A novel mouse model for ataxia-telangiectasia with a N-terminal mutation displays a behavioral defect and a low incidence of lymphoma but no increased oxidative burden.
ABSTRACT: Ataxia-telangiectasia (A-T) is a rare multi-system disorder caused by mutations in the ATM gene. Significant heterogeneity exists in the underlying genetic mutations and clinical phenotypes. A number of mouse models have been generated that harbor mutations in the distal region of the gene, and a recent study suggests the presence of residual ATM protein in the brain of one such model. These mice recapitulate many of the characteristics of A-T seen in humans, with the notable exception of neurodegeneration. In order to study how an N-terminal mutation affects the disease phenotype, we generated an inducible Atm mutant mouse model (Atm(tm1Mmpl/tm1Mmpl), referred to as A-T [M]) predicted to express only the first 62 amino acids of Atm. Cells derived from A-T [M] mutant mice exhibited reduced cellular proliferation and an altered DNA damage response, but surprisingly, showed no evidence of an oxidative imbalance. Examination of the A-T [M] animals revealed an altered immunophenotype consistent with A-T. In contrast to mice harboring C-terminal Atm mutations that disproportionately develop thymic lymphomas, A-T [M] mice developed lymphoma at a similar rate as human A-T patients. Morphological analyses of A-T [M] cerebella revealed no substantial cellular defects, similar to other models of A-T, although mice display behavioral defects consistent with cerebellar dysfunction. Overall, these results suggest that loss of Atm is not necessarily associated with an oxidized phenotype as has been previously proposed and that loss of ATM protein is not sufficient to induce cerebellar degeneration in mice.
Project description:Genomic instability resulting from defective DNA damage responses or repair causes several abnormalities, including progressive cerebellar ataxia, for which the molecular mechanisms are not well understood. Here, we report a new murine model of cerebellar ataxia resulting from concomitant inactivation of POLB and ATM. POLB is one of key enzymes for the repair of damaged or chemically modified bases, including methylated cytosine, but selective inactivation of Polb during neurogenesis affects only a subpopulation of cortical interneurons despite the accumulation of DNA damage throughout the brain. However, dual inactivation of Polb and Atm resulted in ataxia without significant neuropathological defects in the cerebellum. ATM is a protein kinase that responds to DNA strand breaks, and mutations in ATM are responsible for Ataxia Telangiectasia, which is characterized by progressive cerebellar ataxia. In the cerebella of mice deficient for both Polb and Atm, the most downregulated gene was Itpr1, likely because of misregulated DNA methylation cycle. ITPR1 is known to mediate calcium homeostasis, and ITPR1 mutations result in genetic diseases with cerebellar ataxia. Our data suggest that dysregulation of ITPR1 in the cerebellum could be one of contributing factors to progressive ataxia observed in human genomic instability syndromes.
Project description:Evidence suggests that astrocytes play key roles in structural and functional organization of neuronal circuits. To understand how astrocytes influence the physiopathology of cerebellar circuits, we cultured cells from cerebella of mice that lack the ATM gene. Mutations in ATM are causative of the human cerebellar degenerative disease ataxia-telangiectasia. Cerebellar cultures grown from Atm-/- mice had disrupted network synchronization, atrophied astrocytic arborizations, reduced autophagy levels, and higher numbers of synapses per neuron than wild-type cultures. Chimeric circuitries composed of wild-type astrocytes and Atm-/- neurons were indistinguishable from wild-type cultures. Adult cerebellar characterizations confirmed disrupted astrocyte morphology, increased GABAergic synaptic markers, and reduced autophagy in Atm-/- compared with wild-type mice. These results indicate that astrocytes can impact neuronal circuits at levels ranging from synaptic expression to global dynamics.
Project description:Ataxia Telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mitochondrial dysfunction that is thought to contribute to A-T pathogenesis. However, the molecular mechanism leading to mitochondrial dysfunction in A-T remains unclear. Here, we show that lack of ATM leads to reduced mitochondrial DNA (mtDNA) integrity and mitochondrial dysfunction, which are associated to defective mtDNA repair. While protein levels of mtDNA repair proteins are essentially normal, in the absence of ATM levels specifically of DNA ligase III (Lig3), the only DNA ligase working in mitochondria is reduced. The reduction of Lig3 is observed in different A-T patient cells, in brain and pre-B cells derived from ATM knockout mice as well as upon transient or stable knockdown of ATM. Furthermore, pharmacological inhibition of Lig3 in wild type cells phenocopies the mtDNA repair defects observed in A-T patient cells. As targeted deletion of LIG3 in the central nervous system causes debilitating ataxia in mice, reduced Lig3 protein levels and the consequent mtDNA repair defect may contribute to A-T neurodegeneration. A-T is thus the first disease characterized by diminished Lig3.
Project description:Hereditary deficiencies in DNA damage signaling are invariably associated with cancer predisposition, immunodeficiency, radiation sensitivity, gonadal abnormalities, premature aging, and tissue degeneration. ATM kinase has been established as a central player in DNA double-strand break repair and its deficiency causes ataxia telangiectasia, a rare, multi-system disease with no cure. So ATM represents a highly attractive target for the development of novel types of gene therapy or transplantation strategies. Atm tamoxifen-inducible mouse models were generated to explore whether Atm reconstitution is able to restore Atm function in an Atm-deficient background. Body weight, immunodeficiency, spermatogenesis, and radioresistance were recovered in transgenic mice within 1 month from Atm induction. Notably, life span was doubled after Atm restoration, mice were protected from thymoma and no cerebellar defects were observed. Atm signaling was functional after DNA damage in vivo and in vitro. In summary, we propose a new Atm mouse model to investigate novel therapeutic strategies for ATM activation in ataxia telangiectasia disease.
Project description:To assess the role of hepatocyte nuclear factor-3beta (HNF-3beta) in hepatocyte-specific gene transcription, we reported the characterization of the liver phenotype with transgenic mice in which the -3-kb transthyretin (TTR) promoter functioned to increase HNF-3beta expression. During breeding of the TTR-HNF-3beta transgenic mice we noticed that they displayed severe ataxia. In this study, we describe the analysis of our transgenic cerebellar phenotype and demonstrate that ectopic expression of HNF-3beta disrupted cerebellar morphogenesis and caused reduction in cerebellar size. In postnatal cerebellum, the HNF-3beta transgene expression pattern is colocalized to glial fibrillary acidic protein-positive cerebellar astrocytes and Bergmann glial cells. As a result of protracted expression, the transgenic cerebella are impaired in terms of astrocyte dispersal and formation of Bergmann glial cell processes. This caused a disruption in neuronal cell migration to the cortical laminar layers and Purkinje dendritic arbor maturation, thus leading to diminished foliation. Differential hybridization of cDNA arrays was used to identify altered expression of cerebellar genes, which is consistent with the observed defect in transgenic cerebellar morphogenesis and size as well as glial maturation. These include diminished expression of the brain lipid-binding protein, which is required for glial morphological differentiation, and the basic helix-loop-helix NeuroD/Beta2 and homeodomain Engrailed-2 transcription factors, which are required for normal cerebellar morphogenesis and foliation. Undetectable levels of ataxia telangiectasia (ATM), which is required for proper development of the Purkinje dendritic arbor, were found in postnatal transgenic cerebella. Furthermore, the transgenic cerebella displayed levels of insulin-like growth factor binding protein-1 elevated to 22 times greater than those measured for wild-type cerebella, an elevation consistent with the reduction in transgenic cerebellar size.
Project description:Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immune defects and predisposition to malignancies. A-T is caused by biallelic inactivation of the ATM gene, in most cases by frameshift or nonsense mutations. More rarely, ATM missense mutations with unknown consequences on ATM function are found, making definitive diagnosis more challenging. In this study, a series of 15 missense mutations, including 11 not previously reported, were identified in 16 patients with clinical diagnosis of A-T belonging to 14 families and 1 patient with atypical clinical features. ATM function was evaluated in patient lymphoblastoid cell lines by measuring H2AX and KAP1 phosphorylation in response to ionizing radiation, confirming the A-T diagnosis for 16 cases. In accordance with previous studies, we showed that missense mutations associated with A-T often lead to ATM protein underexpression (15 out of 16 cases). In addition, we demonstrated that most missense mutations lead to an abnormal cytoplasmic localization of ATM, correlated with its decreased expression. This new finding highlights ATM mislocalization as a new mechanism of ATM dysfunction, which may lead to therapeutic strategies for missense mutation associated A-T.
Project description:Ataxia telangiectasia (AT) is a progressive multisystem autosomal recessive disorder caused by mutations in the AT-mutated (ATM) gene. Early onset AT in children is characterized by cerebellar degeneration, leading to motor impairment. Lung disease and cancer are the two most common causes of death in AT patients. Accelerated thymic involution may contribute to the cancer, and recurrent and/or chronic respiratory infections may be a contributing factor to lung disease in AT. AT patients have fertility issues, are highly sensitive to ionizing radiation and they present oculocutaneous telangiectasia. Current treatments only slightly ameliorate disease symptoms; therapy that alters or reverses the course of the disease has not yet been discovered. Previously, we have shown that ATM-/- pigs, a novel model of AT, present with a loss of Purkinje cells, altered cerebellar cytoarchitecture and motor coordination deficits. ATM-/- porcine model not only recapitulates the neurological phenotype, but also other multifaceted clinical features of the human disease. Our current study shows that ATM-/- female pigs are infertile, with anatomical and functional signs of an immature reproductive system. Both male and female ATM-/- pigs show abnormal thymus structure with decreased cell cycle and apoptosis markers in the gland. Moreover, ATM-/- pigs have an altered immune system with decreased CD8+ and increased natural killer and CD4+CD8+ double-positive cells. Nevertheless, ATM-/- pigs manifest a deficient IgG response after a viral infection. Based on the neurological and peripheral phenotypes, the ATM-/- pig is a novel genetic model that may be used for therapeutic assessments and to identify pathomechanisms of this disease.
Project description:The disease ataxia-telangiectasia (A-T) has no cure and few treatment options. It is caused by mutations in the ATM kinase, which functions in the DNA-damage response and redox sensing. In addition to severe cerebellar degeneration, A-T pathology includes cancer predisposition, sterility, immune system dysfunction, and bone marrow abnormalities. These latter phenotypes are recapitulated in the ATM null (ATM(-/-)) mouse model of the disease. Since oxidative stress and mitochondrial dysfunction are implicated in A-T, we determined whether reducing mitochondrial reactive oxygen species (ROS) via overexpression of catalase targeted to mitochondria (mCAT) alleviates A-T-related pathology in ATM(-/-) mice. We found that mCAT has many beneficial effects in this context, including reduced propensity to develop thymic lymphoma, improved bone marrow hematopoiesis and macrophage differentiation in vitro, and partial rescue of memory T-cell developmental defects. Our results suggest that positive effects observed on cancer development may be linked to mCAT reducing mitochondrial ROS, lactate production, and TORC1 signaling in transforming double-positive cells, whereas beneficial effects in memory T cells appear to be TORC1-independent. Altogether, this study provides proof-of-principle that reducing mitochondrial ROS production per se may be therapeutic for the disease, which may have advantages compared with more general antioxidant strategies.
Project description:The brains of ataxia telangiectasia (AT) patients display an aberrant loss of Purkinje cells (PCs) that is postulated to contribute to the observed deficits in motor coordination as well as in learning and cognitive function. AT patients have mutations in the ataxia telangiectasia mutated (ATM) gene [Savitsky et al. (1995) Science 268:1749-1753]. However, in Atm-deficient mice, the neurological defects are limited, and the PCs are not deformed or lost as observed in AT patients [Barlow et al. (1996) Cell 86:159-171]. Here we report that PC-specific deletion of the mouse males absent on the first (mMof) gene (Cre(-)), which encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac) and influences ATM function, is critical for PC longevity. Mice deficient for PC-specific Mof display impaired motor coordination, ataxia, a backward-walking phenotype, and a reduced life span. Treatment of Mof(F/F)/Pcp2-Cre(+) mice with histone deacetylase inhibitors modestly prolongs PC survival and delays death. Therefore, Mof expression and H4K16 acetylation are essential for PC survival and function, and their absence leads to PC loss and cerebellar dysfunction similar to that observed in AT patients.
Project description:Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K+ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K+ currents are largely absent in AT fibroblasts in comparison with control cells. These K+ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K+ channel activity and membrane potential.