In mitotic cells, the cyclin-dependent kinase (CDK) subunit protein CKS1 regulates S phase entry by mediating degradation of the CDK inhibitor p27. Although mature neurons lack mitotic CDKs, we found that CKS1 was actively expressed in post-mitotic neurons of the adult hippocampus. Interestingly, Cks1 knockout (Cks1-/-) mice exhibited poor long-term memory, and diminished maintenance of long-term potentiation in the hippocampal circuits. Furthermore, there was neuronal accumulation of cofilin-actin rods or cofilin aggregates, which are associated with defective dendritic spine maturation and synaptic loss. We further demonstrated that it was the increased p27 level that activated cofilin by suppressing the RhoA kinase-mediated inhibitory phosphorylation of cofilin, resulting in the formation of cofilin aggregates in the Cks1-/- neuronal cells. Consistent with reports that the peptidyl-prolyl-isomerase PIN1 competes with CKS1 for p27 binding, we found that inhibition of PIN1 diminished the formation of cofilin aggregates through decreasing p27 levels, thereby activating RhoA and increasing cofilin phosphorylation. Our results revealed that CKS1 is involved in normal glutamatergic synapse development and dendritic spine maturation in adult hippocampus through modulating p27 stability.

Stem and differentiated cells frequently differ in their response to DNA damage, which can determine tissue sensitivity. By exploiting insight into the spatial arrangement of subdomains within the adult neural subventricular zone (SVZ) in vivo, we show distinct responses to ionising radiation (IR) between neural stem and progenitor cells. Further, we reveal different DNA damage responses between neonatal and adult neural stem cells (NSCs). Neural progenitors (transit amplifying cells and neuroblasts) but not NSCs (quiescent and activated) undergo apoptosis after 2 Gy IR. This response is cell type- rather than proliferation-dependent and does not appear to be driven by distinctions in DNA damage induction or repair capacity. Moreover, exposure to 2 Gy IR promotes proliferation arrest and differentiation in the adult SVZ. These 3 responses are ataxia telangiectasia mutated (ATM)-dependent and promote quiescent NSC (qNSC) activation, which does not occur in the subdomains that lack progenitors. Neuroblasts arising post-IR derive from activated qNSCs rather than irradiated progenitors, minimising damage compounded by replication or mitosis. We propose that rather than conferring sensitive cell death, apoptosis is a form of rapid cell death that serves to remove damaged progenitors and promote qNSC activation. Significantly, analysis of the neonatal (P5) SVZ reveals that although progenitors remain sensitive to apoptosis, they fail to efficiently arrest proliferation. Consequently, their repopulation occurs rapidly from irradiated progenitors rather than via qNSC activation.

Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible. Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50-500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.

Anticancer topoisomerase "poisons" exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5'-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1-/- cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1-/-/Tdp2-/-/- DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1-/- DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1-/- mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1-/- mice display an inability to rapidly repair DNA SSBs associated with Top1-DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1-/- mice treated with topotecan, a drug that increases levels of Top1-DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks.

Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5'-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5'-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5'-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3'-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5'-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3'-termini at a subset of single-strand breaks (SSBs), including those with 3'-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1(-/-)/Aptx(-/-) double knockout quiescent mouse astrocytes compared with Tdp1(-/-) or Aptx(-/-) single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1(-/-) and Tdp1(-/-)/Aptx(-/-) double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1(-/-), Aptx(-/-) or Tdp1(-/-)/Aptx(-/-) astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.

Defects in DNA single-strand break repair (SSBR) are linked with neurological dysfunction but the underlying mechanisms remain poorly understood. Here, we show that hyperactivity of the DNA strand break sensor protein Parp1 in mice in which the central SSBR protein Xrcc1 is conditionally deleted (Xrcc1Nes-Cre ) results in lethal seizures and shortened lifespan. Using electrophysiological recording and synaptic imaging approaches, we demonstrate that aberrant Parp1 activation triggers seizure-like activity in Xrcc1-defective hippocampus ex vivo and deregulated presynaptic calcium signalling in isolated hippocampal neurons in vitro. Moreover, we show that these defects are prevented by Parp1 inhibition or deletion and, in the case of Parp1 deletion, that the lifespan of Xrcc1Nes-Cre mice is greatly extended. This is the first demonstration that lethal seizures can be triggered by aberrant Parp1 activity at unrepaired SSBs, highlighting PARP inhibition as a possible therapeutic approach in hereditary neurological disease.

The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C)), ataxia telangiectasia mutated (Atm(-/-)) and double mutant Atm(-/-)/Lig4(Y288C) mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4(Y288C) mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4(Y288C) mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4(Y288C) mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.