Ataxia Telangiectasia is a rare, multi system disease caused by ATM kinase deficiency. Atm knockout mice recapitulate premature aging, immunodeficiency, cancer predisposition, growth retardation and motor defects but not cerebellar neurodegeneration and ataxia. We explored if Atm loss is responsible of skeletal muscle defects by investigating myofiber morphology, oxidative/glycolytic activity, myocyte ultrastructural architecture and neuromuscular junctions.Atm knockout mice showed reduced muscle and fiber size. Atrophy, protein synthesis impairment and switch from glycolytic to oxidative fibers were detected along with an increase of slow and fast myosin expression levels in tibialis anterior and solei muscles isolated from Atm knockout mice. Transmission electron microscopy of tibialis anterior revealed misalignments of z-lines and sarcomeres and mitochondria abnormalities, that were associated with increase of reactive oxygen species. Moreover, neuromuscular junctions appeared larger and more complex compared to Atm wild-type mice, but with preserved presynaptic terminals.In conclusion, we report for the first time that Atm knockout mice have clear morphological skeletal muscle defects that will be relevant to investigate oxidative stress response, motor alteration and the interplay with peripheral nervous system in Ataxia Telangiectasia.

© 2019. Published by The Company of Biologists Ltd.

Ataxia-telangiectasia (AT) is a severe autosomal recessive orphan disease characterized by a number of peculiar clinical manifestations. Genetic diagnosis of AT is complicated due to a large size of the causative gene, ATM. We used next-generation sequencing (NGS) technology for the ATM analysis in 17 children with the clinical diagnosis of AT. Biallelic mutations in the ATM gene were identified in all studied subjects; these lesions included one large gene rearrangement, which was reliably detected by NGS and validated by multiplex ligation-dependent probe amplification (MLPA). There was a pronounced founder effect, as 17 of 30 (57%) pathogenic ATM alleles in the patients of Slavic origin were represented by three recurrent mutations (c.5932G > T, c.450_453delTTCT, and c.1564_1565delGA). These data have to be taken into account while considering the genetic diagnosis and screening for ataxia-telangiectasia syndrome.

Copyright © 2019 Elsevier Masson SAS. All rights reserved.

The ataxia-telangiectasia mutated (ATM) gene regulates DNA damage repair, oxidative stress, and mitochondrial processes. In this issue, Chow et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201806197) connects ATM's oxidative stress response functions to the sensing of metabolic ATP energetics distinctively important in high energy-demanding Purkinje brain cells, which could explain the most distinct A-T patient feature, cerebellar ataxia.

© 2019 Schlacher.

Respiratory disease is a major cause of morbidity and mortality in patients with ataxia-telangiectasia (A-T) who are prone to recurrent sinopulmonary infections, bronchiectasis, pulmonary fibrosis, and pulmonary failure. Upper airway infections are common in patients and S. pneumoniae is associated with these infections. We demonstrate here that the upper airway microbiome in patients with A-T is different from that to healthy controls, with S. pneumoniae detected largely in patients only. Patient-specific airway epithelial cells and differentiated air-liquid interface cultures derived from these were hypersensitive to infection which was at least in part due to oxidative damage since it was partially reversed by catalase. We also observed increased levels of the pro-inflammatory cytokines IL-8 and TNF-α (inflammasome-independent) and a decreased level of the inflammasome-dependent cytokine IL-β in patient cells. Further investigation revealed that the ASC-Caspase 1 signalling pathway was defective in A-T airway epithelial cells. These data suggest that the heightened susceptibility of these cells to S. pneumoniae infection is due to both increased oxidative damage and a defect in inflammasome activation, and has implications for lung disease in these patients.

PURPOSE:

MYC transcription factor has critical roles in cell growth, proliferation, metabolism, differentiation, transformation and angiogenesis. MYC overexpression is seen in about 15% of breast cancers and linked to aggressive phenotypes. MYC overexpression also induces oxidative stress and replication stress in cells. ATM signalling and ATR-mediated signalling are critical for MYC-induced DNA damage response. Whether ATM and ATR expressions influence clinical outcomes in MYC overexpressed breast cancers is unknown.

METHODS:

We investigated ATM, ATR and MYC at the transcriptional level [Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1950)] and at the protein level in the Nottingham series comprising 1650 breast tumours. We correlated ATM, ATR and MYC expressions to clinicopathological features and survival outcomes.

RESULTS:

In MYC over expressed tumours, high ATR or low ATM levels were associated with aggressive breast cancer features such as higher tumour grade, de-differentiation, pleomorphism, high mitotic index, high-risk Nottingham Prognostic Index, triple negative and basal-like breast cancers (all adjusted p values < 0.05). Tumours with low ATM or high ATR levels in conjunction with MYC overexpression also have worse overall breast cancer-specific survival (BCSS) (p value < 0.05).

CONCLUSIONS:

We conclude that ATR/ATM-directed stratification and personalisation of therapy may be feasible in MYC overexpressed breast cancer.

The functional consequences of missense variants are often difficult to predict. This becomes especially relevant when DNA sequence changes are used to determine a diagnosis or prognosis. To analyze the consequences of 12 missense variants in patients with mild forms of ataxia-telangiectasia (A-T), we employed site-directed mutagenesis of ataxia-telangiectasia mutated (ATM) cDNA followed by stable transfections into a single A-T cell line to isolate the effects of each allele on the cellular phenotype. After induction of the transfected cells with CdCl2, we monitored for successful ATM transcription and subsequently assessed: 1) intracellular ATM protein levels; 2) ionizing radiation (IR)-induced ATM kinase activity; and 3) cellular radiosensitivity. We then calculated SIFT and PolyPhen scores for the missense changes. Nine variants produced little or no correction of the A-T cellular phenotype and were interpreted to be ATM mutations; SIFT/PolyPhen scores supported this. Three variants corrected the cellular phenotype, suggesting that they represented benign variants or polymorphisms. SIFT and PolyPhen scores supported the functional analyses for one of these variants (c.1709T>C); the other two were predicted to be "not tolerated" (c.6188G>A and c.6325T>G) and were classified as "operationally neutral." Genotype/phenotype relationships were compared: three deleterious missense variants were associated with an increased risk of cancer (c.6679C>T, c.7271T>G, and c.8494C>T). In situ mutagenesis represents an effective experimental approach for distinguishing deleterious missense mutations from benign or operationally neutral missense variants.

Copyright 2008 Wiley-Liss, Inc.

Ataxia-telangiectasia mutated (ATM) is the gene mutated in the cancer-predisposing disorder ataxia-telangiectasia (A-T). We modeled ATM sequence variants identified in UK A-T patients to determine the stability and kinase activity of the resulting proteins as well as the distribution of these mutations across the coding region. Of 20 missense changes modeled, 10 proteins showed ATM kinase activity and 10 showed none. In the majority of cases the mutant ATM protein was unstable, although this was variable. Reduction in ATM kinase activity can result either from the presence of low levels of unstable mutant protein with relatively normal specific kinase activity or from stable mutant protein with deficient ATM kinase activation. Indeed, ATM mutant proteins without kinase activity toward downstream targets were still able to autophosphorylate on serine 1981, although in a much less efficient manner, suggesting that this was not sufficient for ATM activation. In terms of function, green fluorescent protein (GFP)-tagged kinase inactive ATM proteins could form ionizing radiation (IR)-induced foci (IRIF), at least temporarily, which colocalized with the DNA double-strand break (DSB) marker gammaH2AX. Consistent with this, both kinase active and inactive mutant ATM proteins were able to interfere with phosphorylation of targets by endogenous ATM. Since the majority of missense mutations occurred C-terminal to aa1966, including all 10 mutations with absence of kinase activity, the implication was that mutations N-terminal to this, with exceptions, are less likely to result in loss of kinase activity and therefore, are less likely to be identified in A-T patients.

Ataxia‑telangiectasia (A‑T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM serine/threonine kinase (ATM) gene. Typically, it presents in early childhood with progressive cerebellar dysfunction, accompanied by immunodeficiency and oculocutaneous telangiectasia. In the present study, the clinical and genetic findings of a Chinese family affected with A‑T in two live siblings, the proband (II‑2) and his elder brother (II‑1), as well as a fetus (II‑3) were reported. General health, clinical neurological, electrophysiological (motor and sensory nerve conduction) and magnetic resonance imaging evaluations revealed that patients II‑1 and II‑2 had similar symptoms of ataxia, dysarthria, conjunctival hyperemia and elevated serum α‑fetoprotein, whereas patient II‑1 had earlier A‑T onset at 2 years old and more serious problems with movement and intelligence. Targeted sequencing followed by Sanger sequencing revealed that these two patients carried the compound heterozygotes of a novel nonsense mutation c.5170G>T (p.Glu1724Ter) and a known nonsense mutation c.748C>T (p.Arg250Ter) in the ATM gene. Each mutation was inherited from an asymptomatic parent, which therefore confirmed the diagnosis of A‑T. Given this, proband's mother performed prenatal diagnosis in her third pregnancy. Unfortunately, the fetus had the same causal mutations as its siblings and the pregnancy was terminated. The findings of the present study expanded the mutation spectrum of the ATM gene and may help in understanding the genetic basis of A‑T, in order to guide genetic counseling and prenatal diagnosis.

BACKGROUND:

Ataxia telangiectasia (A-T) is a neurodegenerative disorder. While patients with classic A-T generally die in their 20s, some patients with variant A-T, who have residual ataxia-telangiectasia mutated (ATM) kinase activity, have a milder phenotype. We noticed two commonly occurring ATM mutations that appeared to be associated with prolonged survival and decided to study patients carrying one of these mutations.

METHODS:

Data were retrospectively collected from the Dutch, Italian, German and French A-T cohorts. To supplement these data, we searched the literature for patients with identical genotypes.

RESULTS:

This study included 35 patients who were homozygous or compound heterozygous for the ATM c.3576G>A; p.(Ser1135_Lys1192del58) mutation and 24 patients who were compound heterozygous for the ATM c.8147T>C; p.(Val2716Ala) mutation. Compared with 51 patients with classic A-T from the Dutch cohort, patients with ATM c.3576G>A had a longer survival and were less likely to develop cancer, respiratory disease or immunodeficiency. This was also true for patients with ATM c.8147T>C, who additionally became wheelchair users later in life and had fewer telangiectasias. The oldest patient with A-T reported so far was a 78-year-old patient who was compound heterozygous for ATM c.8147T>C. ATM kinase activity was demonstrated in cells from all patients tested with the ATM c.8147T>C mutant protein and only at a low level in some patients with ATM c.3576G>A.

CONCLUSION:

Compared with classic A-T, the presence of ATM c.3576G>A results in a milder classic phenotype. Patients with ATMc.8147T>C have a variant phenotype with prolonged survival, which in exceptional cases may approach a near-normal lifespan.

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. We found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATMdimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing-deficient C2991L mutant revealed that one function of ATMredox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATMcauses ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. We genetically separated the activation of ATM by DNA damage from that by oxidative stress using separation-of-function mutations. We found that deficient activation of ATM by the Mre11-Rad50-Nbs1 complex and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATMhad minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of a variant ATM incapable of activation by oxidative stress resulted in widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATMactivation and its effects on cellular metabolism and DNA damage responses in human cells and implicate ATM in the control of protein homeostasis.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys⁶³ (K63) linkage bound to DNA through a motif we called the "DNA-interacting patch" (DIP), which is composed of the adjacent residues Thr⁹, Lys¹¹, and Glu³⁴ Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile⁴⁴ patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient-derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition.

ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In the closed state, the PIKK regulatory domain blocks the peptide substrate-binding site, suggesting that this conformation may represent an inactive or basally active enzyme. The active site is held in this closed conformation by interaction with a long helical hairpin in the TRD3 (tetratricopeptide repeats domain 3) domain of the symmetry-related molecule. The open dimer has two protomers with only a limited contact interface, and it lacks the intermolecular interactions that block the peptide-binding site in the closed dimer. This suggests that the open conformation may be more active. The ATMstructure shows the detailed topology of the regulator-interacting N-terminal helical solenoid. The ATM conformational dynamics shown by the structures represent an important step in understanding the enzyme regulation.

The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and 15N-relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the head-group region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe-93 and Trp-96) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations these aggregates are typically used, but impairs interactions with small unilamellar vesicles (SUVs), usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM maybe function as membrane-anchoring unit for other biomolecules.

Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute ex vivo inhibition of ATM protein kinase significantly decreased mitochondrial electron transfer chain complex I-mediated oxidative phosphorylation rate but did not decrease coupling efficiency or oxygen consumption rate during β-oxidation. Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.

Ataxia-telangiectasia (A-T) is a rare autosomal recessive neurodegenerative disorder characterized by progressive cerebellar ataxia, ocular apraxia, immunodeficiency, telangiectasia, elevated serum α-fetoprotein concentration, radiosensitivity and cancer predisposition. Classical A-T is caused by biallelic variants on ATM (ataxia telangiectasia mutated) gene, leading to a loss of function of the protein kinase ATM, involved in DNA damage repair. Atypical presentations can be found in A-T-like disease or in Nijmegen breakage syndrome, caused by deficiency of mre11 or nibrin proteins, respectively. In this report, we present the genetic characterization of a 4-year-old female with clinical diagnosis of A-T. Next-generation sequencing (NGS) revealed two novel heterozygous mutations in the ATM gene: a single-nucleotide variant (SNV) at exon 47 (NM_000051.3:c.6899G > C; p.Trp2300Ser) and ∼90 kb genomic duplication spanning exons 17-61, NG_009830.1:g.(41245_49339)_(137044_147250)dup. These findings were validated by Sanger sequencing and MLPA (multiplex ligation-dependent probe amplification) analysis respectively. Familial segregation study confirmed that the two variants are inherited, and the infant is a compound heterozygote. Thus, our study expands the spectrum of ATM pathogenic variants and demonstrates the utility of targeted NGS in the detection of copy number variation.

© 2019 John Wiley & Sons Ltd/University College London.

The ataxia-telangiectasia mutated (ATM) protein kinase is best known for its critical nuclear roles in the DNA damage response (DDR), cell cycle checkpoints, and the maintenance of gene stability. In this review, we highlight the multifaceted cytoplasmic functions of ATM in autophagy. We focused on the functions of ATM in nonselective autophagy in cancer. An Oncomine database analysis showed a tight association between ATM and autophagy in various cancers. In particular, its mechanisms in nonselective autophagy, those induced by ionizing radiation (IR), are illustrated in detail and involve the MAPK14 pathway, mTOR pathway, and Beclin1/PI3KIII complexes. Recently, an increasing number of studies revealed that autophagy could also be highly selective. We additionally emphasized the novel roles of ATM in selective autophagy, including mitophagy, pexophagy, and lipophagy. The regulation of these processes mainly involves ATM-PEX5, ATM-AMPK-TSC2-mTORC1-ULK1, PPM1D-ATM-MTOR, PINK I/Parkin, and NAD+/SIRT1. We aimed to provide new perspectives on the importance of ATM in the diverse field of autophagy. The intricate regulation of ATM in autophagy still requires further investigation, which would enhance our understanding of its role in cell dynamics and homeostasis. SIGNIFICANCE OF THE STUDY: Our review highlighted the multifaceted cytoplasmic functions of ATM on autophagy. First, we focused on the functions of ATM in nonselective autophagy within cancer especially those induced by IR, involving the MAPK14 pathway, mTOR pathway, and Beclin1/PI3KIII complexes. These provided a theoretical understanding of tumour radiosensitivity and chemosensitivity. In addition, we emphasized the novel roles of ATM in selective autophagy, including mitophagy, pexophagy, and lipophagy. This review provides new perspectives on the importance of ATM in the diverse field of autophagy, which would provide more information on its role in whole cell dynamics and homeostasis.

© 2019 John Wiley & Sons, Ltd.

Seckel syndrome (SS) is a rare spectrum of congenital severe microcephaly and dwarfism. One SS-causative gene is Ataxia Telangiectasiaand Rad3-Related Protein (ATR), and ATR (c.2101 A>G) mutation causes skipping of exon 9, resulting in a hypomorphic ATR defect. This mutation is considered the cause of an impaired response to DNA replication stress, the main function of ATR, contributing to the pathogenesis of microcephaly. However, the precise behavior and impact of this splicing defect in human neural progenitor cells (NPCs) is unclear. To address this, we established induced pluripotent stem cells (iPSCs) from fibroblasts carrying the ATR mutation and an isogenic ATR-corrected counterpart iPSC clone. SS-patient-derived iPSCs (SS-iPSCs) exhibited cell type-specific splicing; exon 9 was dominantly skipped in fibroblasts and iPSC-derived NPCs, but it was included in undifferentiated iPSCs and definitive endodermal cells. SS-iPSC-derived NPCs (SS-NPCs) showed distinct expression profiles from ATR non-mutated NPCs with negative enrichment of neuronal genesis-related gene sets. In SS-NPCs, abnormal mitotic spindles occurred more frequently than in gene-corrected counterparts, and the alignment of NPCs in the surface of the neurospheres was perturbed. Finally, we tested several splicing-modifying compounds and found that TG003, a CLK1 inhibitor, could pharmacologically rescue the exon 9 skipping in SS-NPCs. Treatment with TG003 restored the ATR kinase activity in SS-NPCs and decreased the frequency of abnormal mitotic events. In conclusion, our iPSC model revealed a novel effect of the ATR mutation in mitotic processes of NPCs and NPC-specific missplicing, accompanied by the recovery of neuronal defects using a splicing rectifier.