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Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in NBN or ATM result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show in vivo consequences of conditional inactivation of both genes in neural stem/progenitor cells using Nestin-Cre mice. Genetic inactivation of Atm in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with Nbn deletion was prevented by simultaneous inactivation of Atm in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.

The MRN complex (Mre11/RAD50/NBS1) and ATM (ataxia telangiectasia, mutated) are critical for the cellular response to DNA damage. ATM disruption causes ataxia telangiectasia (A-T), while MRN dysfunction can lead to A-T-like disease (ATLD) or Nijmegen breakage syndrome (NBS). Neuropathology is a hallmark of these diseases, whereby neurodegeneration occurs in A-T and ATLD while microcephaly characterizes NBS. To understand the contrasting neuropathology resulting from Mre11 or Nbs1 hypomorphic mutations, we analyzed neural tissue from Mre11(ATLD1/ATLD1) and Nbs1(DeltaB/DeltaB) mice after genotoxic stress. We found a pronounced resistance to DNA damage-induced apoptosis after ionizing radiation or DNA ligase IV (Lig4) loss in the Mre11(ATLD1/ATLD1) nervous system that was associated with defective Atm activation and phosphorylation of its substrates Chk2 and p53. Conversely, DNA damage-induced Atm phosphorylation was defective in Nbs1(DeltaB/DeltaB) neural tissue, although apoptosis occurred normally. We also conditionally disrupted Lig4 throughout the nervous system using Nestin-cre (Lig4(Nes-Cre)), and while viable, these mice showed pronounced microcephaly and a prominent age-related accumulation of DNA damage throughout the brain. Either Atm-/- or Mre11(ATLD1/ATLD1) genetic backgrounds, but not Nbs1(DeltaB/DeltaB), rescued Lig4(Nes-Cre) microcephaly. Thus, DNA damage signaling in the nervous system is different between ATLD and NBS and likely explains their respective neuropathology.

Apoptosis occurs in the nervous system during normal development, but can also be induced by disease or after exogenous insults such as DNA damage. Depending on the magnitude and timing of the stimulus, apoptosis can be sporadic or widespread. Because of the highly ordered structure of the nervous system, immunohistochemical detection approaches provide a wealth of information about the spatiotemporal nature of apoptosis in this tissue. Therefore, immunohistochemistry offers valuable insights into the neuropathology of disease processes and the identification of specific cell populations that are susceptible to apoptosis. In this chapter, we outline standard approaches for the immunohistochemical analysis of apoptosis in the nervous system, with an emphasis on methodology useful for studies involving DNA damage-induced apoptosis.

Bcl2 is the founding member of a family of proteins that regulates apoptosis by controlling mitochondrial outer membrane integrity. In this issue of Molecular Cell, Wang et al. (2008) propose another function for Bcl2: the inhibition of DNA repair by nonhomologous end-joining.

Scythe (BAT3; HLA-B associated transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (apoptosis-inducing factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe(-/-) cells, which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe(-/-) cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF.

The chromosomal instability syndromes Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) share many overlapping phenotypes, including cancer predisposition, radiation sensitivity, cell-cycle checkpoint defects, immunodeficiency, and gonadal dysfunction. The NBS protein Nbs1 is not only a downstream target of AT mutated (ATM) kinase but also acts upstream, promoting optimal ATM activation, ATM recruitment to breaks, and ATM accessibility to substrates. By reconstituting Nbs1 knockout mice with bacterial artificial chromosomes, we have assessed the contribution of distinct regions of Nbs1 to the ATM-dependent DNA damage response. We find that T cell and oocyte development, as well as DNA damage-induced G2/M and S phase checkpoint arrest and radiation survival are dependent on the N-terminal forkhead-associated domain, but not on the principal residues phosphorylated by ATM (S278 and S343) or on the evolutionarily conserved C-terminal region of Nbs1. However, the C-terminal region regulates irradiation-induced apoptosis. These studies provide insight into the complex interplay between Nbs1 and ATM in the DNA damage response.

Scythe (BAT3 [HLA-B-associated transcript 3]) is a nuclear protein that has been implicated in apoptosis, as it can modulate Reaper, a central apoptotic regulator in Drosophila melanogaster. While Scythe can markedly affect Reaper-dependent apoptosis in Xenopus laevis cell extracts, the function of Scythe in mammals is unknown. Here, we report that inactivation of Scythe in the mouse results in lethality associated with pronounced developmental defects in the lung, kidney, and brain. In all cases, these developmental defects were associated with dysregulation of apoptosis and cellular proliferation. Scythe-/- cells were also more resistant to apoptosis induced by menadione and thapsigargin. These data show that Scythe is critical for viability and normal development, probably via regulation of programmed cell death and cellular proliferation.

Puma encodes a BH3-only protein that is induced by the p53 tumor suppressor and other apoptotic stimuli. To assess its physiological role in apoptosis, we generated Puma knockout mice by gene targeting. Here we report that Puma is essential for hematopoietic cell death triggered by ionizing radiation (IR), deregulated c-Myc expression, and cytokine withdrawal. Puma is also required for IR-induced death throughout the developing nervous system and accounts for nearly all of the apoptotic activity attributed to p53 under these conditions. These findings establish Puma as a principal mediator of cell death in response to diverse apoptotic signals, implicating Puma as a likely tumor suppressor.

We show here that a zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug(-/-) mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug(-/-) mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage.

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. [ABSTRACT FROM AUTHOR]

Abstract: The repair of DNA damage is essential for the prevention of disease. The DNA double-strand break (DSB) is a particularly hazardous lesion. DNA DSBs activate a coordinated cellular response involving cell cycle checkpoint activation and repair of the DNA break, or alternatively apoptosis. In the nervous system the inability to respond to DNA DSBs may lead to neurodegenerative disease or brain tumors. Therefore, understanding the DNA DSB response mechanism in the nervous system is of high importance for developing new treatments for neurodegeneration and cancer. In this regard, the use of mouse models represents an important approach for advancing our understanding of the biology of the DNA damage response in the nervous system. [Copyright &y& Elsevier]

DNA double-strand breaks (DSBs) require a coordinated molecular response to ensure cellular or organism survival. Many factors required for the DSB response, including those involved in non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are essential during nervous system development. Additionally, human syndromes resulting from defective responses to DNA damage often feature overt neuropathology such as neurodegeneration. Thus, appropriate responses to DSBs are critical for the normal development and maintenance of the nervous system. [Copyright &y& Elsevier]

The article presents an abstract of the study "ATM: A Potent Modulator of Neuronal Apoptosis," by Peter J. McKinnon, which will be presented at the 30th annual meeting of the American Society for Neurochemistry to be held from March 14-17, 1999 in New Orleans, Louisiana. To find out about the function of Ataxia Telangiectasia Mutated (ATM) in the nervous system, the researchers generated a mouse model of A-T by inactivation of ATM.