Functional and molecular defects of hiPSC-derived neurons from patients with ATM deficiency.

L Carlessi,D Delia,B Pascucci,C Verpelli,G Bechi,E Fusar Poli,C Sala,D Lecis,L Narciso,M Mantegazza,E Dogliotti

doi:10.1038/cddis.2014.310

Abstract

Loss of ataxia telangiectasia mutated (ATM) kinase, a key factor of the DNA damage response (DDR) pathway, causes the cancer predisposing and neurodegenerative syndrome ataxia-telangiectasia (A-T). To investigate the mechanisms of neurodegeneration, we have reprogrammed fibroblasts from ATM-null A-T patients and normal controls to pluripotency (human-induced pluripotent stem cells), and derived from these neural precursor cells able to terminally differentiate into post-mitotic neurons positive to >90% for β-tubulin III+/microtubule-associated protein 2+. We show that A-T neurons display similar voltage-gated potassium and sodium currents and discharges of action potentials as control neurons, but defective expression of the maturation and synaptic markers SCG10, SYP and PSD95 (postsynaptic density protein 95). A-T neurons exhibited defective repair of DNA double-strand breaks (DSBs) and repressed phosphorylation of ATM substrates (e.g., γH2AX, Smc1-S966, Kap1-S824, Chk2-T68, p53-S15), but normal repair of single-strand breaks, and normal short- and long-patch base excision repair activities. Moreover, A-T neurons were resistant to apoptosis induced by the genotoxic agents camptothecin and trabectedin, but as sensitive as controls to the oxidative agents. Most notably, A-T neurons exhibited abnormal accumulation of topoisomerase 1-DNA covalent complexes (Top1-ccs). These findings reveal that ATM deficiency impairs neuronal maturation, suppresses the response and repair of DNA DSBs, and enhances Top1-cc accumulation. Top1-cc could be a risk factor for neurodegeneration as they may interfere with transcription elongation and promote transcriptional decline.

Highlights

A fraction of ataxia telangiectasia mutated (ATM) is localized in the cytoplasm where it phosphorylates the synaptic proteins VAMP2 and synapsin-1.8 Recently, ATM has been detected in dendrites and dendritic spines, and its activation by neuronal firing and synaptic activity induces the phosphorylation of proteins essential for neuronal function.[9]
Using phospho-specific antibodies for the indicated substrates of ATM and checkpoint kinase 2 (Chk[2]; KAP1-pS473), we found that after 15–30 min of ionizing radiation (IR) these molecules were strongly phosphorylated in Ctrl but not in A-T human neural precursor cells (hNPCs) (Figure 2c)
As a functional analysis of the neuronal cells harboring patient-specific mutations in ATM has not yet been reported, in this study we examined hiPSC-derived A-T hNPCs and terminally differentiated neurons in culture

Summary

Introduction

A fraction of ATM is localized in the cytoplasm where it phosphorylates the synaptic proteins VAMP2 and synapsin-1.8 Recently, ATM has been detected in dendrites and dendritic spines, and its activation by neuronal firing and synaptic activity induces the phosphorylation of proteins essential for neuronal function.[9]. Neurodegeneration in A-T has been linked to epigenetic modifications associated with deregulation of class II histone deacetylase HDAC410 and hyperexpression of the histone-lysine N-methyltransferase EZH2,11 altogether inducing transcriptional repression of multiple neuronal genes and cell death. Fenilindolo; APE1, apurinic/apyridinic endonuclease 1; Pol-b, DNA polymerase b; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; NPCs, neural precursor cells; hiPSCs, human-induced pluripotent stem cells; DDR, DNA damage response; DSBs, double-strand breaks; Top1-cc, topoisomerase 1-DNA covalent complexes; IR, ionizing radiation. It should be noted that germline mutations in ATM severely impair glial cell functionality and vascular integrity, suggesting that Purkinje cell death and cerebellar degeneration in A-T may result from a dysfunctional neuro-astro-vascular unit.[12]

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Results

Conclusion