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.

Analysis of the ataxia telangiectasia mutated-mediated DNA damage response in murine cerebellar neurons.

The DNA damage response is a network of signaling pathways that affects many aspects of cellular metabolism after the induction of DNA damage. The primary transducer of the cellular response to the double-strand break, a highly cytotoxic DNA lesion, is the nuclear protein kinase ataxia telangiectasia (A-T) mutated (ATM), which phosphorylates numerous effectors that play key roles in the damage response pathways. Loss or inactivation of ATM leads to A-T, an autosomal recessive disorder characterized by neuronal degeneration, particularly the loss of cerebellar granule and Purkinje cells, immunodeficiency, genomic instability, radiosensitivity, and cancer predisposition. The reason for the cerebellar degeneration in A-T is not clear. It has been ascribed by several investigators to cytoplasmic functions of ATM that may not be relevant to the DNA damage response. We set out to examine the subcellular localization of ATM and characterize the ATM-mediated damage response in mouse cerebellar neurons. We found that ATM is essentially nuclear in these cells and that various readouts of the ATM-mediated damage response are similar to those seen in commonly used cell lines. These include the autophosphorylation of ATM, which marks its activation, and phosphorylation of several of its downstream substrates. Importantly, all of these responses are detected in the nuclei of granule and Purkinje cells, suggesting that nuclear ATM functions in these cells similar to other cell types. These results support the notion that the cerebellar degeneration in A-T patients results from defective DNA damage response.

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.