms by which the C9orf72 repeat expansion induces motor neuron death are complex and not well understood. Reduced expression levels of C9orf72 have implied loss of function as a pathogenic mechanism (2,3). Transcriptional instability and epigenetic silencing might lead to haploinsufficiency (4–6) as the repeat expansion is methylated (7), and histone trimethylation has been detected in the blood and cerebellum of C9orf72-positive ALS and FTD patients (4,8). Accumulation of toxic RNA transcripts containing the GGGGCC repeat within nuclear foci in C9orf72-ALS/FTD patients (9–12) and repeat-associated non-ATG (RAN) translation to produce dipeptide repeat proteins (DRPs), are also implicated as primary pathogenic mechanisms in C9orf72-ALS/FTD (13–15). RAN translation occurs on both sense and antisense strands, resulting in expression of five DRPs proteins; glycine–alanine (GA), glycine–arginine (GR), proline–alanine (PA), proline–arginine (PR), and glycine–proline (GP). DRP inclusions have been reported in several areas of the CNS in C9orf72-ALS/FTD patients (16,17), although they may not be associated with neuronal loss in human tissue (18). The role of the DRPs in neurodegeneration is controversial, but there is considerable evidence that the arginine-containing peptides; polyGR, produced from the sense strand, and polyPR, from the antisense strand; are the most toxic and may contribute to the progression of C9orf72-mediated ALS/FTD (reviewed recently in (19)). The DRPs form cytoplasmic aggregates in spinal cord motor neurons that co-localise with p62 and TDP43 (16–18). Studies in mammalian models suggest that accumulation of DRPs is upstream and precedes the formation of TDP-43 pathology, as post-mortem C9orf72 tissues from patients who died prematurely revealed the presence of DRP aggregates but minimal TDP43 mislocalization (20). Toxicity can be induced by expression of the DRPs in neuronal cells (10), and polyGR and polyPR are implicated as primary drivers of neurodegeneration (13). These peptides aggregate in the nucleus, leading to impairment of ribosomal biogenesis (21), triggering nucleolar stress, nuclear transport defects, RNA processing alterations, and protein mislocalization (22).

Previously, nucleolar stress was detected in cells expressing the C9orf72 repeat expansion (5,23). Whilst nucleolar stress can induce DNA damage, it remains unknown whether this is induced by the C9orf72 mutation in ALS. DNA damage occurs in many forms, but double-stranded breaks (DSBs) are the most cytotoxic lesions, and the cell activates the DNA damage response (DDR) with the aim of repairing this damage and hence, maintaining cellular homeostasis. The G-rich C9orf72 repeat expansion also forms G-quadruplex structures (24,25) that promote the formation of R loops and RNA-DNA hybrids, which are also an important source of DNA damage (26–28). Phosphorylation of histone H2AX at serine 139 (γ-H2AX) is considered to be one of the most sensitive markers of the DDR (29). γ-H2AX becomes recruited to nuclear foci during DSB repair (30,31), where it subsequently recruits repair proteins to the site of DNA damage (32). H2AX is phosphorylated by the kinase ataxia-telangiectasia mutated (ATM), which also becomes phosphorylated following DNA damage (33) and is another important marker of the DDR. ATM itself is activated by tumour suppressor p53-binding protein (53BP1) (34), which also functions in DNA repair. DNA damage also triggers chromatin modification, including poly-ADP-ribosylation, which is mediated by poly (ADP-Ribose) polymerase (PARP-1). PARP-1 inhibits translation initiation factors, including eIF4G, which mediates translation of specific genes during cellular stress (35,36).

Nucleophosmin (NPM1, also known as B23) is a histone chaperone also induced during the DDR, which is localized in the nucleolus. NPM1 is important in maintaining genomic integrity, hence it can exhibit protective properties. During DNA damage, NPM1 localises to DSBs where it mediates the stability, activity, and accumulation of proteins involved in DNA base excision repair (BER) (37), NPM1 also regulates nucleolar function and ribosome biogenesis (38,39). Importantly, NPM1 also interacts with apurinic/apyrimidinic endonuclease 1 (APE1), a DNA repair protein central to BER, which functions through redox-dependent and independent mechanisms (40). The importance of the NPM1-APE1 interaction is not fully understood, but it appears to regulate multiple cellular functions, including genomic stability and ribosome biogenesis (41), in both the nucleus and nucleoplasm (42). Moreover, the binding of NPM1 to APE1 regulates the activity of APE1 in DNA repair (41,42). NPM1 also activates phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), which subsequently activates mammalian target of rapamycin (mTOR), a kinase that regulates a diverse set of cellular pathways, including cellular growth, survival, proliferation, autophagy, and the cell cycle (43).

Previously, co-localization of NPM1 with the DRPs was detected in cells expressing poly(GR) and poly(PR) (23). Furthermore, up-regulation of APE1 was previously reported in sporadic ALS patient tissue (45) and possible missense mutations in APE1 were detected in sporadic and familial SOD1-linked ALS patients (45). DNA repair activity, as detected by 8-hydroxy-2-deoxyguanosine (OHdG) immunoreactivity, is also increased in the motor cortex of ALS patients (44,46). In addition, FUS interacts with Histone deacetylase 1 (HDAC1) and has a pivotal role in DNA repair, wheras ALS-associated mutant FUS loses this function (47,48).

Given that the C9orf72 repeat expansion promotes stress in the nucleolus and the formation of R loops, we hypothesised that DNA damage may be triggered by the repeat expansion in ALS. In this study, we report significant up-regulation of markers of DNA damage; γ-H2AX, phosphorylated ATM (p-ATM), PARP-1 and 53BP-1; in motor neurons of ALS patients bearing the C9orf72 repeat expansion in comparison to control subjects, revealing that the C9orf72 mutation induces the DDR. We also demonstrate enhanced interaction between APE1 and NPM1 in C9orf72 patients compared to controls. Furthermore, induction of the DDR was confirmed using constructs expressing poly(GR)100 and poly(PR)100 that possess modified nucleotide sequences which do not express C9orf72 RNA, implying that the DRPs rather than the GGGGCC RNA transcript trigger DNA damage. In addition, we also show that overexpression of NPM1 inhibits apoptosis in neuronal cells expressing poly(PR)100 or poly(GR)100, suggesting that depletion of NPM1 is linked to cell death in ALS. Hence, this study demonstrates that the DDR is dysregulated by the C9orf72 repeat expansion in ALS.

Results

Markers of the DNA damage response (DDR) are significantly elevated in C9orf72 patients

Firstly, the presence of DNA damage in patient tissues was investigated by examining the distribution of γ-H2AX in human motor neurons, where the accumulation of γ-H2AX into punctate foci signified DNA damage, as in previous studies (49). Immunohistochemistry of human lumbar spinal cord sections was performed from C9orf72 patients and controls without neurological disease. The mean age of the patients was very similar in both C9orf72 and control populations (63.9 ± 2.6 and 63.6 ± 4.9 respectively), and both populations were predominantly Caucasian, with more males than females (n = 6 for C9orf72 patients and n = 9 for controls). Immunohistochemistry was performed using anti-γH2AX and anti-SMI32 antibodies (Fig. 1A). Low magnification was used to locate motor neuron cell bodies (SMI-32 positive) within the ventral horn. In motor neurons from C9orf72 patients and controls, quantitative analysis of at least 60 motor neurons per group (ALS or control), revealed that a significantly higher percentage of cells contained accumulation of γ-H2AX foci in C9orf72 patients compared to control patients (42.45% increase; P < 0.001) (Fig. 1B). High magnification images shown in Supplementary Material, S1). Control immunocytochemistry experiments using secondary antibodies without primary antibodies were negative, demonstrating the specificity of the antibodies used (not shown). The presence of DNA damage in C9orf72 patient tissues was confirmed by immunoblotting of lysates from human lumbar spinal cords using anti-γ-H2AX antibodies (Fig. 1C). Densitometry analysis revealed significant up-regulation of γ-H2AX in C9orf72 ALS spinal cords compared to controls (30.6% increase; P < 0.001) (Fig. 1D), consistent with the immunohistochemistry results. The blots were stripped and reprobed for β-actin to demonstrate equal loading. In order to examine whether the DNA damage was associated with C9orf72, next we performed fluorescence in situ hybridisation (FISH) using probes specific for C9orf72 RNA, and immunohistochemistry using γ-H2AX antibodies. These data demonstrate that γ-H2AX immunostaining in C9orf72 ALS motor neurons co-localized with the C9orf72 probe (Fig. 1E). Furthermore, quantitative analysis revealed that there were significantly more motor neurons with co-localisation of C9orf72 and γ-H2AX in ALS patients compared to controls as expected (P < 0.05, Fig. 1F). These data confirm that the C9orf72 repeat expansion induces DNA damage.

Figure 1

γ-H2AX accumulates in motor neurons from patients carrying the C9orf72 repeat expansion (A) Paraffin-fixed human spinal cord sections from C9orf72 patients and controls were subjected to immunohistochemistry using anti-phosphorylated (γ-H2AX) and anti-SMI32 antibodies. SMI32 immunostaining was used to locate motor neuron cell bodies in both ALS patients and controls. Accumulation of phosphorylated γ-H2AX (pSer139) foci was detected in motor neurons in the ventral horn regions of C9orf72 patients. Scale bars: 10 μm, (B) Quantitative analysis of accumulated γ-H2AX images in (A), revealed that significantly more motor neurons with γ-H2AX (42.45% increase) were present in C9orf72 patient tissues in comparison to controls. At least 60 motor neurons were scored from each group. Tissues from two C9orf72 patients and six controls were examined (P6 and P7); (C1-C6) (Table 1). Data are represented as mean ± SEM; **P < 0.001 versus controls by unpaired studentt test. (C) Human spinal cord homogenates (30 µg) were subjected to western blotting using anti-γ-H2AX antibodies. Blots were reprobed with anti-β-actin antibodies as a loading control. (D) Densitometry quantification of blots in (C) relative to β-actin revealed that the levels of accumulated γ-H2AX were significantly elevated in patients bearing the C9orf72 repeat expansion compared to controls. (30.6% increase; P < 0.001). Ten patients and ten controls were examined in total (Table 1). (E) Human spinal cord tissues (paraffin-fixed) from ALS patients and control individuals were labelled for C9orf72 using FISH and immunohistochemistry using γ-H2AX antibodies. These studies revealed that DNA damage was present in ALS motor neurons expressing the C9orf72 repeat expansion. (F) Quantification of images shown in (E), represented as a mean +/- SEM, *P < 0.05 by t-student test. 20–30 motor neurons were scored in each group from three individuals. Significantly more motor neurons displayed DNA damage and C9orf72 foci in ALS patients compared to controls.

Next, to confirm that DNA damage is present in C9orf72 tissues, additional markers of the DDR were examined. First, the presence p-ATM was investigated in ALS patients and controls. We could not use the same anti-SMI-32 antibodies to label motor neurons, due to species cross-reactivity with the available p-ATM antibodies. Hence, we used antibodies against vCHAT to locate motor neuron cell bodies, by immunohistochemistry using anti-pATM and anti-vCHAT antibodies in ventral horn regions (Fig. 2A). High magnification images are provided in the Supplementary Material, S2). Quantitative analysis of at least 60 motor neurons per group (ALS and controls), revealed that significantly more (52% increase; P < 0.05) motor neurons stained positive for p-ATM in C9orf72 patients compared to control subjects (Fig. 2B). Secondly, additional markers of the DDR were examined using imunoblotting of tissue lysates from human lumbar spinal cords: 53BP1 and PARP1. Significant up-regulation of both markers in C9orf72 patient tissue lysates compared to control subjects was detected (PARP-1: 84% increase; P < 0.0001; 53BP1: 70% increase; P < 0.001), (Fig. 3A–D). Blots were stripped and reprobed for β-actin to demonstrate equal loading. Hence, these results confirm that multiple markers of the DDR are up-regulated in C9orf72-positive patient tissues compared to controls.

Figure 2

Significantly more motor neurons express phosphorylated ATM in C9orf72 patients (A) Paraffin-fixed human spinal cord sections were subjected to immunohistochemistry using anti p-ATM (phospho S1981) antibodies, and anti-vCHAT antibodies to locate motor neurons in the ventral horn regions. More motor neurons with activated, p-ATM were detected in C9orf72 patients compared to controls. Scale bar 10μm. (B) Quantitative analysis of images in (A) revealed that significantly more motor neurons with activated p-ATM were present in C9orf72 patients in comparison to control tissues (52% increase; P < 0.05). At least 60 motor neuron cells were scored from each group. Data are represented as mean ± SEM; *P < 0.05 versus controls by unpaired student-t test. A total of two C9orf72 patients and two controls were examined (P6 and P7); (C1-C6) (Table 1).

Figure 3

Markers of the DDR are elevated in C9orf72 patients compared to controls (A,B) Human spinal cord homogenates (30 µg) were subjected to western blotting using anti-cleaved PARP-1 and anti-53BP1 antibodies. Blots were reprobed with anti-β-actin antibodies as a loading control. (C,D) Densitometry quantification of blots relative to β-actin revealed that the levels of cleaved PARP-1 and 53BP-1 were significantly elevated in patients bearing the C9orf72 repeat expansion (84% increase; P < 0.0001) (70% increase; P < 0.001). A total of ten C9orf72 and ten control (Table 1) spinal cord lysates were examined and three different repeats of each blot were performed. Data are represented as mean ± SEM; ***P < 0.0001, **P < 0.001, versus controls using unpaired student-t test.

We investigated the DDR further in C9orf72 patients by examing the relationship between NPM1 to APE1, which enhances the activity of APE1 in DNA repair (27,28). First, we performed immunohistochemistry using antibodies against NPM1 and APE1 of spinal cord tissues from human C9orf72 and control patients. From these studies, we found evidence of co-localization between APE1 and NPM1 in motor neurons of ALS patients and controls (Fig. 4A). To investigate the binding of APE1 and NPM1 in more detail, lysates from human C9orf72 patient lumbar spinal cords were subjected to immunorecipitation using anti-NPM1 antibodies, followed by immunoblotting using anti-APE1 antibodies. In control patients, little APE1 precipitated using NPM1 antibodies. However, in contrast, much more APE1 was precipitated in C9orf72 patients (Fig. 4B). Control immunoprecipitations using isotype matched control IgG antibodies were negative, confirming the specificity of the precipitation. Quantification of the immunoprecipitated proteins by densitometry revealed that significantly more (2.3 fold change, P < 0.05) APE1 was precipitated in C9orf72 patients using anti-NPM1 antibodies, compared to control patients (Fig. 4C). Hence, these data imply that the interaction between NPM1 and APE is enhanced in C9orf72 patients compared to controls, consistent with induction of the DDR and enhancement of DNA repair activities.

Figure 4

NPM1 co-precipitates more with APE1 in human C9orf72 patient spinal cord lysates compared to control patients (A) Immunohistochemistry of spinal cord tissues from human C9orf72 patients and control patients was performed using anti-NPM1 and anti-APE1 antibodies. Co-localization of APE1 and NPM1 in motor neurons of control and ALS patients was observed. (B) Human spinal cord lysates (100 μg) were subjected to immunoprecipitation using anti-NPM1 antibodies, followed by western blotting using anti-APE1 antibodies. Isotype-matched IgG antibodies were used as a control for immunoprecipitation. More APE1 was precipitated in C9orf72 lysates (P7-P10; Table 1) compared to control patients (C6-C10; Table 1). Inputs (3%) are also shown. Blots were stripped and reprobed with anti-β-actin antibodies as a loading control. Scale bar 10μm. (C) Densitometry quantification of blots in (B) revealed that significantly more NPM1 was precipitated in lysates from patients bearing the C9orf72 mutation compared to those from control patients (13.36 ± 4.923%, P < 0.05). Data are represented as mean ± SEM; *P < 0.05 versus controls by unpaired student’s-t test. A total of five C9orf72 and five control spinal cords lysates were examined, and each sample was blotted two times. (D,E) Human spinal cord homogenates (30µg) were subjected to western blotting using anti-PI3K and anti-p-elF4G antibodies. Blots were reprobed with anti-β-actin antibodies as a loading control. (F,G) Densitometry quantification of blots in (D,E) relative to β-actin revealed that the levels of PI3K and p-elf4G were significantly reduced in C9orf72 patients compared to controls (40% decrease; P < 0.05; 32.20% decrease; P < 0.001). A total of ten C9orf72 and ten control spinal cord lysates were examined. Data are represented as mean ± SEM; **P < 0.001, versus controls by unpaired student-t test.

NPM1 activates PI3K, which subsequently activates mTOR, the kinase which phosphorylates eukaryotic initiation factors (eIFs), including eIF4G, resulting in active translation. We next examined the levels of PI3K and phosphorylaled-eIF4G in human lumbar spinal cord lysates using immunoblotting Fig. 4D and E). Both PI3K and p-eIF4G were significantly down-regulated in C9orf72 patient lysates compared to controls (40% decrease; P < 0.05, 32.20% decrease; P < 0.001, respectively, Fig. 4F and G). These data therefore imply that the PI3K/protein kinase B (AKT)/mTOR pathway is dysregulated in C9orf72 patients.

DNA damage is present in neuronal cells expressing poly(GR)100 or poly(PR)100

Next, SH-SY5Y human neuroblastoma cells were transfected with previously described FLAG-tagged constructs, encoding arginine-rich DRPs; poly(GR)100 or poly(PR)100 (17). These constructs contain synthetic cDNAs encoding 100 repeats of each DRP with an ATG start codon. However, the nucleotide sequence has been modified so they do not contain GGGGCC repeats: hence the effect of the DRP can be examined in the absence of the normal RNA transcript. Consistent with previous studies, poly(GR)100 and poly(PR)100 were expressed either diffuse or as intra-nuclear aggregates (17) (Fig. 5A). The presence of DNA damage in the nucleus was then assessed by immunocytochemistry using anti-γ-H2AX antibodies, and counter-staining with DAPI, where the presence of more than five nuclear γ-H2AX foci per cell was considered to be positive, and 50 cells from each group were scored. These studies revealed that a significantly higher percentage of cells expressing poly(GR)100 or poly(PR)100 contained γ-H2AX foci, compared to control cells expressing empty vector alone, (polyGR: 85% increase; polyPR:86% increase, P < 0.0001), where the distribution of γ-H2AX was predominantly diffuse (Fig. 5A and B). To confirm these observations, further studies were carried out to examine whether DNA damage was present in primary mouse cortical neurons expressing the C9orf72 repeat expansion. Immunocytochemistry was performed using anti-FLAG and γ-H2AX antibodies (Fig. 5C). Quantification revealed that the % of cells displaying γ-H2AX foci was significantly increased in neurons expressing poly(GR)100 or poly(PR)100 compared to neurons transfected with empty vector only (P < 0.001, P < 0.01 respectively) (Fig. 5D). These data therefore confirm that the C9orf72 repeat expansion triggers nuclear DNA damage in ALS. Furthermore, because poly(GR)100 and poly(PR)100 are expressed using codon-altered constructs without GGGGCC repeats, this implies that the DRPs rather than RNA transcripts activate the DDR in neuronal cells. We confirmed the presence of DNA damage in poly(GR)100 and poly(PR)100 transfected cells by immunocytochemistry using antibodies against p-ATM. Significant up-regulation of p-ATM in cells expressing poly(GR)100 or poly(PR)100 compared to cells transfected with empty vector only was detected (polyGR: 55% increase; polyPR: 57% increase, P < 0.0001, P < 0.05, respectively) (Fig. 6A and B).

Figure 5

γ-H2AX foci accumulate in the nucleus of SH-SY5Y cells and primary cortical neurons expressing poly (GR)100 and poly (PR)100 (A) SH-SY5Y cells were transfected with either FLAG-tagged poly (GR)100, poly (PR)100 constructs or empty vector (PCMVIE) for 48 h. Immunocytochemistry using anti-γ-H2AX antibodies revealed that more cells formed nuclear γ-H2AX foci in cells expressing poly (GR)100 or poly (PR)100 compared to cells transfected with vector only. Scale bar, 10μm applied to all fields. Cells with accumulation of more than 5 foci of γ-H2AX in the nucleus were counted as positive. (B) Quantification of transfected cells in (A) revealed that significantly more cells with nuclear γ-H2AX foci were present in transfected cells expressing poly (GR)100 or poly (PR)100 compared to control cells transfected with empty vector only (Vec). Data are represented as mean ± SEM; ***P < 0.0001, versus untransfected cells by one-way ANOVA followed by Tukey’s post-test. 50 cells were scored in each group from two different experiments. (C) Primary cortical neuronal cultures were prepared from mouse C57BL/6 E16 brains. Neurons were transfected with either FLAG-tagged poly (GR)100, poly (PR)100 constructs or empty vector (PCMVIE), and fixed with 4% paraformaldehyde after 24 h. Immunocytochemistry was performed using γ-H2AX and anti-FLAG antibodies, and nuclei were counterstained with Hoechst 33342. Scale bar 10μm. (D) Quantification of the percentage of cells with activated γ-H2AX revealed that significantly more cells with nuclear γ-H2AX foci were present in primary neurons expressing poly (GR)100 or poly (PR)100 compared to control neurons transfected with empty vector only (Vec). Data are represented as a mean +/- SEM; γ-H2AX activation: ***P < 0.001, **P < 0.01; versus control cells (Vec) by one-way ANOVA followed by Sidak’s post-test. At least 20 cells in each group were included from three different experiments.

Figure 6

More SH-SY5Y cells expressing poly (GR)100 and poly (PR)100 display phosphorylated ATM. (A) SH-SY5Y cells were transfected with either FLAG-tagged poly (GR)100, poly (PR)100 or empty vector (PCMVIE) for 48 h. Immunocytochemistry using anti-p-ATM antibodies revealed that p-ATM was activated in cells expressing poly (GR)100 or poly (PR) 100. Scale bar, 10μm applied to all fields. (B) Quantification analysis of transfected cells in (A) revealed that significantly more cells with p-ATM were present in cells expressing poly (GR)100 or poly (PR)100 compared to cells transfected with empty vector only (Vec). Data are represented as mean ± SEM; *P < 0.05, versus untransfected cells by one-way ANOVA followed by Tukey’s post-test. 50 cells were scored from two different experiments.

Apoptosis is inhibited by co-expression of NPM1 with poly(GR)100 and poly(PR)100

NPM1 is a well characterised marker of nucleolar stress, which is protective against DNA damage by inhibiting pro-apoptotic pathways (50). We performed initial immunocytochemistry experiments to investigate the intracellular distribution of endogenous NPM1 in cells expressing poly(GR)100 and poly(PR)100. Consistent with previous studies, and indicative the presence of nucleolar stress, in cells expressing poly(GR)100 or poly(PR)100, the nucleolus appeared fractured and NPM1 was dispersed throughout the nucleus, in contrast to cells transfected with empty vector only, where NPM1 was more compact (Fig. 7A) (5). Quantification of the area of NPM1 distribution in the nucleus revealed a much greater area in cells expressing poly(GR)100 or poly(PR)100 compared to vector only, indicating significant shift of NPM1 away from the nucleolus and a dispersed localization (Fig. 7B). Furthermore, intranuclear DRP aggregates co-localized with NPM1 in the nucleolus in every cell examined (Fig. 7C). Hence, these findings are consistent with previous descriptions of DRP expression in the nucleolus (23), and they imply a possible association between NPM1 and the C9orf72 DRPs. We next examined whether over-expression of NPM1 is protective in neuronal cells expressing poly(GR)100 or poly(PR)100. NPM1 tagged with GFP was overexpressed with poly(GR)100 and poly(PR)100, and induction of apoptosis was examined by activation of caspase 3, using immunocytochemistry. Consistent with previous studies, 12-15% of cells expressing poly(GR)100 or poly(PR)100 only displayed caspase 3 activation, confirming that the DRPs induce toxicity in vitro (23). In contrast, caspase 3 was not activated in control cells expressing vector only at this time point (Fig. 7C and D). However, co-expression with NPM1 resulted in significantly fewer cells expressing poly(GR)100 or poly(PR)100 with activated caspase 3 in comparison to cells expressing each DRP without NPM1 overexpression (P < 0.0001, P < 0.001 respectively) (Fig. 7A and C). In fact, no cells co-expressing NPM1 with poly(GR)100 or poly(PR)100 were detected that displayed activated caspase-3. To examine the toxicity further, we also performed immunoblotting of cell lysates from cells expressing NPM1 with poly(GR)100 or poly(PR)100 compared to controls using anti-cytochrome c antibodies (Fig. 7E). Significantly lower levels of cytochrome c were present in lysates from cells co-expressing NPM1 with poly(GR)100 compared to those expressing empty vector alone (P < 0.01), although there was no statistically significant difference between cells co-expressing NPM1 with poly(PR)100 compared to those expressing empty vector alone, possibly due to the high background of untransfected cells in these populations (Fig. 7F). This result is consistent with the greater levels of toxicity of the polyGR compared to polyPR constructs used in this study. These data therefore imply that overexpression of NPM1 is protective in C9orf72-ALS.

Figure 7

Overexpression of NPM1 inhibits apoptosis in cells expressing poly (GR)100 and poly (PR)100 (A) To examine the normal distribution of NPM1, human SH-SY5Y cells expressing FLAG-tagged poly (GR)100 or poly (PR)100 or vector only were fixed and stained with anti-NPM1 (green) and anti-FLAG (red) antibodies. NPM1 was more dispersed throughout the nucleus in cells expressing the DRPs and the nucleolus was more fractured compared, indicting nucleolar stress, than in cells transfected with vector only. Scale bar 10μm. (B) Quantification of the area of NPM1 staining relative to the size of the nucleus (stained by Hoechst) of the images in (A) was performed using Image J. A significantly increased number of cells with dispersed localization of NPMN1 was detected in cells expressing poly (GR)100 or poly (PR)100 compared to cells transfected with vector only. Data are represented as mean ± SEM; **P < 0.001 versus cells transfected with vector only (vec), by one-way ANOVA followed by Tukey’s post-test. (C) Human SH-SY5Y cells were cotransfected with constructs encoding GFP-tagged NPM1 and either FLAG-tagged poly(GR)100 or poly(PR)100. Cells were fixed and immunocytochemistry was performed using anti-activated caspase 3 (magenta) and/or anti-FLAG antibodies (red). Scale bar, 10μm. (D) Quantification analysis of cells in (C) revealed that significantly fewer cells co-expressing NPM1 with either poly(GR)100 or poly(PR)100 displayed activated caspase-3, compared to control cells expressing poly(GR)100 or poly (PR)100 alone. Data are represented as mean ± SEM; ***P < 0.0001, **P < 0.001 versus cells expressing each DRP only, by oneway ANOVA followed by Tukey’s post-test. 50 cells were scored from two different experiments. (E) Immunoblotting of lysates using anti-cytochrome c antibodies. Blots were reprobed with anti-β-actin antibodies as a loading control. (F) Densitometry quantification of blots in (A) relative to β-actin revealed that the levels of cleaved cytochrome c were significantly decreased in cells expressing poly (GR)100. Data are represented as mean ± SEM; **P < 0.001 versus cells expressing each DRP only, by one-way ANOVA followed by Tukey’s post-test.

Discussion

Maintaining the stability and integrity of the genome is essential for normal cellular viability, and damage to DNA can arise from both endogenous and exogenous sources. In this study, we demonstrate that nuclear DNA damage and activation of the DDR is triggered by the C9orf72 repeat expansion in ALS. Several markers of the DDR, including γ-H2AX, p-ATM, 53BP1, and PARP-1, were up-regulated in C9orf72 patient spinal cords, as detected by immunohistochemistry or immunoblotting. This was confirmed by the up-regulation of γH2AX and p-ATM in neuronal cells expressing poly(GR)100 or poly(PR)100.

Our study is consistent with previous studies demonstrating that DNA damage is present in ALS, as elevated levels of 8-OHdG have been identified in both sALS and fALS human spinal cords (51). However, these studies linked DNA damage to oxidative stress which often occurs in mitochondrial rather than nuclear DNA. Also, they were performed prior to the discovery of the C9orf72 mutation in ALS/FTD, hence it is unknown if any of the previously examined patients expressed the C9orf72 repeat expansion or not. Similarly APE1 was previously shown to be elevated in tissues from sporadic ALS patients and mutant SOD1 mice, although APE1 elevation was previously linked to oxidative stress (52,53). A recent study demonstrated elevated oxidative stress and DNA damage in iPSC-derived motor neuron from C9orf72 patients (54), consistent with our findings.

Dysfunctional DNA repair and genomic instability have also been described in relation to mutant FUS in ALS. Wildtype FUS is recruited to sites of DNA damage in neurons where it plays a protective role in the DDR, but ALS associated-FUS mutants demonstrate impaired activity in DNA repair (48). Similarly, DNA damage is present in transgenic mice expressing ALS-associated mutant FUS-R521C in cortical neurons and spinal motor neurons (47,48). In addition, a recent study suggested that TDP43 has a role in DNA repair (55). Furthermore, DNA damage has also been detected in neuronal cells expressing G93A mutant SOD1 (56). Activation of DNA repair processes has been previously implicated in motor neuron degeneration (45,53) and mice lacking the gene encoding ERCC1, which is essential for nucleotide excision repair and repair of DSBs, show age–related motor dysfunction (57).

Two recent studies identified mutations in proteins involved in DNA damage/repair as a risk factor for sporadic ALS: NEK1 and C21ORF2 (58,59). NEK 1 is involved in the early DNA damage response to ionising radiation, and in chromosomal stability (60). C21ORF2 interacts with NEK1 and appears to be involved in the same DNA damage/repair pathway (58). Taken together, these findings therefore highlight the importance of DNA damage in ALS.

Whilst the mechanisms by which DNA damage are triggered remain unclear, the accumulation of abnormal DNA/RNA hybrids and the formation of R-loops by the C9orf72 repeat expansion have been previously reported in ALS (5,61). The presence of DNA-repair proteins within the nucleolus is also well established (62,63), and several studies now implicate nucleolar dysfunction in C9orf72-ALS. A recent study demonstrated that nucleolin, an important nucleolar protein involved in the synthesis and maturation of ribosomes, binds specifically to G-quadruplexes formed by the C9orf72 repeat expansion. Furthermore, nucleoin was depleted from the nucleolus in C9orf72 patient motor neurons, inducing nucleolar stress (5), and polyGR and polyPR expression resulted in nucleolar swelling and reduction of the levels of 18S rRNA/28S rRNA in HEK293 and NSC-34 cells (23). Hence, expression of poly(GR)100 or poly(PR)100 may induce ribosomal dysfunction and nucleolar stress, thus triggering DNA damage. However, R loops by themselves are a major source of genomic instability and DNA damage, and their formation inhibits normal transcription (64). The constructs used in our study express DRPs but do not contain GGGGCC repeats, demonstrating that the poly(GR)100 or poly(PR)100 DRPs trigger DNA damage in the cells observed in this study. However, we cannot rule out the possibility that R loop formation also may directly impair genomic stablility in the C9orf72 ALS human patients examined here. Hence, it is possible that both the presence of R loops as well as the expression of DRPs, contributes to the DNA damage we detected in human patient tissues.

We also detected significantly more co-precipitation between NPM1 and APE1 in C9orf72 patients compared to controls. The interaction between NPM1 and APE1 is poorly understood, but it appears to be important in maintaining genomic stability, and the binding of NPM1 to APE1 enhances the repair activity of APE1 (42). Hence, these data are consistent with induction of the DDR in C9orf72 patients. APE1 also acts as a growth factor, mediating neurite outgrowth and cellular proliferation (65), and its dysregulation can arrest the cell cycle and inhibit the PI3K/Akt pathway (66). NPM1 regulates transcription, the activity of DNA repair, and cell growth, and it also plays a role in the repair and clearance of DSBs (67,68). We also demonstrated down-regulation of PI3K and p-eIF4G in C9orf72 patient tissues compared to controls, consistent with these data, thus implying inhibition of the PI3K/AKT pathway in ALS-C9orf72 (69,70). Similarly, nucleolar stress also triggers down-regulation of the mTOR pathway (71). It is possible that G-quadruplexes created by the C9orf72 repeat expansion interfere with the normal function of NPM1, thus disrupting RNA processing (41) and triggering DNA damage. Interestingly, we detected significant inhibition of apoptosis when NPM1 was over-expressed in cells expressing poly(GR)100 or poly(PR)100. Hence, it is possible that overexpression of NPM1 restores the normal function of the nucleolus (72), thus reducing toxicity. However, further experiments are required to determine whether this is the case. Our findings therefore suggest that inhibition of nucleolar stress and over-expression of NPM1 should be investigated in more detail as a protective strategy in ALS.

Conclusions

Based on our findings, we propose a possible mechanism by which the C9orf72 repeat expansion induces toxicity in ALS. The DRPs accumulate in the nucleolus, where they disturb its normal function. This impacts on many downstream cellular functions, which together with the formation of R loops, perturbs DNA repair mechanisms, leading to DNA damage and cell death (Fig. 8). Understanding the molecular mechanisms underlying the pathogenesis of ALS will facilitate the identification of novel therapeutic avenues, and our study implies that modulation of the DDR and enhancement of DNA repair processes may be novel neuroprotective strategies in ALS.

Figure 8

Proposed model for the induction of DNA damage by the C9orf72 repeat expansion in ALS. The formation of R loops by the C9orf72 repeat expansion, as well as expression of the DRPs, disturbs nucleolar NPM1 (B23) function, which in turn impairs APE1 endonuclease function. This perturbs normal DNA repair mechanisms and RNA metabolism, leading to cell death and neurodegeneration.

Materials and Methods

Human tissue samples

Details of the patients used in this study are summarised in Table 1.

Table 1

Patient clinical information

Age = age at death, PMI = post mortem interval; L =lumbar; C= Cervical, M = Male and F = Female.

Table 1

Patient clinical information

Age = age at death, PMI = post mortem interval; L =lumbar; C= Cervical, M = Male and F = Female.

Constructs

Synthetic cDNAs encoding 100x poly-GR or poly-PR, linked to a FLAG tag, were as previously described (poly(GR)100 or poly(PR)100) (49). A construct encoding NPM1 tagged with GFP was obtained from Addgene (17578).

Immunohistochemistry

Immunohistochemistry was used to examine the expression of γ-H2AX and p-ATM in motor neurons from ALS patients bearing the C9orf72 mutation and controls. Paraffin sections (14 µm thick) from two patients (P6, P7) and six controls (C1-C7) (Table 1) were treated as previously described (18). Briefly, post-mortem spinal cords were blocked with normal goat or horse serum for 30 min and then stained with anti-SMI32 (1:50, Abcam, ab28029), Abcam), anti-VCHAT (1:200, Chemicon, Ab 1578), anti-γ-H2AX (pSer139), 1:100, Calbiochem, DR1017), or anti p-ATM (S1981) antibodies (1:100, Abcam, ab36810) for 48 h at 4°C, and after three washes with PBS, the tissues were incubated with AlexaFluor 488/564 (1:200, Molecular Probes) conjugated secondary antibodies. Images were acquired using an inverted fluorescent Zeiss microscope (Zeiss Instruments, Jena, Germany).

Cell culture and transfection

SH-SY5Y human neuroblastoma cells were grown at 37 °C in Dulbecco's medium supplemented with 10% fetal calf serum, 40 mg of penicillin/L, and 100 mg of streptomycin/L, in an atmosphere of 5% CO2. Cells were transfected transiently with empty CMV vector only or with poly(GR)100 and poly(PR)100 (500ng DNA per well) using lipofectamine 2000 (Invitrogen/LifeTechnologies), according to the manufacturer’s instructions. After 72 h, cells were examined using an inverted fluorescent Zeiss microscope (Zeiss Instruments, Jena, Germany).

Nucleolar stress and NPM1 distribution

SH-SY5Y human neuroblastoma cells were transfected with empty CMV vector only or with poly(GR)100 and poly(PR)100 using lipofectamine 2000, and immunocytochemistry using anti- NPM1 antibodies was performed to examine the distribution of NPM1; nuclei were stained with Hoechst 33342 (Sigma-Aldrich). To quantify the difference in area of NPM1 distribution relative to the size of the nucleus, imageJ was used to measure the pixel area of NPM1 immunostaining relative to the area of nuclear staining by Hoechst (73), and as previously described (5).

Growth of primary neurons and transfection

Primary cortical neuronal cultures were prepared from mouse C57BL/6 E16 brains. After isolation, cells were plated on glass coverslips covered with poly-L-lysine (Sigma-Aldrich) in B27 neurobasal medium supplemented with 1% glutamate and 1% Pen/Strep, and incubated at 37°C and 5%CO2. Neurons were transfected with Lipofectamine 2000 at 5 DIV and fixed with 4% paraformaldehyde after 24 h. After washing with PBS, primary cortical neurons were permeabilised in 0.1% Triton X-100 in PBS for 3 min and blocked in 1% BSA in PBS for 30 min. Immunocytochemistry was performed using antibodies against γ-H2AX (Novus Biologicals), and FLAG (Sigma-Aldrich) overnight at 4°C, followed by incubation with secondary anti-rabbit Alexa Fluor 488 (Life Technologies), and anti-mouse Alexa Fluor 594 (Life Technologies) antibodies for 2 h. Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich).

Immunocytochemistry

SH-SY5Y neuroblastoma cells were grown on coverslips, washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min. Cells were permeabilised in 0.1% Triton X-100 in PBS for 2 min, blocked for 30 min with 1% BSA in PBS, and incubated with primary γ-H2AX (pSer139)(1:500, Calbiochem), or NPM1 (1;500, Santa Cruz, sc-271737) antibodies for 16 h at 4 °C. Secondary AlexaFluor-594/488 conjugated anti-mouse or anti-rabbit antibodies (1:200, Molecular Probes) were then incubated for 1 h at room temperature, and cells were counter-stained with DAPI and mounted. Images were acquired using a Zeiss microscope (Zeiss Instruments, Jena, Germany)

Protein extraction

Cells were lysed in Tris-NaCl (TN) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% (v/v) NP-40, 0.1% (w/v) SDS, while human spinal cord lysates were extracted with RIPA buffer (10 mM Tris-Cl (pH 7.6), 1 mM EDTA,1% Triton X-100) and 1% (v/v) protease inhibitor mixture (Sigma) for 10 min on ice. Cellular lysates were clarified by centrifugation at 16 g for 10 min. Proteins were quantified using the BCA assay kit (Pierce).

Fluorescence in s itu hybridaization (FISH)

After deparaffinisation of human spinal cord sections, antigen retrieval was performed (30 min. at 80°C in citrate buffer, pH = 6). Slides were blocked in 5% Normal Donkey Serum with 0.1% Triton X-100 for 1 h. Staining was performed with primary anti γ-H2AX antibodies (Novus Biologicals) overnight, followed by incubation with secondary anti-rabbit Alexa Fluor 488 antibodies (Life Technologies) for 2 h. Next, slides were dehydrated and dried. Fluorescence in situ hybridization using a C9 probe (Alexa Flour 546-O-(CCCGG) 2) (PNA Bio), at 200 nM for 2 h in the dark at room temperature, with prior preheating of slides and hybridisation buffer to 85% for 5 min. After washing with 2x SSC, 0.1% Tween-20 buffer nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich).

Immunoblotting

Twenty μg of protein per well was separated on 12.5% SDS-PAGE gels. Proteins were then electrophoretically transferred onto nitrocellulose membranes (Bio-Rad). These membranes were subsequently blocked with 5% (w/v) non-fat milk powder in tris-buffered saline (TBS) or with 3% bovine serum albumin (BSA) in tris-buffered saline containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h, followed by incubation with the following primary antibodies, diluted in 1% BSA/TBS-T overnight at 4 °C; anti-γ-H2AX (pSer139) (1:1000, Calbiochem), PI3K (1:1000, Santa Cruz, sc-7248), cleaved PARP-1 (1:500, SantaCruz, sc-56196), phosphor-eIF4G (Ser1108) (1:500, Cell Signaling), 53BP1 (1:500, Novus, NB100-304), cytochrome c (1:500, Cell Signaling) or β-actin (1:5000, Sigma). Membranes were washed with TBS-T and then incubated with for 1 hr at room temperature with secondary antibodies (1:4000, HRP-conjugated goat anti-rabbit, or goat anti-mouse antibodies, Chemicon). Protein bands were detected using enhanced chemiluminescence (ECL) reagents (Roche), as described by the manufacturer.

Statistical analysis

All analyses were performed using Graph Pad Prism 5 software. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-test or unpaired Student-t test, from two or three independent experiments, and P < 0.05 was considered to be significant. Data are presented as mean ± standard error of the mean (SEM).

Ethics approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964. Helsinki declaration and its later amendments or comparable ethical standards. Ethics approval was granted; #FHEC10/R28 within the Faculty of Science, Technology and Engineering, La Trobe University and # CT15056 at Macquarie University.

Supplementary Material

Supplementary Material is available at HMG online.

Acknowledgements

Human tissue was kindly provided by Professor Pam Shaw and Dr. Claire Troakes, London Neuro-degenerative Diseases Brain Bank, NSW Brain Banks, University of Sydney, and Professor Catriona McLean Victorian Brain Bank Network, Australia.

Conflict of Interest statement. None declared.

Funding

National Health and Medical Research Council of Australia [Project grants 1006141, 108290 and 1030513] and MND Research Institute of Australia Cure for MND Foundation Research Grant, 2016.

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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Topic:

• apoptosis
• mutation
• amyotrophic lateral sclerosis
• cell nucleolus
• dna damage
• dna repair
• motor neurons
• poly(adp-ribose) polymerases
• up-regulation (physiology)
• pulmonary atmospheric pressure
• npm1 gene
• c9orf72 gene
• tumor suppressor p53-binding protein 1

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