Functional and molecular defects of human induced pluripotent stem cell-derived neurons fron poatiens with the neurodegenerative disorder ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a rare hereditary, early onset neurodegenerative disorder caused by inactivation of the ATM serine/threonine protein kinase, which is the major regulator of the DNA damage response to double-strand breaks (DSBs) and works in sensing and signaling oxidative stress. Disease models are essential for unraveling the mechanisms underlying the neuropathology, but ATM knockout mice do not recapitulate the central nervous system phenotype and neural stem cells are very heterogeneous. In this work we applied a reprogramming approach to generate an in vitro human A-T model in order to investigate the outcome of ATM ablation in neurons. We derived induced pluripotent stem cells from A-T and normal control fibroblasts by introducing the reprogramming factors OCT3/4, KLF4, SOX2 and l-MYC and from these we obtained expandable nestin-positive neural precursor cells (NPCs) via embryoid body and neural rosette formation. Following differentiation, NPCs gave rise to a high percentage of mature neurons expressing ;-tubulin III and microtubule-associated protein 2. Our results show that electrophysiological properties were similar in control and A-T cells. Also, base excision repair, which is important for the removal of DNA single strand breaks caused by oxidative stress, and single strand break repair were normal in A-T cells. On the other hand, AT neurons displayed attenuated DNA damage response and repair following irradiation and were resistant to apoptosis induced by genotoxic agents, but not by oxidative agents. Moreover, mutant neurons displayed deficits in the expression of the synaptic markers synaptophysin and postsynaptic density protein 95 as well as of the neuronal growth-associated protein SCG10 and K+ channel-interacting proteins KChIP. Notably, A-T neurons exhibited abnormal accumulation of topoisomerase 1-DNA covalent complexes, which may cause impairment of transcription elongation. These findings reveal that, in neural cells, ATM deficiency suppresses the response and repair of DNA DSBs, impairs neuronal maturation and could promote transcriptional decline, possibly contributing to neurodegeneration in A-T. This previously undocumented functional and molecular characterization of patientderived neuronal cells represents the first step in the employment of these cells as the most informative in vitro model to investigate neurodegeneration in A-T. Moreover, this model represent a powerful tool for further exploring the mechanisms that cause degeneration of human ATM-deficient neurons and it may be exploited for identifying therapeutic targets and for screening molecules with potential neuroprotective properties