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

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 ß-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.

Murine neural stem cells model Hunter disease in vitro: glial cell-mediated neurodegeneration as a possible mechanism involved.

Mucopolysaccharidosis type II (MPSII or Hunter Syndrome) is a lysosomal storage disorder caused by the deficit of iduronate 2-sulfatase (IDS) activity and characterized by progressive systemic and neurological impairment. As the early mechanisms leading to neuronal degeneration remain elusive, we chose to examine the properties of neural stem cells (NSCs) isolated from an animal model of the disease in order to evaluate whether their neurogenic potential could be used to recapitulate the early phases of neurogenesis in the brain of Hunter disease patients. Experiments here reported show that NSCs derived from the subventricular zone (SVZ) of early symptomatic IDS-knockout (IDS-ko) mouse retained self-renewal capacity in vitro, but differentiated earlier than wild-type (wt) cells, displaying an evident lysosomal aggregation in oligodendroglial and astroglial cells. Consistently, the SVZ of IDS-ko mice appeared similar to the wt SVZ, whereas the cortex and striatum presented a disorganized neuronal pattern together with a significant increase of glial apoptotic cells, suggesting that glial degeneration likely precedes neuronal demise. Interestingly, a very similar pattern was observed in the brain cortex of a Hunter patient. These observations both in vitro, in our model, and in vivo suggest that IDS deficit seems to affect the late phases of neurogenesis and/or the survival of mature cells rather than NSC self-renewal. In particular, platelet-derived growth factor receptor-α-positive (PDGFR-α+) glial progenitors appeared reduced in both the IDS-ko NSCs and in the IDS-ko mouse and human Hunter brains, compared with the respective healthy controls. Treatment of mutant NSCs with IDS or PDGF throughout differentiation was able to increase the number of PDGFR-α+ cells and to reduce that of apoptotic cells to levels comparable to wt. This evidence supports IDS-ko NSCs as a reliable in vitro model of the disease, and suggests the rescue of PDGFR-α+ glial cells as a therapeutic strategy to prevent neuronal degeneration.

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

Ataxia-telangiectasia (A-T) is a neurodegenerative disorder caused by defects in the ATM kinase, a component of the DNA-damage response (DDR). Here, we employed an immortalized human neural stem-cell line (ihNSC) capable of differentiating in vitro into neurons, oligodendrocytes and astrocytes to assess the ATM-dependent response and outcome of ATM ablation. The time-dependent differentiation of ihNSC was accompanied by an upregulation of ATM and DNA-PK, sharp downregulation of ATR and Chk1, transient induction of p53 and by the onset of apoptosis in a fraction of cells. The response to ionizing radiation (IR)-induced DNA lesions was normal, as attested by the phosphorylation of ATM and some of its substrates (e.g., Nbs1, Smc1, Chk2 and p53), and by the kinetics of gamma-H2AX nuclear foci formation. Depletion in these cells of ATM by shRNA interference (shATM) attenuated the differentiation-associated apoptosis and response to IR, but left unaffected the growth, self-renewal and genomic stability. shATM cells generated a normal number of MAP2/beta-tubulin III+ neurons, but a reduced number of GalC+ oligodendrocytes, which were nevertheless more susceptible to oxidative stress. Altogether, these findings highlight the potential of ihNSCs as an in vitro model system to thoroughly assess, besides ATM, the role of DDR genes in neurogenesis and/or neurodegeneration.