NFAT3/c4-mediated Excitotoxicity In Hippocampus Cell Apoptosis During Radiation-induced Brain Injury

ObjectiveRadiotherapy,an indispensible treatment for head and neck tumor,may cause progressive brain injury,especially injury to the hippocampus,an area important for learning and memory,compromising the quality of life for survivors.However,little is known about the precise molecular pathways for the decline in cognitive function.Recently,the N-methyl-D-aspartate receptor(NMDAR)is widespread in the CNS and is associated with the occurrence of excitotoxic neuronal cell death.Moreover,nuclear factor of activated T cells isoform 4(NFAT3/ c4)signaling pathway is vital to excitotoxic hippocampus cell apoptosis induced by radiation.In this regard,it has been assumed that radiation-induced hippocampal-related cognitive dysfunction is associated with NMDAR/NFAT3/c4/Bax pathway.Materials and MethodsSprague–Dawley male rats(21-day old)weighing 50-60 g were divided into four groups: the sham group(Sham),the sham with 11R-VIVIT peptide injection(Sham +11R-VIVIT peptide),the irradiation group(IR),the irradiation and 11R-VIVIT peptide injection group(IR +11R-VIVIT peptide),to receive the whole brain irradiation(WBI)treatments with a single dose of 0(control)or 20 Gy by using 4 MeV electron beam.The animals were sacrificed and the hippocampus were removed at 1h,3h,6h,12 h,1d,3d after irradiation(n=3 in each group for each time point).Then we use Western blot method to detect the expression of NMDAR subunits(NR2A,not NR1 or NR2B),calcineurin,NFATc4/3,GSK-3β,and apoptosis marker Bax in the rat hippocampal.And we use immunofluorescence staining method to count the number of hippocampal proliferation and neuron survival,mainly in DG cells.Once treated with the NFAT3/c4 inhibitor 11R-VIVIT peptide pre-radiation,we observe the expression of NMDAR subunits,NFATc4/3 and Bax.Then we detect the number of Brdu and NeuN.ResultsThe results of Morris and immunofluorescence staining indicated that WBI can induce cognitive dysfunction and hippocampal proliferation and neuron survival cells.Western blot indicated that WBI significantly increased NMDAR subunits expression at 6h post-radiation compared with the sham group.While expression levels of CaN and GSK-3β had no difference.With stimulate of radiation,NFAT3/c4 rapidly accumulated in the nucleus,then promoted expression of the apoptosis marker Bax and decreased level of the anti-apoptosis marker Bcl-2.The treatment of 11R-VIVIT peptide prevented nuclear accumulation of NFAT3/c4,then inhabited the appearance of the apoptosis marker Bax.Lastly,the 11R-VIVIT peptide alleviated the decline in the DG proliferating cells and mature neurons.ConclusionsOur study has confirmed that WBI can induce cognitive decline detected by behavioral testing as Morris water maze.Moreover,the result of immunofluorescence staining indicated that the number of hippocampal proliferation and neuron survival,mainly in DG cells decreased after whole brain irradiation.And Western Blot clearly showed that Whole-brain radiation activated NMDAR/NFAT3/c4/Bax pathway and induced apoptosis,suggesting that NFATc4/3 transcription is involved in radiation-induced neurogenesis impairment.While the inhibitor,11R-VIVIT peptide rescued the activation of NFAT3/c4-dependent apoptosis and alleviated the decline in the DG proliferating cells and mature neurons after radiation.Based on these results,we believe that radiation-induced cognitive decline is associated with NMDAR-mediated non-conventional apoptotic response.