Nbs1 Deletion Mutations Lead To A Small Cranial Deformities Molecular Mechanism Study

Nijmegen breakage syndrome (NBS), caused by mutation in NBS1gene (known as Nbn in mice), is a chromosomal instability syndrome and belongs to so called DNA repair disorder. Patients with NBS show microcephaly and cereberal developmental defects. Nbsl protein encoded by NBS1gene is a key molecule in DNA damage response signaling. Nbs1with Mre11and Rad50forms MRN complex, which is critical for DNA damage response, DSB repair, regulation of cell cycle checkpoints and maintenance of chromesome stability. During the early stage of development of central nervous system, the rapidly proliferative neuroprogenitors are susceptible to be damaged by endogenous DNA damage. Therefore, the precise system of DNA damage response and DNA repair is critical for normal development of brain. After we specifically deleted Nbn in mouse central nervous system (CNS) using Cre-loxp, these mice (NbnCNS-del) exhibited growth retardation, early onset of ataxia caused by the agenesis of the cerebellum and cataracts. In addition, NbnCNS-del displayed microcephaly and severely affected white matter integrity, retina and astrocyte functionality. However, the nature of cerebral reduction and the underlying molecular mechanism in NbS-del mice have not been investigated.The present study showed that NbnCNS-delmice exhibited microcephaly characterized by the reduction of cerebral cortex and corpus callosum, recapitulating neuronal anomalies in human NBS. In the NbnCNS-del cortex, the thickness of layer â…¡/â…¢, which contains small pyramidal cells, was reduced with less neuron cellularity; layer â…£/â…¤, where granule cells and large pyramidal cells localize, respectively, was less recognizable and its thickness and the cellularity was also decreased, and the density and thickness of MBP-stained neural fibers were markedly decreased, suggesting the thickness of the corpus callosum (CC) was reduced. In contrast to NbnCNS-ctr neocortex, yH2AX was accumulated in NbnCNS-del brain, implying that Nbs1deficiency led to accumulation of endogenous DNA damage in neocortex. The western blotting results showed that ATR-mediated phosphorylation of Chkl at serine-345(Chk1-S345p) in NbnCNS-del embryonic cortex (E15.5-E18.5) was largely compromised. Nbs1deficiency impaired focus formation of DNA damage response (DDR) molecules like phosphorylated Chk1, BRCA1, Mre11, Mcphl at DNA damage sites by analyses of cellular immunofluorescence. Nbsl is also important to keep the stability of Mcphl protein. Notably, in contrast to massive apoptotic cell death in Nbsl-deficient cerebella, activation of p53mediated by ATM-Chk2pathway leads to a defective neuroprogenitor proliferation in neocortex, likely via specific persistent induction of hematopoietic zinc finger protein (Hzf) that preferentially promotes p53-mediated cell cycle arrest whilst inhibiting apoptosis. Moreover, Trp53mutations substantially rescue the microcephaly in Nbsl-deficient mice.Thus, our study reveals the first clue that developing neurons at different regions of brain selectively respond to endogenous DNA damage, and underscores an important role for Nbsl in neurogenesis. Nbsl-deficient mice present a useful model for further dissection of the molecular mechanisms of human NBS.