Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.     

Contribution of the Atm protein to maintaining cellular homeostasis evidenced by continuous activation of the AP-1 pathway in Atm-deficient brains

Maintenance of genome stability is essential for keeping cellular homeostasis. The DNA damage response is a central component in maintaining genome integrity. Among of the most cytotoxic DNA lesions are double strand breaks (DSBs) caused by ionizing radiation or radiomimetic chemicals. ATM is missing or inactivated in patients with ataxia-telangiectasia. Ataxia-telangiectasia patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Additional features are immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. Disruption of the mouse Atm locus creates a murine model of ataxia-telangiectasia that exhibits most of the clinical features of the human disease but very mild neuronal abnormality. The ATM protein is a multifunctional protein kinase, which serves as a master regulator of cellular responses to DSBs. There is growing evidence that ATM may be involved in addition to the DSB response in other processes that maintain processes in cellular homeostasis. For example, mounting evidence points to increased oxidative stress in the absence of ATM. Here we report that the AP-1 pathway is constantly active in the brains of Atm-deficient mice not treated with DNA damaging agents. A canonical activation (increased phosphorylation of mitogen-activated protein kinase kinase-4, c-Jun N-terminal kinase, and c-Jun) of the AP-1 pathway was found in Atm-deficient cerebra, whereas induction of the AP-1 pathway in Atm-deficient cerebella is likely to mediate elevated expression of c-Fos and c-Jun. Although Atm(+/+) mice are capable of responding to ionizing radiation by activating stress responses such as the AP-1 pathway, Atm-deficient mice display higher basal AP-1 activity but gradually lose their ability to activate AP-1 DNA-binding activity in response to ionizing radiation. Our results further demonstrate that inactivation of the ATM gene results in a state of constant stress.     

Nuclear ataxia-telangiectasia mutated (ATM) mediates the cellular response to DNA double strand breaks in human neuron-like cells

The protein kinase ATM (ataxia-telangiectasia mutated) activates the cellular response to double strand breaks (DSBs), a highly cytotoxic DNA lesion. ATM is activated by DSBs and in turn phosphorylates key players in numerous damage response pathways. ATM is missing or inactivated in the autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterized by neuronal degeneration, immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. The predominant symptom of A-T is a progressive loss of movement coordination due to ongoing degeneration of the cerebellar cortex and peripheral neuropathy. A major deficiency in understanding A-T is the lack of information on the role of ATM in neurons. It is unclear whether the ATM-mediated DSB response operates in these cells similarly to proliferating cells. Furthermore, ATM was reported to be cytoplasmic in neurons and suggested to function in these cells in capacities other than the DNA damage response. Recently we obtained genetic molecular evidence that the neuronal degeneration in A-T does result from defective DNA damage response. We therefore undertook to investigate this response in a model system of human neuron-like cells (NLCs) obtained by neuronal differentiation in culture. ATM was largely nuclear in NLCs, and their ATM-mediated responses to DSBs were similar to those of proliferating cells. Knocking down ATM did not interfere with neuronal differentiation but abolished ATM-mediated damage responses in NLCs. We concluded that nuclear ATM mediates the DSB response in NLCs similarly to in proliferating cells. Attempts to understand the neurodegeneration in A-T should be directed to investigating the DSB response in the nervous system.     

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.     

Accumulation of DNA damage and reduced levels of nicotine adenine dinucleotide in the brains of Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a human genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype, in which a major neurological feature is progressive ataxia due to degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical and cellular features of the human disease, but the neurological phenotype is barely expressed. We present evidence for the accumulation of DNA strand breaks in the brains of Atm(-/-), supporting the notion that ATM plays a major role in maintaining genomic stability. We also show a perturbation of the steady state levels of pyridine nucleotides. There is a significant decrease in both the reduced and the oxidized forms of NAD and in the total levels of NADP(T) and NADP(+) in the brains of Atm(-/-) mice. The changes in NAD(T), NADH, NAD(+), NADP(T), and NADP(+) were progressive and observed primarily in the cerebellum of 4-month-old Atm(-/-) mice. Higher rates of mitochondrial respiration were also recorded in 4-month-old Atm(-/-) cerebella. Taken together, our findings support the hypothesis that absence of functional ATM results in continuous stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.     

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H₂O₂, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm^−/− mice had significantly less hepatic fibrosis than Atm^+/+ or Atm^+/− mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.     

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H₂O₂, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm^?/? mice had significantly less hepatic fibrosis than Atm^+/+ or Atm^+/? mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.     

MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

A new role for ATM: regulating mitochondrial function and mitophagy

The various pathologies in ataxia telangiectasia (A-T) patients including T-cell lymphomagenesis have been attributed to defects in the DNA damage response pathway because ATM, the gene mutated in this disease, is a key mediator of this process. Analysis of Atm-deficient thymocytes in mice reveals that the absence of this gene results in altered mitochondrial homeostasis, a phenomenon that appears to result from abnormal mitophagy engagement. Interestingly, allelic loss of the autophagic gene Becn1 delays tumorigenesis in Atm-null mice presumably by reversing the mitochondrial abnormalities and not by improving the DNA damage response (DDR) pathway. Thus, ATM plays a critical role in modulating mitochondrial homeostasis perhaps by regulating mitophagy.     

Investigation of the functional link between ATM and NBS1 in the DNA damage response in the mouse cerebellum

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are related genomic instability syndromes characterized by neurological deficits. The NBS1 protein that is defective in NBS is a component of the Mre11/RAD50/NBS1 (MRN) complex, which plays a major role in the early phase of the complex cellular response to double strand breaks (DSBs) in the DNA. Among others, Mre11/RAD50/NBS1 is required for timely activation of the protein kinase ATM (A-T, mutated), which is missing or inactivated in patients with A-T. Understanding the molecular pathology of A-T, primarily its cardinal symptom, cerebellar degeneration, requires investigation of the DSB response in cerebellar neurons, particularly Purkinje cells, which are the first to be lost in A-T patients. Cerebellar cultures derived from mice with different mutations in DNA damage response genes is a useful experimental system to study malfunctioning of the damage response in the nervous system. To clarify the interrelations between murine Nbs1 and Atm, we generated a mouse strain with specific disruption of the Nbs1 gene in the central nervous system on the background of general Atm deficiency (Nbs1-CNS-Δ//Atm(-/-)). This genotype exacerbated several features of both conditions and led to a markedly reduced life span, dramatic decline in the number of cerebellar granule neurons with considerable cerebellar disorganization, abolishment of the white matter, severe reduction in glial cell proliferation, and delayed DSB repair in cerebellar tissue. Combined loss of Nbs1 and Atm in the CNS significantly abrogated the DSB response compared with the single mutation genotypes. Importantly, the data indicate that Atm has cellular roles not regulated by Nbs1 in the murine cerebellum.     

Mechanisms of non-canonical activation of ataxia telangiectasia mutated

ATM is a master regulator of the cellular response to DNA damage. The classical mechanism of ATM activation involves its monomerization in response to DNA double-strand breaks, resulting in ATM-dependent phosphorylation of more than a thousand substrates required for cell cycle progression, DNA repair, and apoptosis. Here, new experimental evidence for non-canonical mechanisms of ATM activation in response to stimuli distinct from DNA double-strand breaks is discussed. It includes cytoskeletal changes, chromatin modifications, RNA–DNA hybrids, and DNA single-strand breaks. Noncanonical ATM activation may be important for the pathology of the multisystemic disease Ataxia Telangiectasia.     

ATM, the Mre11/Rad50/Nbs1 complex, and topoisomerase I are concentrated in the nucleus of Purkinje neurons in the juvenile human brain

The genetic disease ataxia telangiectasia (AT) results from mutations in the ataxia telangiectasia mutated (ATM) gene. AT patients develop a progressive degeneration of cerebellar Purkinje neurons. Surprisingly, while ATM plays a criticial role in the cellular reponse to DNA damage, previous studies have localized ATM to the cytoplasm of rodent and human Purkinje neurons. Here we show that ATM is primarily localized to the nucleus in cerebellar Purkinje neurons in postmortem human brain tissue samples, although some light cytoplasmic ATM staining was also observed. No ATM staining was observed in brain tissue samples from AT patients, verifying the specificity of the antibody. We also found that antibodies against components of the Mre11/Rad50/Nbs1 (MRN) complex showed strong staining in Purkinje cell nuclei. However, while ATM is present in both the nucleoplasm and nucleolus, MRN proteins are excluded from the nucleolus. We also observed very high levels of topoisomerase 1 (TOP1) in the nucleus, and specifically the nucleolus, of human Purkinje neurons. Our results have direct implications for understanding the mechanisms of neurodegeneration in AT and AT-like disorder.     

Genotype-phenotype relationships in ataxia-telangiectasia and variants.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition. A-T cells are sensitive to ionizing radiation and radiomimetic chemicals and fail to activate cell-cycle checkpoints after treatment with these agents. The responsible gene, ATM, encodes a large protein kinase with a phosphatidylinositol 3-kinase-like domain. The typical A-T phenotype is caused, in most cases, by null ATM alleles that truncate or severely destabilize the ATM protein. Rare patients with milder manifestations of the clinical or cellular characteristics of the disease have been reported and have been designated "A-T variants." A special variant form of A-T is A-TFresno, which combines a typical A-T phenotype with microcephaly and mental retardation. The possible association of these syndromes with ATM is both important for understanding their molecular basis and essential for counseling and diagnostic purposes. We quantified ATM-protein levels in six A-T variants, and we searched their ATM genes for mutations. Cell lines from these patients exhibited considerable variability in radiosensitivity while showing the typical radioresistant DNA synthesis of A-T cells. Unlike classical A-T patients, these patients exhibited 1%-17% of the normal level of ATM. The underlying ATM genotypes were either homozygous for mutations expected to produce mild phenotypes or compound heterozygotes for a mild and a severe mutation. An A-TFresno cell line was found devoid of the ATM protein and homozygous for a severe ATM mutation. We conclude that certain "A-T variant" phenotypes represent ATM mutations, including some of those without telangiectasia. Our findings extend the range of phenotypes associated with ATM mutations.     

DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture

Significant progress has been made in recent years in elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. Much of our understanding of the mechanisms involved in cellular DNA damage response pathways has come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. Ataxia-telangiectasia mutated (ATM), the gene mutated in the disorder ataxia-telangiectasia, codes for a protein kinase that is a central mediator of responses to DNA double-strand breaks (DSB) in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the response of the cell to the DNA damage. We recently developed a novel system to create DNA DSBs at defined endogenous sites in the human genome and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation. Results from this system showed the functional importance of ATM kinase activity and phosphorylation in the response to DSBs and supported a model in which ordered chromatin structure changes that occur after DNA breakage and that depend on functional NBS1 and ATM facilitate DNA DSB repair. Insights about these pathways provide us with opportunities to develop new approaches to benefit patients. Examples and opportunities for developing inhibitors that act as sensitizers to chemotherapy or radiation therapy or activators that could improve responses to cellular stresses, such as oxidative damage, are discussed. Relevant to the latter, we have shown benefits of an ATM activator in disease settings ranging from metabolic syndrome to cancer prevention.     

A multifaceted role for ATM in genome maintenance

The pleiotropic nature of the clinical phenotypes of patients with ataxia-telangiectasia (A-T)--which encompass cerebellar degeneration (leading to ataxia), gonadal atrophy, and cancer predisposition--suggests multiple functions of the gene responsible for the disease. The ataxia-telangiectasia mutated gene product (ATM), whose loss of function is responsible for ataxia-telangiectasia, is a protein kinase that interacts with several substrates and is implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control and telomere maintenance. This review focuses on the critical roles that ATM appears to play in cell-cycle checkpoints, DNA repair, telomere metabolism and oxidative stress, indicating how defects in these processes might lead to ataxia-telangiectasia.     

CHFR is important for the survival of male premeiotic germ cells

DNA damage response is required for male fertility. DNA damage repair mediates recombination between homologous chromosomes in meiotic prophase, which is essential for proper chromosome segregation during meiotic division. Interestingly, some DNA damage response proteins are also required for the survival of premeiotic germ cells, but their roles in these cells are still unclear. CHFR was recently shown to participate in DNA damage response, but it remains to be established if CHFR is required for male fertility. In this study, we characterized Chfr knockout male mice and found that around 30% of them were infertile. The onset of spermatogenesis was delayed and there was significant increase in apoptosis in premeiotic germ cells. This resulted in complete loss of germ cells in testes in 3 months and azoospermia in these mice. We further demonstrated that ATM activation was compromised in the testes of these mice. Therefore, CHFR is important for the survival of male premeiotic germ cells, which is likely through maintaining genomic stability in spermatogonial stem cells.     

Genetic interactions of the Aspergillus nidulans atmAATM homolog with different components of the DNA damage response pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for DeltaatmA, DeltauvsB, DeltachkA, and DeltachkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APC1), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.     

Differential localization of ATM is correlated with activation of distinct downstream signaling pathways

ATM, the gene mutated in the genetic disease ataxia telangiectasia (AT), is a well-known protein involved in the DNA double-strand break response, where it plays an important role in sensing damage and signaling to DNA repair machinery and cell cycle checkpoints. However, a number of recent papers, including ours have found that ATM also plays important roles outside of the nucleus, which may explain some of the phenotypic features seen in AT patients. Our research into mechanisms of TSC2 regulation helped uncover a pathway upstream of TSC2 that is regulated by cytoplasmic ATM in response to ROS initiated by ATM activation of LKB1 and AMPK. We found that TSC2 activation results in mTORC1 repression and subsequent induction of autophagy. Elucidation of this stress response pathway provides a molecular mechanism for ATM signaling in the cytoplasm, and lays the groundwork for further studies on how ATM activity is regulated beyond DNA damage in different cellular compartments.