TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. Here, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers. Similar results were observed in a non-neuronal model of HeLa cells depleted of TDP-43. These results uncover a new role of TDP-43 in the control of co-transcriptional R-loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R-loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.


Introduction
TDP-43 is a nuclear RNA binding protein (RBP) with a repressor role of HIV-1 transcription. It binds to the trans-active response element DNA sequence of the viral genome [1,2]. Like other hnRNP proteins, TDP-43 binds to nascent pre-mRNA molecules when they are released from the RNA Polymerase II (RNApol II) and regulates RNA maturation either through sequential interactions with or in collaboration/antagonism with specific RNA binding factors [3]. TDP-43 is also involved in the regulation of non-coding RNAs like miRNAs and lncRNAs [4,5]. Thanks to its ability to recognize single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) with a preferential binding to (UG)nenriched sequences [6], TDP-43 is involved in different steps of mRNA metabolism and in several mechanisms of genome integrity [7], consistent with the idea that RNA metabolism and DNA damage response (DDR) may be functionally interconnected [8].
Mutations in TDP-43 are associated to sporadic and familial cases of Amyotrophic Lateral Sclerosis (ALS), an adult onset, progressive neurodegenerative disease, caused by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord [9,10]. TARDBP is a major pathological gene for the ALS susceptibility and their mutations are found in 3% of familial and 2% of sporadic ALS cases [11,12].
Particularly, homozygous p.A382T TARDBP variation (A382T TDP-43) is one of the most common missense mutation in familial patients. A382T TDP-43 accumulation in the cytoplasm can reduce its physiological nuclear function, such as transcription regulation, mRNA splicing and transport [13,14,15] and miRNAs biogenesis [5,9]. Subsequent to this, the formation of oligomers and aggregates of TDP-43 in the cytoplasm may recruit native TDP-43 or other interactors proteins [16], constituting a gain of toxic function associated with neurodegeneration [17]. TDP-43 aggregates are identified as a major component of the ubiquitinated neuronal cytoplasmic inclusions deposited in spinal motor neurons both in familiar and sporadic ALS patients [18].
In addition to transcriptional autoregulation, TDP-43 can be cleaved into smaller Cterminal fragments before being enzymatically degraded to maintain its physiological levels [9,19] by a range of cysteine proteases, including caspases and calpains. Moreover, lines of evidence suggest that these CTFs can be produced via translation of an alternative transcript which is upregulated in ALS [20]. Recent studies proved that increased cytosolic 4 sequestration of the poly-ubiquitinated and aggregated forms of mutant TDP-43 correlates with higher levels of DNA strand breaks, activation of DDR factors such as phospho-ataxiatelangiectasia mutated (ATM), phospho-53BP1, γH2AX in SH-SY5Y lines expressing wildtype (WT) or Q331K-mutant TDP-43 [21]. TDP-43 depletion leads to increased sensitivity to various forms of DNA damage and mutation in the C-terminus glycine-rich lowcomplexity region (LC domain) associates with the loss of its nuclear function [22]. In addition, TDP-43 colocalizes with active RNA polymerase II at sites of DNA damage along with the DDR protein, BRCA1, participating in the prevention and/or repair of R loopassociated DNA damage [23].
Evidence indicate that a major source of spontaneous DNA damage comes from the accumulation of R-loops, consisting in DNA-RNA hybrids and a displaced single strand DNA (ssDNA) [8]. Non-physiological R-loops occur as unscheduled events formed cotranscriptionally that can compromise genome integrity. Increasing evidence [16,24] has highlighted a common association of increased R-loops with a variety of genetic diseases, including neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS) [25].
R-loop formation is enhanced in genomic regions containing highly repetitive DNA, which could facilitate the thermodynamic stabilization of RNA-DNA hybrids [26,27] and in cells mutated in genes encoding factors controlling R-loop homeostasis. Such factors are generally related to RNA processing and export or have DNA-RNA unwinding (helicase) or hybrid-specific ribonuclease (RNase H) activities [28,29]. However, a crucial role in prevention of R-loop formation is also played by the DDR. It is particularly notorious the role of BRCA2 and BRCA1 DSB repair factors or the Fanconi Anemia pathway (FA), especially FANCD2, involved in the repair of the inter-strand crosslinks (ICLs) and replication fork blockages [30,31]. Deficiency on any of these factors lead to harmful Rloop accumulation in human cells [8].
All this, together with the fact that a number of neurodegenerative diseases highlight a particular sensitivity of the nervous system and motor neurons are associated with deficiencies in RNA metabolism and DDR, prompted us to investigate whether TDP-43 deficiency, as found in ALS cells, have a role in R-loop homeostasis that could explain previously described DDR defects of ALS cells. We show that TDP-43 plays a role in preventing R-loop accumulation and R loop-mediated DNA breaks in neuronal and non-5 neuronal cells and in patient cell lines, thus opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.

TDP-43 depletion leads to activation of DDR and of Fanconi Anemia pathway
A key regulatory role of TDP-43 in essential metabolic processes was previously suggested since silencing of TDP-43 in HeLa cells lead in dysmorphic nuclear shape, misregulation of the cell cycle, apoptosis, increase in cyclin-dependent kinase 6 (Cdk6) transcript and protein levels [40]. As a major readout associated with RNA transcription metabolic defects, we analysed accumulation of nuclear DNA-RNA hybrids in TDP-43 depleted HeLa cells (siTDP-43 HeLa cells).
Genomic DNA-RNA hybrids in siTDP-43 HeLa cells were first assessed by immunofluorescence microscopy (IF) using the anti-DNA-RNA hybrid S9.6 antibody, and determining the levels of the S9.6 signal in the nucleoplasm after subtracting the nucleolar contribution [41,42]. As controls we used HeLa cells transiently transfected with a mock control vector expressing GFP (siCTRL) or overexpressing the RNaseH1 enzyme, which specifically degrades the RNA moiety of hybrids [41]. A slight but significant increase of R-loops was observed in siTDP-43 HeLa cells in comparison to the siCTRL that was reduced upon RNaseH1 overexpression, which confirmed that S9.6 signal detected corresponded to R-loops ( Fig 1A). Next, we determined R-loop accumulation by the more accurate method of DNA-RNA immunoprecipitation (DRIP)-qPCR, based specifically on the purification of genomic DNA-RNA hybrids of different sizes. In this case the S9.6 signal was determined for the highly expressed APOE and RPL13A genes, which have been established to be regions prone to form R-loops [31,41,43], and the SNRPN gene used as negative control [43,44]. We detected accumulation of DNA-RNA hybrids in the analysed genes in siTDP- 43 HeLa cells compared to the siCTRL HeLa cells, obtaining a significative result on RPL13A gene. Importantly, RNaseH treatment induced a dramatically signal decrease confirming that it was R-loop dependent ( Fig 1B).
Then, we investigated the functional impact of nuclear DNA-RNA hybrid enrichment on DDR, given that hybrids have been shown to enhance transcription-replication conflicts 6 [45]. As can be seen in Fig 2A, γH2AX foci, as determined by IF, were significantly increased in siTDP-43 compared to siCTRL HeLa cells. γH2AX foci significantly decreased after RNaseH1 overexpression, indicating that the damage caused by TDP-43 depletion is R-loop mediated. It has been shown that the Fanconi Anemia (FA) repair pathway is a critical pathway to resolve R-loop mediated DNA breaks as the result of transcription-replication collisions and that the FA factors works at the collisions [30,31,34,46,47]. Therefore, we tested whether the damage generated by TDP-43 depletion was signalled by the FA pathway, for which we used the FANCD2 component [34]. As it can be seen in Fig 2B, FANCD2 foci were significantly increased in siTDP-43 HeLa cells compared to siCTRL HeLa. Importantly, this increase was reduced by RNaseH1 overexpression, proving that TDP-43 depletion is responsible for an activation of Fanconi Anemia repair factor caused by R-loop accumulation. The result is consistent with the idea that FANCD2 accumulates at R loopcontaining sites at which the replication fork is blocked, similarly to inactivation of other RNA metabolic factors that lead to R-loop accumulation [32,34].

Cytoplasmic mislocalisation of mutated TDP-43 causes R-loop accumulation and leads to activation of DDR and of Fanconi Anemia pathway
In ALS patients harbouring TDP-43 mutations, TDP-43 mislocalises from the nucleus to the cytoplasm in detergent-resistant aggregated forms either full-length (43 KDa) and fragmented forms (35KDa, 25KDa), which can be ubiquitinated and hyperphosphorylated [48]. We hypothesized that TDP-43 mislocalisation due to missense mutations could have an impact on R-loop accumulation and DNA damage in ALS disease. To determine TDP-43 cellular localisation, we performed IF microscopy in basal SH-SY5Y, SH-TDP+ (overexpressing a GFP-tagged TDP-43 WT form) and SH-TDP382 (expressing the GFPtagged p.A382T TDP-43 mutant form; SH) cells using an anti-TDP-43 or anti-GFP antibody, able to detect the wild-type nuclear protein and the cytoplasmic full length and fragmented forms. In SH-TDP+, the RBP was localised preferentially in the perinuclear area compared to non-transfected SH-SY5Y cells, in which localisation was predominantly nucleoplasmic.

TDP-43 nuclear localization was significantly decreased in SH-TDP382 cells compared both
to non-transfected SH-SY5Y and SH-TDP+, confirming that the A382T mutation is linked to TDP-43 cytoplasmic mislocalisation with formation of inclusions or aggregates (S1 Fig)   7 as previously reported [49,50,51]. Results were the same when using the anti-TDP43 or anti-GFP antibodies.
Next, we tested whether mislocatisation of this RBP could impact onto genomic integrity and R-loop accumulation. We first assayed whether overexpression of wild-type TDP-43 and mutated and mislocalised TDP-43 affected R-loop accumulation. Since both mutated and overexpressed TDP-43 may affect its physiological role in miRNA biogenesis, we added in this case an ulterior treatment with RNaseIII, which degrades specifically dsRNAs, to counteract the reported ability of S9.6 to detect dsRNAs [35,52]. A significant increase of nucleolar S9.6 intensity was detected both in SH-TDP+ and SH-TDP382 cells compared to SH-SY5Y ( Fig 3A). However, RNaseH1 overexpression caused nucleolar S9.6 signal decrease in SH-TDP382 cells only, but not in SH-TDP+ cells. The extra signal seen in TDP+ cells correspond to dsRNAs rather than DNA-RNA hybrids as shown by the sensitivity of such a signal to RNase III treatment. The reason why overexpression of TDP43 increases dsRNAs would need to be investigated further, and is not related to this study.
Therefore, we conclude that mislocalisation of TDP-43 also causes R-loop accumulation.
We performed DRIP-qPCR in the neuroblastoma cell lines in the previously reported target genes [41, 43,44], to confirm the results involving TDP-43 role in preventing R-loop accumulation in human cells. RNaseH treatment dramatically decreased the levels of the signal in all cases, confirming that the signal detected was specific for nuclear DNA-RNA hybrids. Whereas a significant R-loop accumulation was observed at the RPL13A gene in the SH-TDP382 mutant cells compared both to SH-SY5Y and SH-TDP+ (Fig 3B), this was not evident for APOE confirming the same tendency observed in siTDP-43 HeLa cells.
To test whether R-loop accumulation observed in SH-TDP382 cell lines causes DNA damage we determine the levels γH2AX foci by IF microscopy and their dependence of Rloops by testing whether RNaseH1 overexpression reduced them. As can be seen in Fig 4A, γH2AX foci were significantly increased in SH-TDP382 cells whereas this was not the case in SH-SY5Y and SH-TDP+ cells expressing the wild-type form of TDP-43. The increase in damage was suppressed by RNaseH1 overexpression, indicating that DNA break accumulation caused by mutant TDP43 was mediated by DNA-RNA hybrids.
Next, we tested whether the origin of such DNA damage was due to an increase in transcription-replication collisions enhanced by R-loops. We determined the levels of 8 FANCD2 foci as previously reported. Notably, FANCD2 foci were significantly increased in SHSY-TDP382 mutant cells compared to SHSY-TDP+ and this increase was reduced by RNaseH1 overexpression (Fig 4B). Therefore, the ALS pathogenic TDP-43 mutation in the analysed neuronal model leads to a comparable functional effect to that observed in silenced HeLa cells. The pathogenic TDP43 mutation causes an increase in DNA breaks derived from R-loop accumulation that promotes transcription-replication collisions that are processed by the FA pathway, as reported for other cases of recombinogenic R-loops [30,31,34,46,47]. causing the loss of its nuclear physiological function [53]. Moreover, there is a colocalization of S9.6 signal with TDP-43 in the perinuclear area of LCL-TDP382 cells in comparison to LCL-CTL and LCL-SALS. R-loop quantification was also determined in LCLs by flow cytometry, in which case the analysis reported an increased positivity of S9.6 intensity in the orange peak associated with LCL-TDP382, in comparison to the blue peak associated with LCL-CTL ( Fig 5B). The positive signal in LCL-TDP382 represented by the orange peak was clearly suppressed by RNase H treatment in the same sample detected as green peak, confirming that the detected signal corresponds to DNA-RNA hybrids (Fig 5C). S9.6 mean fluorescence of LCL-TDP382 in a triplicate experiment was significantly increased compared to LCL-CTL and LCL-SALS, and this increase was reverted by RNaseH treatment (Fig 5D).
Finally, we investigated the possibility that TDP43 could have a role on DNA-RNA hybrids directly, in which case we should expect some kind of physical association. 9 Therefore, we wondered whether TDP-43 and genomic DNA-RNA hybrids colocalize by performing a co-immunoprecipitation (coIP) in chromatin (Chr) fractions from the three cell lines. At the same time, we extracted whole lysate (WL) fractions from the same samples as control for the cytoplasmatic fraction (Fig 6A-B). In the Chr fraction, coimmunoprecipitation could be observed with the S9.6 antibody. In LCL-TDP382, the TDP-43 mutant protein showed lower levels of co-IP, while in the WL fraction of the same sample the co-IP signal was similar to WT, which suggests that the mutant full length TDP-43 was not able to interact with R-loops due to its sequestration at the cytosolic compartment in the cell [53], as previously seen in Fig 5A. Interestingly, the truncated TDP-35 form detected in Chr fraction of LCLs show high levels of S9.6 co-IP in the WL fraction of LCL-TDP382 in comparison with control LCL-CTL and LCL-SALS. This specific C-terminal mutation may predispose TDP-43 to fragmentation into CTFs, which as reported in literature are transported out of the nucleus and accumulated into complexes with RNA transcripts [49].
The result suggests that a fraction of cellular TDP-43 is present in chromatin in association with DNA-RNA hybrids and both wild-type and truncated forms can be found in the cytoplasm, Therefore, we can conclude that TDP-43 has a role in RNA metabolism from nucleus to cytoplasm that help prevent cells to accumulate co-transcriptional harmful Rloops.

Discussion
Mislocalisation of TDP-43 causes a gain of neurotoxic function characteristic of the neurodegeneration process in ALS patients [54]. Moreover, TDP-43 aggregation induced by double-site mutations and TDP-43 knockdown have a common set of differentially expressed proteins, behaving in a similar way [55]. Here we show that mislocalisation of mutated TDP-43 can sequester full length TDP-43 form in cytoplasmic inclusions preventing its physiological nuclear function. Importantly, this function is related to R-loop homeostasis. Nuclear depletion of TDP43, as achieved by either mislocalisation to the cytoplasm or siRNA depletion in different cell types, causes a significant increase in harmful R-loops that leads to DNA breaks and FANCD2 foci. The results suggest that the TDP-43 RNA-binding protein has a key role in preventing R-loop accumulation as a safeguard of genome integrity. 10 Silencing TDP-43 by siRNA in HeLa cells led to a significative increase of R-loop signal by S9.6 IF compared to the siCTRL control. This was confirmed by reversion of signal in case of RNaseH1 overexpression. DRIP-qPCR revealed an important R-loop presence on the tested RPL13A gene encoding a highly expressed ribosomal protein involved in pathways of viral mRNA translation and of rRNA processing, whose impairment could lead to alteration of protein homeostasis as well as modifications of RNA metabolism [56]. It is worth noting that TDP-43 interacting proteins largely cluster into two distinct interaction networks and RPL13A in particular is implicated in TDP-43 cytoplasmic interactome cluster, resulting fundamental for RNA translation regulation [57]. Also, experimental studies reported that both C9orf72 mut ALS patients' derived iPSCs and TDP43-EGFP overexpressing iPSCs presented a set of commonly destabilized RNAs involved in the ribosomal and in the oxidative phosphorylation pathways. As consequence of RNA instability in iPSCs it was detected an increase of cytoplasmic ribosome proteins including RPL13A, suggested as cellular compensatory response for preservation of protein synthesis capacity. Moreover, the analysis of 3′UTRs of transcripts in the same cells showed a high enrichment in motifs recognized by RBPs and involved in the formation of cytoplasmic inclusions, that in turn exhibited an alteration of their stability [58]. We do not believe that the particular effect observed in accumulation of R-loops in RPL13A is related to these phenotypes, since ribosomal protein genes are highly transcribed, thus favouring R-loop formation. However, it is certainly possible that indirectly R-loop accumulation itself in ribosomal protein genes, may be linked to the misbehaviour of the translation and UTR function.
The absence of nuclear TDP-43 affects the DDR, consistent with previous reports [23]. We showed that siTDP-43 HeLa cells had a significative increase of DSBs as determined by γH2AX foci. Importantly, this DSB increase was R-loop-dependent, as it could be fully reverted by RNaseH1 overexpression, and it was accompanied by an accumulation of FANCD2 foci that was also R loop-dependent. The result indicates that TDP-43 prevents the co-transcriptional accumulation of harmful R-loops that promote transcription-replication conflicts that have to be resolved by the FA pathway, consistent with the previously reported role for the FA pathway [45].
Notably, our assay of the impact of the TDP-43 pathogenic ALS mutation and overexpression in a neuroblastoma cell line, SH-SY5Y, revealed that the pathogenic A382T mutation in SH-SY5Y cells also affected the TDP-43 role controlling R-loops homeostasis, leading to a higher detection of genomic RNA-DNA hybrids, as detected by IF and DRIP-qPCR. As expected DSBs and replication blockage detected by γH2AX and FANCD2 foci, respectively, were also increased in an R loop-dependent manner. It is worth noticing that when TDP-43 was overexpressed (SH-TDP+), a significant increase of an S9.6 signal that was sensitive to RNase III treatment but not to RNaseH1 overexpression, in contrast to A382T mutant. Knowing that S9.6 can also detect dsRNAs [35,52] this result indicates that TDP-43 overexpression leads to an accumulation of dsRNA molecules. Interestingly, TDP-43 has been reported to co-localize with Dicer and Ago2, but their interaction is inhibited by aggregates formation in response to cellular stressors or also by overexpression of human TDP-43 [60]. There is no evidence that overexpression and mutation of TDP-43 lead to double hairpin pre-miRNA accumulation in neuronal models, so it is formally possible that dsRNA accumulation observed in SH-SY5Y overexpressing TDP-43 could be associated to inhibition of Dicer processing function responsible for the loss of maturation of pre-miRNAs, presented as dsRNAs hairpin structure, in mature miRNAs. However, it may also be possible that excess of RBPs would prevent normal RNA metabolism by excess of cellular RBPs that would bind to any RNA molecule having secondary structure segments. A negative impact of RBP overexpression on RNA metabolism has been reported in other cases [37]. These are possibilities to explore in the future.  [20]. Due to the lack of nuclear localization signal (NLS), CTFs TDP-35 mislocalise to the cytoplasm, where may associate with RNA forming cytoplasmic inclusions [61]. Indeed, the biochemical analysis suggests that TDP-35 facilitates aggregate assembly promoting inclusion formation [62] and might transport different types of RNA structures. It is known that TDP-35 can also recruit full-length TDP-43 to cytoplasmic deposition from functionally nuclear localization [63] and TDP-43 continuously shuttles between nucleus and cytoplasm in a transcription-dependent manner [64]. The higher S9. 6  14 Immunofluorescence microscopy For S9.6 IF analysis in HeLa and SH-SY5Y, cells were fixed with cold methanol for 10 minutes at -20°C according the literature [34]. SH-SY5Y cells were treated with 40 U/ml RNaseIII (1 U/µl, Thermo Fisher Scientific) for 30 minutes at 37° using 1X RNase III Reaction Buffer. For γH2AX and FANCD2 IF analysis in HeLa and SH-SY5Y, cells were incubated with a fixation solution (PFA 4%, Triton-X 0,1%) for 10 minutes at room temperature (RT) as previously described [35]. For S9.6 and TDP-43 IF microscopy analysis in LCLs, cells were fixed with two methods: one using formaldehyde and cold acetone and the other using cold methanol.

DNA-RNA immunoprecipitation-qPCR
DNA-RNA immunoprecipitation (DRIP) was performed on HeLa and SH-SY5Y cells as already described in literature [36,37]. The amount of R-loop levels was quantified as a function of input DNA, that for each sample was 10% of the entire amount. anti-CD19 antibody for B lymphocytes recognition. Cells were fixed and permeabilized using a kit based on saponin permeabilization (Fixation/Permeabilization Solution Kit, BD) following a protocol described by Schauer U. et al. [38]. As negative control for R-loop presence, cells were treated with 60 U/ml of ribonuclease H (RNase H, 5.000 units/ml, NEB) using RNase H buffer at 37°C for 1 hour. As negative control for ssRNAs, cells were treated with 100 µg/mL ribonuclease A (RNase A, 10 mg/mL, Thermofisher) in 0.3M NaCl buffer at 37°C for 1 hour. At the end, cells were stained for one hour with conjugated anti-S9.6 antibody (PE/R-Phycoerythrin Conjugation Kit, Abcam) and analysed by flow cytometry (BD FACS Canto II). Logarithmic amplification was used for all channels and FACSDIVA was used for the analysis.