In the present study, we have shown that neural inactivation of Nbs1 leads to microcephaly in mice characterized by reduced size of cerebral cortex and thickness of CC, resembling neuronal abnormalities of human NBS patients 31
Development of the cerebral cortex is a strictly regulated process whereby neural progenitor cells from the proliferative pseudostratified ventricular zone go through massive expansion before they exit the cell cycle and form cortical neurons 32
. Neuroprogenitors are rapidly proliferating and potentially generate high level of oxidative damage, which may lead to a high level of lesions encountered at the replication fork 33
. Several pathways may execute to recognize and repair these lesions, unrepaired DNA strand breaks can cause detrimental outcome, including chromosomal instability, cell death, developmental defects and neoplastic transformation 34
. In particular, the nervous system is susceptible to DNA repair deficiency, which can lead to neurodegeneration, microcephaly or brain tumors 33
. Our study using developmental brain provides direct evidence that endogenous DNA damage caused by SSBs/replication fork blockage or DSBs activates ATR-Chk1 and ATM-Chk2-p53 pathways. Thus, it is likely that both ATM and ATR pathways participate in monitoring endogenous DNA damage during neurogenesis (see ).
Figure 7 Working model for Nbs1 function and regulation in neocortex. Endogenous DNA damage during rapid development of neocortical neurons activates ATM-Chk2 and ATR-Chk1 signaling. However, defective Nbs1 impairs ATR-Chk1 pathway rather than ATM-Chk2 signaling. (more ...)
It has been shown that Nbs1 is required to modulate ATM or ATR activation in vitro
and in vivo
3, 8, 11, 25, 35
. Notably, we show in the present study that Nbs1 dysfunction impairs the ATR-Chk1 signaling (see and ), rather than the ATM-Chk2 pathway (see Supplementary information, Figure S2
). It is likely that during neurogenesis, endogenous DNA damage caused by Nbs1 deficiency mainly leads to SSBs, such as replication fork blockage that activate ATR pathway. Although MRN complex activate ATM kinase 3
, deletion of Nbs1 retained accumulation of Mre11 at cytoplasmic compartment, presumably inactivating the MRN function 36
(see ). However, it has been shown recently that in neuroprogenior cells, ATM activation responding to DNA damage may not require Nbs1 37
. Thus, defects in ATR-Chk1 signaling in Nbs1-deficient neocortical neurons may convert unrepaired replication fork to DSBs and result in a progressive accumulation of persistent DSBs throughout the brain, which in turn activate cell cycle checkpoint mediated by ATM-p53 pathway, leading to proliferation defects and microcephaly 38
(see ). In supporting this hypothesis, inactivation of Trp53
largely rescues microcephaly in NbnCNS-del
We have shown previously that Nbs1-deficient cerebellar neuroprogenitors are susceptible to DNA damage and postmitotic neurons with accumulation of DNA damage and chromosomal aberrations are cleared by massive cell death through activation of ATM-p53 signaling that leads to agenesis of cerebellum 18
. In the present study, although Nbs1-deficient cortical neuroprogenitors are susceptible to endogenous DNA damage that slows down proliferation (see ), distinct from cerebellar and medulla oblongata neurons, neocortical postmitotic neurons are more tolerable to DNA damage-induced cell death (see ). Thus, the cerebrum continues to develop into microcephaly, which is likely due to proliferation defects. This functional difference of neurons in a specific region to endogenous DNA damage may be caused by a specific overactivation of Hzf upon ATM-p53 signaling in neocortex (see ), which favors cell cycle arrest over apoptosis by selectively recruiting p53 to promoters of pro-arrest target gene such as p21
, resulting in proliferation arrest 27
. In contrast, in Nbs1-deficient cerebella endogenous DNA damage enhanced p53-mediated cell death and proliferation arrest. Therefore, Hzf acts both as a p53 target () and as an important regulator in p53-mediated cell fate determination during cortical neurogenesis (see ). Thus, this study may partly explain the respective neuropathology in human that neurons at different regions of brain respond differently to endogenous DNA damage during neurogenesis.
In conclusion, the present study shows a role of Nbs1 in neocortical neurons by regulating ATR-Chk1 pathway in monitoring endogenous DNA damage. Neuronal Nbs1 deletion causes defective proliferation by activation of p53-Hzf signaling in neocortex, but not p53-mediated proapototic pathway, leading to microcephaly, and neurons display a region-specific response to endogenous DNA damage during neurogenesis. Thus, Nbs1-deficient mice present a useful model for further dissection of the molecular mechanisms of human NBS.