DNA Damage Response (DDR) and Synaptic Activity-Dependent Transcriptional Regulation in A-T Neurons

To explore the mechanisms of neurodegeneration in A-T, using as model system functionally mature postmitotic neurons from iPSCs derived from A-T patients and controls (WT), we have examined the DDR and transcriptional response to genotoxic and pharmacological stimuli. We focused on transcription prompted by the evidence that ATM loss impairs the removal of Topoisomerase 1-DNA covalent complexes (Top1ccs), potentially interfering with transcription. We found that the Top1cc poison camptothecin (CPT) elicits both an ATM-dependent DDR as well as an ATM-independent transcription repression of especially long genes and induction of the immediate early genes (IEGs) cFos, NPAS4, EGR1 and ARC, which in the CNS are rapidly induced by synaptic activity and crucial for synaptogenesis and plasticity, learning and memory. Moreover, nascent RNA-seq analysis showed an essentially similar transcriptional elongation and recovery after Top1cc block-release in WT and A-T neurons, excluding an effect on transcription of even long genes by the over-accumulation of Top1ccs in ATM-deficient neurons. Remarkably, pharmacological stimulation of ionotropic glutamate receptors with AMPA and NMDA ligands, or membrane depolarization by KCl, activated a DDR only in WT neurons, but induced a similar level of IEGs expression (mRNA and protein) both in WT and A-T neurons. Finally, RNA-seq analysis revealed the down-regulation in unperturbed ATM-deficient neurons of a set of genes typically functioning in the development, maintenance and physiology of central nervous system, underscoring their potential involvement in the neurodegenerative process in A-T patients.