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

Abstract

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 c-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/b-tubulin III þ neurons, but a reduced number of GalC þ 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.

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

“…H2AX was revealed to be phosphorylated at serine 139 (-H2AX) in response to Chk1 inhibition particularly when followed by treated with DNA replication inhibitors (Ewald et al, 2007;Syljuasen et al, 2005). Since the increased level of -H2AX is recognised as a marker in response to DNA double-strand breaks, it is not surprising that loss of Chk1 kinase activity could result in DNA damage and further cell apoptosis (Carlessi et al, 2009;Durkin et al, 2006). However, instead of playing a direct role in the Chk1 associated signalling transduction pathway, -H2AX foci in Chk1-depleted cells were supposed to point out the sites of where persistent replication fork defect occurs (Gagou et al, 2010).…”

ATM protein kinase: the linchpin of cellular defenses to stress

“…Cells from the adult hipppocampus of Atm -/-mice failed to differentiate into neurons or oligodendrocytes in vitro [237]. However, studies of immortalized human neural stem cell line (see below) after knockdown of ATM expression showed almost normal differentiation of progenitors into all three neuronal lineages [238]. Ito et al (2004) [239] discovered that ATM can protect hematopoietic stem cells (HSC) from oxidative stress damage and allow them to maintain their self-renewal potential.…”

Molecular Bases of Ataxia Telangiectasia: One Kinase Multiple Functions

“…They could show that ATM plays a central role in terminal differentiation of ihNSCs through its function in DDR [158]. All these data support a role of ATM in the control of neuronal differentiation though its DDR dependent functions and oxidative stress dependent functions and suggest that defective proliferation of NSC could be in part responsible of the neurodegenerative phenotype in Atm -/-mice and A-T patients.…”