Nuclear Ataxia-Telangiectasia Mutated (ATM) Mediates the Cellular Response to DNA Double Strand Breaks in Human Neuron-like Cells

Abstract

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.

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

“…However, in the earlier work [9], control studies using brain tissue from AT patients as a negative control were not employed, thus raising questions about the specificity of the ATM staining. Here, using an antibody previously demonstrated to specifically stain ATM in human neuronal-like cells in culture [12], we show that ATM is predominantly localized in the nucleus of Purkinje neurons in postmortem tissue from the juvenile human brain. Importantly, our conclusion is supported by control experiments demonstrating the absence of staining in Purkinje neurons from AT patients, which represents the most conclusive evidence for the specificity of the staining.…”

“…Biton et al [12] found that the antibody Y170 could be used to specifically localize the ATM protein in the nucleus of cultured human neuronal cells. Therefore we utilized this antibody to detect the ATM protein in the cerebellum of human postmortem brain samples from two normal donors.…”

“…However, the question of whether the neurodegeneration observed in AT patients results from a loss of this DNA damage response function of ATM has been controversial, since several authors have found that ATM protein is located in the cytoplasm of Purkinje neurons in the human [9] and rodent [10,11] brain. Recently however, Shiloh and co-workers [12,13] found that the ATM protein is present in the nucleus of neuronal derived human cells maintained in tissue culture. Also, Barzilai and co-workers [14] showed that nuclear ATM protein is phosphorylated in Purkinje neurons of mice exposed to ionizing radiation.…”

DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression

“…[20][21][22] In ATM-deficient cells, the phosphorylation of the ATM targets is markedly attenuated. 23 To determine the effect of ATM depletion in our cells, we analyzed the genotoxic response on western blots using antibodies specific for ATM phosphoresidues.…”

Blood metal levels and related antioxidant enzyme activities in patients with ataxia telangiectasia

“…ATM phosphorylates several key proteins that initiate the activation of DNA 60 damage checkpoints, leading to cell cycle arrest, DNA repair or apoptosis. Although the 61 neurodegenerative phenotype has been attributed to a defective response to DNA breaks in pre-and 62 post-mitotic neurons (Lee et al, 2001), oxidative stress and reduced anti-oxidant defence may also 63 play a role (Biton et al, 2006). 64…”