ataxia - telangiectasia ( a - t ) is an inherited neurodegenerative disorder arising from inactivating mutations in atm , a gene encoding a protein kinase regarded as a key regulator of the dna damage response ( ddr ) to double - strand breaks ( dsbs ) [ 13 ] . concordant with this function , cells from a - t patients are hypersensitive to ionizing radiation and defective in cell cycle checkpoint arrest . the most prominent neuropathological finding in a - t is cerebellar ataxia , which involves a significant loss of purkinje and granule cells ; to some extent , loss of neurons in striatum and substantia nigra is also found . although the neurodegenerative phenotype has been attributed to a defective response to dna breaks in pre- and post - mitotic neurons , oxidative stress and reduced anti - oxidant defence may seemingly play a role . for instance , a - t patients show persistent oxidative stress at cellular level , whereas brains and dna from atm knock - out mice exhibit increased signatures of reactive oxygen species ( ros ) , and furthermore dietary supplementation of nac ( n - acetyl - cysteine ) ameliorates oxidative dna damage   and protects purkinje cells from redox - induced death . at molecular level , the persistent oxidative stress in a - t cells was recently attributed to the capacity of atm to undergo disulfide bond formation and activation in response to oxidants , a mechanism clearly distinct from that elicited by dna breaks . hence , atm works in redox sensing and signalling , and the loss of redox balance in a - t may be central to the neuropathological phenotype . moreover , unscheduled re - entry of terminally differentiated post - mitotic neurons into the cell cycle following exposure to stimuli such as chronic inflammation , hypoxia and oxidative stress initiates an apoptotic response , and this cell cycle reactivation may be particularly noxious to purkinje and granule cells in a - t . while mostly nuclear , in neurons a fraction of atm is localized in the cytoplasm , where it functionally interacts with the synaptic proteins vamp2 and synapsin-1 . together with the defective long term potentiation and synaptic vesicle recycling in atm - deficient brains , these findings lead to hypothesize that the neurological symptoms of a - t may also arise from an impaired cytoplasmic activity of atm separate from its dna damage response . most recently , neurodegeneration in a - t has been linked to the loss of the cytoplasmic histone deacetylase hdac4 and its nuclear accumulation , with consequent suppression of neuronal gene expression . interestingly , the evidence that the malfunction of atm severely impairs glial cell functionality and vascular integrity , has led to the hypothesis that the cerebellar degeneration in a - t is a reflection of a dysfunctional neuro - glio - vascular unit . in summary , several factors appear implicated in the neurodegenerative phenotype of a - t , but which of them are most crucial is still debated , primarily because of the unavailability of human model systems able to recapitulate the neurological disease in vitro . a potent experimental tool to study neurodegeneration is offered by genetically stable neural stem cells ( nscs ) derived from multiple locations within the mammalian brain , endowed with in vitro self renewal capacity and able to differentiatiate into neurons , astrocytes and oligodendrocytes , in proportions that mirror their physiological distribution [ 2022 ] . human nscs ( hnscs ) , for instance , can generate neurons with a gabaergic , glutamatergic or dopaminergic phenotype and electrophysiological action potentials , and upon transplantation can improve sensorimotor functions and generate synaptic junctions . ablation of atm expression in these cells can be achieved by shrna silencing , as we have recently shown . the downregulation of atm expression in human embryonic stem cells ( hescs ) , which can be directed to differentiate down specific neural cell lineages , also offers an in vitro model to study neurodegeneration . a prominent advance in stem cell biology is represented by the possibility of generating human induced pluripotent stem ( ips ) cells by reprogramming fibroblasts and other somatic cells . owing to their capacity to differentiate into pure populations of specific cell types , ips cells provide a unique opportunity for disease modelling , which is of paramount importance for neuropathological disorders . in the case of a - t , the direct reprogramming of patients fibroblasts into ips cells and conversion to functional neurons has been recently reported . here , we review the human cell systems that have been developed in order to perform in vitro analysis regarding the outcome of atm inactivation in relation to dna damage response and repair , susceptibility to oxidative stress and neural differentiation , with the overall goal to get an insight into the molecular mechanisms of neurodegeneration in a - t . nscs are self - renewing , multipotent precursor cells that reside in specialized regions of the embryonic and adult central nervous system ( cns ) and participate in its homeostasis . primary cultures of human nscs have been isolated from anatomically distinct fetal brain regions ( e.g. midbrain and cortex ) and grown in vitro as neurospheres ( n ) in the presence of egf and fgf-2 . these cells can be maintained long - term in a proliferative undifferentiated state and can be induced to differentiate into the three major cns cell types , i.e. neurons , astrocytes , and oligodendrocytes , in proportions that mirror their physiological distribution ( fig . to overcome some limitations ( e.g. slow growth ) , primary nscs have been immortalized with a retroviral vector encoding the v - myc oncogene ( ihnscs ) , and have shown to retain self - renewal and multipotential capacity . the impact of atm on the biological properties of these cells has been generally studied in two ways , either by chemical inhibition of atm with ku-55933   or by lentiviral shrna - mediated stable knockdown of atm ( shatm ) , although it has been argued that inhibition of atm activity and knockdown of the protein do not fully mirror the broad spectrum of a - t - causing mutations , in line with recent findings showing that mice expressing a kinase - inactive atm die during embryonic development , whereas atm - null mice are viable . pluripotent human embryonic stem cells ( hescs ) , which can be differentiated into neural progenitors and mature neurons and astrocytes , provide another powerful model system to assess the effects of loss or inactivation of atm . also for these studies , atm has either been stably knockdown by transducing hescs with lentiviruses harbouring shrna against atm , or chemically inhibited with ku-55933 . interestingly , neuronal models of a - t based on the use of patients tissues are currently being developed . for example , olfactory mucosa - derived neurospheres ( ons ) have recently been established from several a - t patients and have been shown to differentiate in vitro into neurons , astrocytes and oligodendrocytes . finally , patient - derived induced pluripotent stem ( ips ) cells provide a powerful tool for disease modelling . to address the role of atm in neurodegeneration , we and others   have generated ips cells by reprogramming fibroblasts from a - t patients and normal donors by the introduction of pluripotency factors ( oct4/klf4/sox2/cmyc ) . moreover , we have also established conditions to generate from ips cells stable proliferating neural precursor cell ( npc ) lines expressing nestin and sox2 , which can be induced to differentiate into -tubulin iii and map2 positive neurons ( fig . 1b ) expressing gabaergic and glutamatergic markers , and which acquire electrophysiological function upon maturation . although the differentiation in a complete range of neuronal subtypes has not yet been achieved , this model system is extremely promising as it allows to study patient - derived cells which retain the original disease - causing mutation and can be differentiated in virtually all cell types . upon terminal differentiation , rapidly proliferating neuroprogenitors exit the cell cycle and become postmitotic . as the repair of dna dsbs occurs through either homologous recombination ( hr ) or non - homologous end joining ( nhej ) , the former operating in dividing cells while the latter also in non - dividing cells , the cellular answer to genotoxic damage may differ according to the cell cycle phase . moreover , it has been shown that in human neuron - like cells the levels of atm and other ddr components , e.g. atr and nbs1 , are downregulated during neuronal differentiation , with consequent attenuation of the damage response in non - dividing versus dividing cells   ( fig . clearly , the availability of hnscs offers the opportunity to study these phenomena in relation to atm deficiency . we have previously demonstrated that the ddr of dividing ihnscs is defective upon ablation of atm by shrna interference , as evident from the reduced phosphorylation of atm substrates . moreover , the disappearance of -h2ax , an indicator of ongoing dsb repair , is markedly delayed in shatm cells , indicating that atm deficiency , like in other dividing cell types , delays the dna repair also in hnscs . by contrast , in terminally differentiated shatm ihnscs , the number of -h2ax foci 30  min following dna damage is greater than in control cells , while the resolution of the foci is only modestly delayed ( fig . it should be noted that , compared to atm - proficient neurons , non - dividing neurons from atm - deficient neural progenitors obtained from hescs are virtually negative for -h2ax expression . oxygen regulates the homeostasis , apoptosis and differentiation of stem cells and committed progenitors in the central nervous system , where its levels range from about 0.558% . low oxygen tension ( hypoxia ) , a stress condition encountered in a variety of neuropathological states such as cerebral ischemia , is known to promote an undifferentiated state in several stem cells and progenitor cells   and to induce a paradoxical increase in the production of mitochondrial ros , which further increase intracellular stress . in fact , hypoxia activates adaptive mechanisms involving the stabilization of the hypoxia - inducible factor 1 ( hif-1 ) and the transcription of hif-1-regulated genes . a link between hypoxia and atm was recently established in studies demonstrating that atm mediates the phosphorylation of hif-1 on ser696 , and this modification causes the downregulation of the sensor of nutrient and energy sufficiency mtorc1 , which acts as a restriction point under stress conditions . loss of hypoxia - dependent inhibition of mtorc1 sensitizes atm - deficient cells to p53-dependent apoptosis . hypoxia - induced ros are currently believed to be the key cytosolic signal that elicits the accumulation of hif-1 by inhibiting prolyl - hydroxylase ( phd ) activity through an unknown mechanism . deregulated ros can be neuropathological and given that ros can activate atm   to downregulate mtorc1 , it is possible that atm mediates certain cellular stress responses through the hypoxia - induced ros . however , little is known about the role of hypoxia in neural cells in relation to atm . we have recently investigated this aspect and one of the most striking findings in atm - proficient ihnscs was the dramatic upregulation by hypoxia ( 1% o2 ) of the levels of atm and of its family members atr , dna - pkcs and mtor ( fig . 3a , left ) , but not of the atm substrates smc1 , nbs1 , mre11 , chk2 and chk1 ( not shown ) . we also found that atm deficiency reduces the accumulation of hif-1 and carbonic anhydrase ix ( caix ) in proliferating ihnscs grown under hypoxia ( fig . this reduced accumulation of hif-1 and especially caix is also observed in non - dividing terminally differentiated cells ( fig . 3b ) , indicating that the expression of these molecules is partially dependent on atm . atm deficiency appears to impact on hnsc differentiation . indeed , while the yield of neurons expressing map2 and -tubulin iii markers is unaffected by atm deficiency , that of gabaergic neurons falls by 50% ( fig . 4a ) . notably , gaba signaling plays an important role in adult neurogenesis , through a mechanism in which gaba induces the phosphorylation of h2ax by atm / atr , and this epigenetic change within the subventricular zone niche restricts the proliferation of neural stem cells and neuronal output . whether the reduced yield of gabaergic neurons in atm - deficient ihnscs arises from a dysfunctional gaba signaling owing to atm deficiency and somehow limiting a specific neuronal output , remains unknown nevertheless , this in vitro result agrees with pathological and clinical findings showing a gaba deficiency in the cerebellum of an a - t patient , and amelioration of the ataxia manifestation by treatment with a gaba - analog . concordant with this , we have observed that the amount of neurons expressing -tubulin iii is markedly reduced in ihnscs differentiating under hypoxia , although to the same extent in atm - deficient and  proficient cells . hypoxia also affects the yield of gabaergic neurons , but to a greater extent in atm - deficient than in control ihnscs , and the potassium channel - interacting protein-1 ( kchip-1 ) , predominantly expressed by gabaergic neurons , also appears to be markedly dependent on oxygen tension , since the fraction of positive cells falls substantially under hypoxia , and to a higher degree in atm - deficient ihnscs ( fig . atm - deficiency also attenuates the yield of galc- and cnpase - expressing oligodendrocytes   and furthermore , when ihnscs are induced to differentiate under oxidative stress conditions achieved by the glutathione - depleting agent bso ( buthionine sulfoximine ) or by hypoxia , a remarkable vulnerability of oligodendrocytes to atm deficiency is observed ( fig . these results indicate that atm deficiency does not appear to impair overall neurogenesis , though it partially affects oligodendrogenesis . as animal models of a - t do not faithfully reproduce the human syndrome , attempts have been made to generate and use human neural stem cell lines derived from embryos , fetal brain or adult brain tissues , to study the mechanisms that underlie the neurodegenerative disease . hnscs are a renewable source of undifferentiated cells , can be induced to differentiate into functional neurons and glia , and are amenable to experimental genetic manipulation . studies with such cells are certainly providing important clues on the role of atm in ddr and neuronal differentiation progression and maturation , as well as on the vulnerability to genotoxic and oxidative agents . however , since the characteristics of hnscs either derived from es cells or isolated from different regions of the brain can differ , possibly reflecting normal developmental patterns , it must be recognized that despite many advantages , the use of these stem cells for disease modelling has limitations . human ips cells generated from reprogrammed a - t fibroblasts and capable of generating a variety of functional neurons offer an additional tool to study neurodegeneration also as function of the disease - associated genetic mutation . a current challenge is to direct the neural differentiation of ips cells towards specific neuronal subtypes , e.g. purkinje cerebellar neurons , which are specifically sensitive to atm deficiency . this would allow the investigation of the effects of a - t - causing mutations using the cell population which is directly involved in the neurodegenerative phenotype of a - t .
loss of atm kinase , a transducer of the dna damage response and redox sensor , causes the neurodegenerative disorder ataxia - telangiectasia ( a - t ) . 
 while a great deal of progress has been made in elucidating the atm - dependent dna damage response ( ddr ) network , a key challenge remains in understanding the selective susceptibility of the nervous system to faulty ddr . 
 several factors appear implicated in the neurodegenerative phenotype in a - t , but which of them plays a crucial role remains unclear , especially since mouse models of a - t do not fully mirror the respective human syndrome . 
 therefore , a number of human neural stem cell ( hnsc ) systems have been developed to get an insight into the molecular mechanisms of neurodegeneration as consequence of atm inactivation . 
 here we review the hnsc systems developed by us an others to model a - t .