Chronic Oxidative Damage together with Genome Repair Deficiency in the Neurons is a Double Whammy for Neurodegeneration: Is Damage Response Signaling a Potential Therapeutic Target?

2.1. Endogenous causes of genome damage in the CNS

Due to the protection by blood brain barrier from external genotoxins, endogenous reactive oxygen species (ROS) -induced damage is the most critical threat to the CNS genome. In the brain, constitutively expressed enzymes such as the neuronal isoform of nitric oxide synthase (nNOS) contribute to the production of nitric oxide (NO°) radicals, which react with superoxide (O₂^·−) to produce peroxynitrite (ONOO⁻), a reactive nitrogen species (RNS) [16]. Trace levels of ROS/RNS are required for the normal functioning of the CNS, and serve as signaling molecules for regulating synaptic plasticity and adult neurogenesis [17]. Excessive ROS and other pro-oxidant species induce a variety of damage in DNA that include, oxidized and alkylated base lesions, abasic (AP) sites, inter-strand cross links, and single strand breaks (SSBs) [18]. The most common oxidized DNA lesion is 8-oxoguanine (8-oxoG), derived from guanine. Highly mutagenic/toxic but less common oxidized bases are formamidopyrimidines (FapyG and FapyA), 7,8-dihydro-8-oxoadenine (8-oxoA), 5-Hydroxyuridine (5-OHU), thymine glycol, 5-hydroxycytosine (5-OHC) etc [18]. SSBs may be induced directly by ROS or as intermediates during oxidized lesion repair. It is likely that persistent unrepaired bi-stranded oxidative damage in close proximity could result in secondary double strand breaks (DSBs) [18]. This phenomenon could be more prevalent in promoter sequences of actively transcribing genes, where a localized generation of ROS due to CpG demethylation-mediated promoter activation has been recently shown to generate transient strand breaks [19]. DSBs may also be generated in neuronal genome from accidental or diagnostic/therapeutic exposure to ionizing radiation (IR) and chemotherapeutic drugs [20]. A recent study reveals that DSBs occur in mouse brain cells during normal physiological/metabolic processes involving gene expression and chromosome remodelling associated with learning and memory [21]. Endogenous metabolic by-products such as homocysteine (Hcy), a non-essential sulfur-containing amino acid derived from methionine metabolism, has been implicated in CNS disorders with cystathionine beta synthase deficiency in AD. Elevated Hcy level disturbs the DNA methylation/demethylation cycle [22] and contributes to DNA damage by inducing thymidine depletion and promoting uracil mis-incorporation [23].

2.2. Excision repair pathways in the CNS genome

Excision repair pathways, which resolve the majority of endogenous genome damage including modified base or large bulky lesions are the most versatile among DNA repair processes. Small base lesions including oxidized or alkylated bases and AP sites are repaired via BER while larger bulky lesions like inter-strand cross-links are repaired via NER, both of which are conserved from bacteria to humans and are ubiquitous in most tissues including the CNS [24].

2.3. DNA strand break repair

Repair of DNA strand breaks in a timely and error-free manner is vital for the preservation of genomic integrity. The importance of neuronal SSBR is evident in several NDDs, namely, AOA1, SCAN1 and microcephaly with early-onset, intractable seizures and developmental delay, which are caused by inherited mutations in SSB end processing factors [26, 37]. SSBR, which includes many overlapping sub-pathways, share the later steps of repair with BER such as end processing of damaged termini, gap-filling, and nick ligation [26, 38]. However, in addition to the 3′ P and 3′ dRP groups generated at SSB termini during BER, SSBR could involve diverse end processing enzymes to remove a variety of blocked termini generated by ROS [26]. Specifically, APE1 removes the most common block at ROS-induced SSB like 3′ phosphoglycolate or 3′ phosphoglycolaldehyde, another 3′ end-processing enzyme tyrosyl DNA phosphodiesterase 1 (TDP1) cleaves Top1(Tyr)-crosslinked to 3′ P generated by an aborted DNA topoisomerase 1 (TOP1) reaction. The resulting 3′ P is then removed by PNKP [39, 40], which also phosphorylates the 5′OH generated at SSB. Furthermore, abortive DNA ligation could form a unique type of 5′ blocking group, namely, adenylate linked via 5′-5′ bond at the 5′P terminus, which is processed by aprataxin (APTX) to restore the 5′ P terminus at the SSB [41, 42]. Two other key SSBR components include poly(ADP-ribose) polymerase 1 (PARP-1) and X-ray repair cross-complementing protein 1 (XRCC1), which not only serve as scaffolding factors for recruiting and stabilizing other SSBR proteins, but also play a structural and regulatory role as damage sensors [43–45].

The repair of DNA DSBs in mammalian genomes could occur via three sub pathways: error-free pathway homology directed recombination (HDR) that occurs in only S/G₂ cells requiring homologous DNA sequence (sister chromatid) as templates; and error-prone pathway non-homologous end-joining (NHEJ) or short microhomology-dependent alternative end-joining (Alt-EJ) throughout cell cycle. Although HDR may be critical during neurodevelopment, NHEJ has been suggested as the predominant DSBR pathway in mature neurons [46, 47]. Besides, it is likely that Alt-EJ may step into repair these secondary DSBs (generated from unrepaired bi-stranded SSBs) since elevated Alt-EJ activity has been observed in neuroblastoma cell lines [48], whereas its role in primary neurons has not been established.

NHEJ primarily requires DNA-PK holoenzyme (DNA-PKcs/Ku70/Ku80) and XRCC4/DNA LigIV complex, while Alt-EJ requires a distinct set of components including PARP-1, XRCC1/DNA LigIII (co-opted from SSBR) to initiate the repair [49, 50]. In any case, mutations in key DSBR proteins (e.g., ataxia telangiectasia mutated (ATM) in A-T, NBS1 in Nijmegen breakage syndrome, and meiotic recombination 11 (MRE11) in ataxia-telangiectasia-like disorder (A-TLD) have been shown to predispose humans to various neurodegenerative phenotypes, suggesting that DSBR is critical for post-mitotic neurons [47, 51–54].

2.4. DNA Damage Response (DDR) signaling in the CNS

DDR consists of a kinase cascade based network of coordinated signaling pathways, activated primarily in response to DSBs. In dividing cells (including neural progenitor cells during development, trace number of neural stem cells and dividing glial cells in adult CNS, the cell cycle is arrested at critical stages, particularly in response to DSBs, which is termed as cell cycle or DNA damage checkpoint activation, to provide sufficient time for optimal DNA damage repair and to prevent replication or segregation of DNA with unrepaired damages [55]. DNA strand breaks in mammalian genome typically activate phosphoinositide-3-kinase related (PIKK) family of serine/threonine kinases, ATM and ATM/RAD3-related (ATR), that play a central role in signal amplification by phosphorylating multiple downstream proteins to regulate checkpoint activation, DNA repair or cell death. ATM is predominantly activated in response to DSB-induced by radiation and other genotoxins, while ATR is activated by DNA replication stress, SSBs and UV-mediated damage, besides playing a back-up role to ATM [56].

As a central component of DDR cascade, ATM also plays an essential role in maintaining genomic stability in neuronal cells, and the loss of ATM associated DNA repair deficiency has been linked to neuronal dysfunction or neurodevelopmental defects as in A-T patients. A number of unique regulators of ATM and p53 in neurons have been identified. Tian et al. have demonstrated that cyclin-dependent kinase 5 (Cdk5) directly phosphorylates ATM at Ser794, which is required for the activation of its kinase activity and the phosphorylation of its substrates- p53 and histone H2A.X [57]. Another factor involved in DDR regulation, NAD+-dependent lysine deacetylase sirtuin 1 (SIRT1), is recruited at DNA break sites in an ATM dependent manner to stimulate ATM auto-phosphorylation and its activity. Phosphorylation of H2A.X (γH2AX) is significantly reduced in SIRT1 knockout neurons, which indicates the importance of SIRT1-initiated DNA damage signaling in neurons [58]. Furthermore, an RNA/DNA binding protein, fused in sarcoma/translocated in liposarcoma (FUS/TLS), whose toxicity has been implicated in motor neuron diseases, was identified as a substrate of ATM in response to DNA damage and has been shown to participate in both HDR- and NHEJ- mediated DSB repair pathways [13, 59]. The coordination between ATM and p53 appears to be critical for DNA damage signaling in neurons as in cycling cells. ATM and p53 are also involved in triggering persistent damage-induced neuro-inflammation via nuclear factor-kappa B (NF-κB) activation, which is discussed in section 5.2. Herzog et al. showed that the p53 level is upregulated in wild type mouse neurons but significantly reduced in some brain regions of ATM^−/− mouse in response to irradiation and post-mitotic neurons in ATM^−/− mouse showed resistance to p53-mediated irradiation induced apoptosis [60], which provided some insight in to the possibility of preventing DNA damage related neuronal cell death by targeting the ATM-p53 pathway.

2.5. Mitochondrial DNA repair in the CNS

It has long been demonstrated that the increase of oxidative mtDNA damage is associated with neurodegenerative diseases and normal aging process [61]. Compared with nuDNA, human mtDNA is more susceptible to oxidative damage in a variety of tissues including brain [62], since mitochondria generates the majority of energy for cells through oxidative phosphorylation, producing abundant ROS [63, 64] and lacks protection from histones, [65]. SN-BER is recognized as a major pathway for mtDNA damage repair [66], although growing evidence shows that LP-BER, mismatch repair, HDR and NHEJ are also involved in mtDNA damage processing [67–69]. As in nuclear genome, the BER in mitochondria is initiated by DNA glycosylase including OGG1, Uracil DNA glycosylase (UNG), Muty Homologue (MYH), NTH1 and NEILs, followed by the processing of AP site by the only endonuclease in mitochondria, APE1. The knockout of APE1 is embryonic lethal in mouse [70, 71], while another study demonstrated that the loss of APE1 in mitochondria but not in nucleus is believed to trigger cellular apoptosis [72]. The final step of the mitochondrial BER is the ligation of the DNA nick by Ligase III but independent of XRCC1 [73, 74], unlike in nucleus. Interestingly, Gao et al. developed a mouse model in which the Ligase III or XRCC1 was conditionally inactivated in the developing neurons, and revealed that the Ligase III is important for mtDNA integrity but not for XRCC1 dependent, H₂O₂ or irradiation induced nuDNA damage repair in astrocytes [75, 76], which underscores the critical role of Ligase III mediated mtDNA repair in neuroprotection. Finally, human mtDNA encodes only 13 proteins, and mitochondria BER is highly dependent on nuclear encoded proteins [77].

The mtDNA repair capacity varies among different CNS cell types. For example, the oxidative mtDNA damage repair capacity was observed to be significantly decreased in cultured oligodendrocytes and microglia compared with astrocytes, and the difference is correlated with the apoptosis induction [78, 79].

3.1. DNA damage accumulation in AD, PD and ALS

AD, the most common progressive NDD and the major cause of dementia in adults, is strongly age-associated and characterized by progressive loss of memory and cognitive capacity. AD is histopathologically associated with extracellular deposition of amyloid β (Aβ) peptide, intracellular aggregates of hyper-phosphorylated tau and neurofibrillary tangles (NFTs) [84]. It was hypothesized around 30 years ago that increased DNA damage may be associated with AD due to the observation of specific chromatin structure alteration in AD patients [85, 86] and Mullart et al. subsequently quantified at least 2-fold increase in DNA breaks in cortex of AD patients [87], underscoring the contribution of accumulated genome damage. Other studies suggested strong association between the extent of oxidative damage in the neuronal genomes in affected brain regions with severity and progression of dementia in AD patients [11, 88–94]. Among the oxidized bases, increased levels of 8-OHA and Hcy were observed in DNA from AD parietal lobe, whereas increased thymine glycol, FapyA and FapyG, and 5-OHU were found in other affected brain regions [95]. Hippocampal DNA from late stage AD was shown to have a 2-fold acrolein/guanosine DNA adducts compared to that from unaffected brain, the repair implications of which are unknown [96]. Aβ seems to elicit DNA damage directly. In addition to the stimulation of ROS production [97–99], Aβ42 was shown to have DNA nicking activities similar to nucleases [100]. A recent study indicated that pathologically elevated level of Aβ increases neuronal DSB at baseline in mouse brain sections and cultured primary neurons, and enhanced the DSB that physiologic brain activity caused by eliciting aberrant synaptic activity [21].

As the most frequent neurodegenerative movement disorder, PD is characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc) [101–104] as well as the presence of proteinaceous cytoplasmic inclusions known as Lewy bodies in the brain of patient with either sporadic or familial form [105]. Although the pathological mechanisms underlining PD are complex and remains largely unknown, oxidative DNA damage (specifically, the production of 8-oxo-dG) has been demonstrated to be extensively generated in PD patients compared with age-matched control [106, 107]. PD-linked α-Synuclein (α-Syn) expressed transgenic mice show accumulation DNA damage within neurons [108]. Our later study also showed that both SSB and DSB accumulate in postmortem midbrain region of PD patients and the isolated DNA exhibited increased susceptibility to DNAse I treatment compared to control DNA [103]. Additionally, DNA damage-dependent activation of p53 and PARP-1 was shown to play a role in the death of DAergic neurons [109]. The MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced PD mouse model shows DNA damage together with the activation of PARP1, which correlated with SNpc neuronal degeneration [110].

ALS or Lou Gehrig’s disease is an adult onset motor neuron disease, about 10% of which are inherited while ~90% occurs sporadically. Mutations in genes like superoxide dismutase (SOD-1) [111, 112], TAR-DNA binding protein-43 (TDP-43) [113, 114], FUS/TLS [115, 116], ubiquilin 2 (UBQLN2) [117], optineurin [118], valosin-containing protein (VCP) [119], C9orf72 [120], profilin1 (PFN1) [121] and matrin 3 (MATR 3) [122] have been identified in ALS patients. Similar to AD and PD pathologies, ALS affected motor neurons (frontal cortex, motor cortex, spinal cord, striatum) accumulate oxidized DNA bases in both nuclear and mitochondrial genomes [123]. For instance, ALS linked SOD1 G93A mutant expressed transgenic mice, consistently showed higher DNA damage compared to mice with wild type SOD1 [124]. In ALS patients harboring FUS/TLS mutations, increased DSBs were observed, and FUS/TLS knockdown in cultured human and rodent neurons leads to significant accumulation of DSBs [13]. TDP-43 is functionally similar with FUS, while its involvement in genome damage accumulation has not been investigated yet, although substantial increase in DNA damage has been observed in ALS patients with TDP-43 pathology [125].

3.2. DNA damage accumulation in Stroke

Stroke results from sudden interruption of blood supply to the brain, which could be due to abrupt blockage of arteries leading to the brain (ischemic stroke) or due to bleeding into brain tissue when a blood vessel bursts (hemorrhagic stroke). There is substantial evidence for the formation of oxidative genome damage after hypoxia followed by ischemic reperfusion and the release of iron and toxic hemoglobin by-products in brain parenchyma following hemorrhagic stroke [126–128]. Lesions including oxidative bases and strand breaks have been reported to remain unrepaired in post-stroke brain tissue, which could chronically predispose neurons to damage-mediated dysfunction or defective damage repair. It appears that BER is particularly important in protecting neuronal oxidative DNA damage after stroke. A mouse study indicated that activation of the BER pathway protects ischemic induced injury in brain by enhancing the endogenous oxidative DNA damage repair [129]. OGG1^−/− mouse exhibited elevated oxidative DNA base lesions under ischemic conditions [130]. NEIL1 deficiency results in increased brain damage in a focal ischemia/reperfusion model of stroke [126]. While APE1 was shown to be greatly reduced specifically in rat hippocampus after ischemic insult, which suggests that the specific APE1 deficiency may involve in the extent of cellular apoptosis in response to ischemia [131]. During ischemia, damage to nucleic acids may also be caused by nucleases causing DNA fragmentation [132]. Extensive oxidative damage was detected in the mtDNA and nuDNA of astrocytes, neurons and vascular endothelial cells within 15–30 min of ischemia/reperfusion [133]. In intracerebral hemorrhage (ICH), hemin and iron promotes accumulation of DNA strand breaks and base oxidation in the affected cell genomes [134]. This is supported by the observation that intra-hippocampal injection of iron or hemoglobin in rats induces significant genome damage and neuronal death [135].

4.1. Inherited DDR defects

A substantial subset of familial NDDs have been found to be inherited or acquired from defects in one or more key genes involved in DDR signaling. Mutation of DSBR proteins involved in the predisposition to familial NNDs include ATM, NBS1 and MRE11. For example, neurodegeneration is a syndrome of A-T patient carrying ATM mutation [149]. Mutations in another DSBR factor MRE11 is associated with ATLD phenotype [150]. In Nijmegen breakage syndrome, a 657Δ5 frameshift mutation of NBS1 results in the formation of truncated product, affecting its ability to act as a scaffold between γH2AX, and MRE11, which is required for DNA damage signaling in HDR, NHEJ and telomere maintenance [144]. While some syndromes characterized by ataxia are deficient in SSBR, for example, SCAN1 and AOA1 are caused by mutations in TDP1 and APTX, respectively [37, 151, 152]. TDP1 is required for efficient SSBR in mouse neurons and in lymphoblastoid cells from SCAN1 patient [153], [39]. Homozygous mutation in TDP1 (H493R), causes 25-fold reduction in its activity, leading to the accumulation of covalent Tdp1-DNA intermediates, thereby, inhibiting SSBR [39, 154]. While APTX was shown to protect DNA damage by interacting PARP-1, APE1, XRCC1 and XRCC4 [155, 156]. Caglayan et al. demonstrated that APEX coordinates with Polβ and flap endonuclease 1 (FEN1) in processing of the 5′-adenylated dRP-containing BER intermediate [157]. Interestingly, mouse models lacking both of TDP1 and APTX shows synergistic decrease in SSBR [158]. Homozygous mutation in PNKP with reduced SSBR capacity was demonstrated in progressive polyneuropathy characterized by underdevelopment and neurodegeneration [159]. Mutation in ATR in Seckel syndrome leads to reduced DNA repair capacity [160]. Other rare syndromes like LigIV, and NHEJ syndromes are directly linked to repair defects; mutations in LigIV affects its ability to interact with XRCC4 [161] whereas mutations in the NHEJ-associated gene encoding XRCC4-like factor (XLF) affects its ability to form a scaffold essential for XRCC4 and LigIV [162]. Taken together, familial NDDs are predominantly associated with DNA repair defects, or with compromised DDR signaling. We have listed various CNS diseases associated with inherited DDR defects and associated DNA repair proteins in Table 1.

4.2. Neurodegeneration-linked etiological factors contribute to DNA damage/repair imbalance: A common basis for sporadic NDDs?

Reduced DNA repair is increasingly linked to a number of sporadic NDDs. General reduction in DNA repair capacity during ageing and in sporadic NDDs could involve multiple mechanisms including, (i) altered transcriptional regulation causing reduced expression of a repair protein (e.g., reduced levels of MRN, DNA-PKcs [143, 163] in CS; reduced OGG1 (in mitochondria) [164] and Polβ in AD [165], reduced breast cancer associated gene 1 (BRCA1) in AD brain [166]); (ii) inhibition of catalytic activity of repair proteins, whose levels are otherwise comparable to healthy neurons in the CNS, primarily by NDD-linked etiological factors like exposure to excessive pro-oxidant metals and ROS (e.g., metal and ROS-mediated inhibition of NEIL1, NEIL2, LigIII, APE1 etc., in AD and PD [167, 168]; reduced catalytic activity of OGG1 in AD [136, 169] and reduced Polβ activity in AD [136, 165] and (iii) abnormal degradation and/or mis-localization of repair proteins (e.g., degradation of ATM and PARP1 by caspases/Matrix-Metallo-Proteinases (MMPs) [170], nuclear to cytoplasmic mis-localization of FUS in ALS [13]. Most of the NDDs have complex etiologies including oxidative stress, pro-oxidant metal and other toxic free radicals, and the misfolded/aggregating protein response, together with extensive accumulation of various types of DNA damages [18]. However, the relative contribution of these factors towards progressive neurodegeneration and their precise mechanistic role in causing persistent genome damage in specific CNS regions remains unclear. Here, we present the cumulative evidence regarding the interplay among these etiologies in inducing genome damage, in inhibiting genome damage repair, affecting the dynamics of DDR signaling, and thereby causing damage-repair imbalance in neuronal genomes (Figure 1, 2).

4.3. CNS mitochondrial DNA damage and repair defects

Mitochondrial dysfunction, one of key events contributing to NDDs and aging, can result from mtDNA point mutations or deletions [197]. For example, mutations in the subunits of mitochondrial respiratory chain Complex I occurs in approximately 95% of Leber Hereditary Optic Neuropathy [198]. Some mtDNA mutations have also been associated with AD [199, 200] and PD [201–203], although no clear causative mtDNA mutations are linked with these diseases.

Consistently, studies have revealed elevated mtDNA damage accumulation or mtDNA repair defects in patients with NDDs. In AD, a three-fold increase in 8-oxoG lesion in mtDNA from frontal, temporal, and parietal lobes and cerebellar brain tissues have been observed compared to age matched controls [204]. Increased DNA fragmentation and reduced DNA content/mass in brain mitochondria have been linked to the extent of hippocampal degeneration in AD [205]. In HD, mutant huntingtin is associated with elevated mtDNA oxidation [206], as well as higher levels of 8-oxoG in the striatum of HD patients and in a transgenic mouse model (R6/2) [207, 208]. mtDNA damage was suggested as an early biomarker for HD-associated neurodegeneration [209]. Patients with XP group C (XPC), accumulate pyrimidine dimers, 8-oxoG, and thymine glycol in the mtDNA of CNS [210]. In children with autism, elevated mtDNA damage was observed [211]. Neurofibromatosis type 1 (NF1) with somatic mitochondrial mutations [212], Spinal and Bulbar muscular atrophy or Kennedy disease, (a motor neuron disorders) also exhibit mtDNA damage [213]. Somatic mutations in neuronal mtDNA causes ATP depletion, increased oxidative stress, and in turn leading to nuclear genome damage [214]. Patients with XP accumulate pyrimidine dimers, 8-oxoG, and thymine glycol in the mtDNA of CNS tissue. These observations provide important insights into the impact of mtDNA somatic mutations in neuronal mitochondria.

Elevated mtDNA AP sites were observed in nigral dopamine neurons in PD patients, suggesting persistent BER defects [215]. A transgenic mouse model expressing Mito-PstI, an endonuclease targeted exclusively into DAergic neuronal mitochondria to generate mtDNA DSBs, demonstrates strong patterns of SN degeneration similar to that observed in PD patients [12]. In support of the above, mtDNA in DAergic neurons possesses higher oxidized base lesions [106, 216] together with unrepaired strand breaks, AP sites, and deletions in SN of PD brain [217]. Presence of unrepaired SSBs and DSBs in the mitochondrial genome of familial α-Syn mutant (A53T) transgenic mice correlated with mitochondrial degeneration and PD like pathology [108].

As previously mentioned, mtDNA repair is highly dependent on nuclear encoded proteins, indicating that a continuous interplay between nuDNA and mtDNA is essential. Nuclear encoded proteins are imported into mitochondria through mitochondria membrane translocases: translocase of the mitochondrial outer (TOM) and inner (TIM) membrane protein complex. Brain regions such as hippocampus, SN, and cortex which are significantly affected in AD, PD, and HD show altered levels of TOM and TIM [218]. Mid brain tissue studies from PD patients and wild type and mutant (A53T) α-Syn transgenic mice model revealed that the protein level of TOM40, an important transmembranous domain of the TOM complex involve in the import of nuclear encoded proteins, is significantly reduced along with high level of oxidative damage and deletions in mtDNA, which could be rescued by overexpressing of TOM40 [218]. Although accumulated mtDNA damage may be induced by elevated ROS observed in α-Syn accumulating cells, reduction of TOM40 levels may also contribute to the mtDNA damage by inefficient import of nuclear encoded mtDNA repair proteins. These studies opened up new avenues towards understanding the mitochondrial protein import defects in NDDs as well as search for novel biomarkers of degraded TOM/TIM peptide fragments.

The regulation of brain mitochondrial BER varies through the lifespan. DNA glycosylases (UNG1, OGG1, and NTH1), are upregulated in the cortical mitochondria of brain tissues from young mice, but downregulated in the brain tissues from ageing mice [219, 220].

5.1. DNA damage induced transcriptional inhibition

With about three to five times more actively transcribing genes than elsewhere in the body [6], damage and repair at transcriptional sites are critical for neuronal survival. Neuronal transcription machinery has been shown to closely collaborate with DNA damage sensing and repair as well as chromatin organization [223]. Many DNA lesions (e.g., 5′, 8-purine cyclo-deoxynucleosides, purine dimers, 8-oxoG, thymine glycol and DNA strand breaks) stall RNA polymerase II in transcribing genes. Furthermore, it has been proposed that the inhibition of RNA polymerase II can be via ATM dependent or DNA-PK dependent pathways [224]. The accumulation of 8-oxoG in redox-sensitive transcribing genes containing GC-rich promoter sequences renders them particularly vulnerable to oxidative modification-mediated transcriptional inhibition [225]. Consistently, recent studies have reported the accumulation of R-loop structures (presumably formed by damage-induced inhibition of transcription) in AD and ALS affected brain tissues [226–228]. Transcriptional stalling together with DNA repair inhibition is a recipe for exacerbating genomic instability and enhancing the hyper-methylation of gene promoters leading to gene silencing [229]. Taken together, these recent developments underscore the implications of defective DNA repair and DDR in neuronal cell death.

5.2. DDR and neuro-inflammation

The activation of the neuro-inflammatory response has been linked to ageing and NDDs, and its connection to unrepaired genome damage has been recently postulated [230]. Several studies have shown that factors like NF-κB are activated in response to stroke [231], kainite-induced seizures [232] and Aβ-treated fetal rat cortical neurons [233]; however, the functional role of RelA/NF-κB in neuronal death/survival remains unclear [234]. As mentioned before, DDR-induced signaling activates the ATM-p53 and ATM-IKKα/β-interferon (IFN)-β signaling pathways. The latter involves the expression of IFN-inducible IFI16 gene, which stimulates STING-dependent activation of IFN-β, activation of IFI16 inflammasome, and the production of pro-inflammatory cytokines (e.g., IL-1β and IL-18). Because DDR activates the expression of the IFI16 gene, thereby, increased levels of the IFI16 protein enhances p53-mediated transcriptional activation of many genes including the ones involved in DNA repair. At the same time, persistent DDR-mediated neuro-inflammation contributes to ageing-associated progressive neuronal dysfunctions. However, a complete understanding of how the accumulation of DNA damage, and DDR in various NDDs contribute to inflammation requires further investigations.

5.3. Cell cycle re-entry of neurons – role of unrepaired DNA strand breaks?

Neuronal cycle re-activation was first linked to DNA damage in 2004, when Kruman et al. [235] found that DNA damage causing agents including irradiation, etoposide, Hcy, methotrexate and Aβ1-42 were able to induce cell cycle re-entry followed by neuronal apoptosis, in which p53 played a critical regulatory role [235]. Post-mitotic neurons remain in a quiescent G₀ state and are normally incapable of re-entering cell cycle. However, aberrant cell cycle re-entry has been extensively observed in several NDDs, supported by the expression of cyclins (e.g., Cyclin B1 and D), cyclin-dependent kinases (e.g., CDK3 and 4) and other cell cycle mediators (e.g., Cdc2) in AD, PD, ALS and HD [58, 236–238]. Growing evidence indicate that the re-entry into the cell cycle leads to cell death instead of neuronal proliferation. The cell cycle-regulated protein kinase Cdc2 expressed in rat post-mitotic granular neurons mediates apoptosis, suggesting that cell cycle re-entry may function as a common pathological basis for neuronal cell death [239]. Despite the mystery of how cell cycle re-entry induces neuronal apoptosis, DNA damage has been recognized as a trigger of unscheduled cell cycle re-entry. Consistently, an aberrant cell cycle with accumulated DNA damage is frequently observed in cultured cells or mouse models of neurodegeneration [240]. However, the mechanism of DNA damage-activated cell cycle in post-mitotic neuronal cells remains unclear. It has been proposed that neurons re-enter the cell cycle in order to repair DSBs via HDR [235].