DNA damage response defects induced by the formation of TDP-43 and mutant FUS cytoplasmic inclusions and their pharmacological rescue

Abstract

Formation of cytoplasmic inclusions (CIs) of TDP-43 and FUS, along with DNA damage accumulation, is a hallmark of affected motor neurons in Amyotrophic Lateral Sclerosis (ALS). However, the impact of CIs on DNA damage response (DDR) and repair in this pathology remains unprobed. Here, we show that CIs of TDP-43 and FUS^P525L, co-localizing with stress granules, lead to a dysfunctional DDR activation associated with physical DNA breakage. Inhibition of the activity of the DDR kinase ATM, but not of ATR, abolishes DDR signaling, indicating that DNA double-strand breaks (DSBs) are the primary source of DDR activation. In addition, cells with TDP-43 and FUS^P525L CIs exhibit reduced DNA damage-induced RNA synthesis at DSBs. We previously showed that the two endoribonucleases DROSHA and DICER, also known to interact with TDP-43 and FUS during small RNA processing, contribute to DDR signaling at DSBs. Treatment with enoxacin, which stimulates DDR and repair by boosting the enzymatic activity of DICER, restores a proficient DDR and reduces DNA damage accumulation in cultured cells with CIs and in vivo in a murine model of ALS. In Drosophila melanogaster, Dicer-2 overexpression rescues TDP-43-mediated retinal degeneration. In summary, our results indicate that the harmful effects caused by TDP-43 and FUS CIs include genotoxic stress and that the pharmacological stimulation of the DNA damage signaling and repair counteracts it.

Fig. 1. Cells with TDP-43 or FUS^P525L cytoplasmic inclusions (CIs) show elevated γH2AX levels and increased DSB formation.

A Analysis by immunofluorescence (IF) of γH2AX signals (red) in damaged (NCS) or undamaged HeLa cells transfected with plasmids expressing TDP-43, FUS or FUS^P525L. Cells transfected with an empty vector (EV) were used as a control. TDP-43 and FUS signals are shown in orange; nuclei were counter-stained with DAPI (blue). Arrowheads mark cells with CIs. B Super-plot showing the quantification of γH2AX intensity in HeLa cells with or without cytoplasmic inclusions (± CI) determined in (A). Red dots represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. C Representative images of comet assay experiments performed in undamaged HeLa cells transfected as in (A). D Tail moment analysis of HeLa cells shown in (C). The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments.

Fig. 2. ATM is aberrantly activated, and its inhibition abolishes pan-nuclear γH2AX formation in cells with TDP-43 and FUS^P525L CIs.

A Analysis by immunofluorescence (IF) of γH2AX levels (red) in HeLa cells expressing TDP-43 or FUS^P525L and treated with ATMi or ATRi. Cells treated with DMSO were used as a control. TDP-43 and FUS are labeled in orange; nuclei were counter-stained with DAPI. Arrowheads mark cells with CIs; scale bar = 10 µm. B Quantification of γH2AX intensity in HeLa cells with or without cytoplasmic inclusions (±CI) described in (A). The red dots of the super-plots represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. C IF analysis of pATM signal (red) in HeLa cells transfected with plasmids expressing an empty vector (EV), TDP-43 or FUS^P525L. TDP-43 and FUS are labeled in orange; nuclei were counter-stained with DAPI. Arrowheads mark cells with CIs; scale bar = 10 µm. D, E Quantification of diffused and focal pATM signal (D, E, respectively) in HeLa cells with or without cytoplasmic inclusions (± CI) determined in (C). The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments.

Fig. 3. DDR signaling and 53BP1 activation is impaired in cells with TDP-43 and FUS^P525L CIs.

A, C Representative images of 53BP1 and p53BP1 (red) in damaged (NCS) or undamaged HeLa cells transfected with plasmids encoding for TDP-43, FUS or FUS^P525L. Cells transfected with an empty vector (EV) were used as a control. TDP-43 and FUS are shown in orange; nuclei were counter-stained with DAPI. Arrowheads mark cells with CIs; scale bar = 20 µm. B, D Quantification of DDR marker signals in cells with or without cytoplasmic inclusions (± CI) determined in (A, C), respectively. The red dots of the super-plots shown in (B, D) represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments.

Fig. 4. Transcription is halted in cells with TDP-43 and FUS^P525L CIs.

A HeLa cells expressing TDP-43, FUS or FUS^P525L were pulse-labeled with EU and analyzed by immunofluorescence; TDP-43 and FUS, EU and γH2AX are shown in orange, green and red, respectively. Nuclear DNA was visualized using Hoechst dye; arrowheads mark cells with CIs. B Quantification of EU signal in cells with or without cytoplasmic inclusions (± CI) shown in (A). The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. C Damage-induced long non-coding RNA (dilncRNA) expression was studied by strand-specific RT-qPCR in cut (I-SceI +) or uncut (I-SceI -) I-HeLa111 cells selected for the presence of FUS or FUS^P525L CIs through FACS. The histogram shows dilncRNA levels normalized over RPLP0 mRNA and shown as relative to the uncut FUS-expressing sample; values are the means ± SEM of three independent experiments. D Representative images of DROSHA expression (green) in damaged (NCS) or undamaged HeLa cells transfected with plasmids encoding for TDP-43, FUS or FUS^P525L; cells transfected with an empty vector (EV) were used as a control. TDP-43 and FUS are labeled in orange; nuclei were counter-stained with DAPI. Arrowheads mark cells with CIs; scale bar = 10 µm. E Quantification of DROSHA protein levels in cells with or without cytoplasmic inclusions (± CI) from panel (D). The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. F DROSHA mRNA levels were monitored by RT-qPCR in HeLa cells selected for the presence of FUS or FUS^P525L CIs through FACS. Values are the averages ± SEM of three independent experiments.

Fig. 5. TDP-43 and FUS cytoplasmic aggregation correlates with an altered DDR, DNA damage accumulation and reduced DROSHA levels in neuronal cell lines.

A Analysis by immunofluorescence (IF) of γH2AX (red) and 53BP1 (green) signals in damaged (NCS) or undamaged HT-22 cells expressing TDP-43, FUS or FUS^P525L; cells transfected with the empty vector (EV) were also examined. TDP-43 and FUS are shown in orange; nuclei were counter-stained with DAPI (blue); arrowheads mark cells with CIs. Scale bar = 20 µm. B Dot-plot showing γH2AX intensity (left) and 53BP1 foci (right) in HT-22 cells with or without cytoplasmic inclusions (± CIs), as determined in (A); red bars indicate the averages ± SEM; at least 40 cells were scored for each condition in two independent experiments. C Analysis by IF of γH2AX (green) and DROSHA (red) signals in hMNPs derived from sALS or a healthy (CTRL) donor; endogenous TDP-43 (orange) is also shown. Nuclei are counter-stained with DAPI (blue); scale bar = 20 µm. D Quantification of γH2AX foci (left) and DROSHA levels (right) in hMNPs, as determined in (C). The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. E TUBB3-GFP-expressing mMNs carrying wild-type or mutant Fus were stained for γH2AX. The dot-plot shows γH2AX foci in each nucleus; red bars indicate the averages ± SEM; at least 150 cells were scored for each condition in three independent experiments. F IF analysis of p53BP1 foci in mMNs. The dot-plot shows p53BP1 foci in each nucleus; red bars indicate the averages ± SEM. At least 60 cells were scored for each condition in two independent experiments. G IF analysis of DROSHA protein levels in mMNs. The dot-plot shows DROSHA intensity in each nucleus; red bars indicate the averages ± SEM. At least 130 cells were scored for each condition in three independent experiments. E–G nuclei were counter-stained with DAPI; scale bar = 100 µm.

Fig. 6. Enoxacin rescues foci formation and partially restores transcription in cells with TDP-43 and FUS^P525L CIs.

A, C HeLa cells expressing TDP-43 or FUS^P525L were treated or not with enoxacin prior to NCS administration and analyzed by immunofluorescence for DDR activation; TDP-43 and FUS are labeled in orange. Nuclei were counter-stained with DAPI; arrowheads mark cells with CIs. Scale bar = 10 µm. B, D Quantification of γH2AX intensity and 53BP1 foci in cells with or without cytoplasmic inclusions (±CI) shown in (A, C), respectively. The red dots of the super-plots represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. E Representative images of comet assay experiments performed in undamaged HeLa cells transfected with the plasmid expressing TDP-43, FUS^P525L, or an empty vector (EV) and treated or not with enoxacin; scale bar = 100 µm. F Tail moment analysis of HeLa cells shown in E. The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments. G HeLa cells expressing TDP-43 or FUS^P525L were treated or not with enoxacin and pulse-labeled with EU prior to immunofluorescence analysis; TDP-43 and FUS are shown in orange. Nuclear DNA was visualized using Hoechst dye; arrowheads mark cells with CIs. Scale bar = 10 µm. H Quantification of EU signal in cells with or without cytoplasmic inclusions (±CI) determined in (G). The red dots of the super-plot represent the mean values of each biological replicate; red bars indicate the averages ± SEM of three independent experiments.

Fig. 7. Enoxacin stimulates DDR and reduces DNA damage accumulation in hFUS murine spinal cords. Dicer-2 overexpression counteracts retina degeneration in hTDP-43 Drosophila melanogaster eyes.

A Representative IHC images of spinal cords of hFUS-expressing mice, treated with enoxacin or vehicle and stained for γH2AX and 53BP1. B Quantification of the percentage of cells positive for the indicated markers as determined in (A). Values are the means ± SEM. At least three mice were studied for each condition; the precise number is indicated below each histogram. C Representative images of Drosophila melanogaster eyes from adult flies expressing the indicated constructs under control of eyeless-GAL4. D Dot plot showing the quantification of mean eye area of the flies with the indicated genotypes; more than 70 eyes per genotype were analyzed in three independent experiments; red bars represent the means ± SEM.