Effects And Mechanism Of Histone Acetylation Modification Mediated By CBP In Vitamin A Deficiency-Induced Learning And Memory Impairment

Part Ⅰ Effects of vitamin A deficiency beginning from pregnancy on the learning and memory function and histone acetylation modification in adult young ratsObjective:To investigate the effects of vitamin A deficiency beginning from pregnancy on the learning and memory function and histone acetylation modification in adult young rats, and the connections of the changes in RA signaling pathway and histone acetylation modification.Methods:The rat models of vitamin A deficiency beginning from pregnancy were established and the serum retinol levels of the young rats in VAN and VAD groups were detected by real-time monitoring; The learning and memory function of rats at 8 weeks of age in VAN and VAD groups were tested using the water maze tests. At the same time, real-time PCR and western blot were used to detect the RARa gene and protein expressions in the rat hippocampus, respectively; HAT activity was examined with the HAT assay kit; The expression levels of acetylated histone H3, H4 protein were measured by western blot. The expressions of learning and memory related genes (Zif268, cFOS, FosB) were dected using real-time PCR; CHIP-qPCR was usd to examine the enrichment of Ac-H3 and Ac-H4 on the promoters of the learning and memory genes in the rat hippocampus of the two groups. And then real-time PCR and western blot were used to detect the gene and protein expressions of the histone acetyltransferases (CBP, P300, PCAF); Finally, the interaction between the RARa and CBP proteins in the rat hippocampus of the two groups was examined using Co-IP and laser confocal microscopy.Results:(1) Serum retinol concentrations at P1D, P3D, P7D, P2W, P4W, and P8W of the offspring in both VAN and VAD groups:The levels in the VAD group were significantly lower than those in the VAN group (p<0.001). The Morris water maze tests performed in the pups at 8 weeks of age:Both groups could learn the location of the platform (p<0.001) but the learning and memory function of the rats in the VAD group were significantly worse than those of the VAN group (p<0.05); (2) The mRNA and protein expression levels of RARa:Both were significantly lower in the VAD group compared with the VAN group (p<0.01/0.001); (3) The global HAT activity and acetylation levels of two typical histone proteins obtained from the hippocampus:Both showed significant decreases in VAD rats when compared with VAN rats (p<0.05/0.001). The mRNA expression levels of three memory-related genes (Zif268, cFos, and FosB) in the rat hippocampus:They were also significantly decreased in VAD group (p<0.01/0.05). The enrichment levels of Ac-H3 and Ac-H4 on the promoters of these three genes:There were comparatively significant decreases in the VAD group than in the VAN group (p<0.01/0.05); (4) Expression levels of three HATs (CBP, p300, PCAF):The mRNA levels of CBP and p300 were significantly decreased in the hippocampus of the VAD group in comparison with the VAN group (p<0.01/0.05), but no significant change in PCAF expression was observed (p=ns). CBP protein expression was significantly decreased in the hippocampus of VAD rats in contrast with the VAN rats (p<0.001). And the levels of p300 and PCAF proteins in the VAD group were somewhat reduced, but no significant difference was observed between the two groups (p=ns); (5) Immunoprecipitation and laser-scanning confocal microscopy were used to test the interaction of RARa and CBP proteins:Immunoprecipitation experiments showed that their interaction was weaker in the VAD group than that in the VAN group (p<0.001); the colocalization of RARa and CBP was observed weaker in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus in the VAD group (p<0.001).Conclusions:(1) We have successfully established the rat models with VAD from pregnancy and confirmed that VAD beginning from pregnancy could cause learning and memory impairments in adult rats; (2)The mRNA and protein expression levels of RARa are significantly lower in the VAD group compared with the VAN group; (3) VAD significantly decreases the global HAT activity, acetylation levels of histone proteins and expression levels of learning-and memory-related genes; (4) The expressions of histone acetyltransferase CBP, p300, PCAF are decreased in different degrees in VAD rat hippocampus, and the expression of CBP is seriously damaged. The significant reductions of HAT activity, expressions of Ac-H3, Ac-H4, as well as the learning-and memory-related genes in VAD may be closely related to the decline of the CBP expressions; (5) VAD causes significant changes in the RA signal transduction pathway and histone acetylation modification. Both the expressions of RARa and CBP in the rat hippocampus are significantly decreased with an interaction between the two, and this interaction is significantly weaker in VAD group than that of VAN group. The abnormal interaction between RARa and CBP in VAD may play an important role in learning and memory dysfunction.Part Ⅱ The mechanism of CBP-dependent histone acetylation modification related to learning and memory regulated by RARa-mediated RA signaling pathwayChapter Ⅰ The mechanism of learning- and memory-related histone acetylation modification regulated by RARaObjective:To further investigate the mechanism by which RARa-mediated RA signaling pathway affects the CBP, HAT activity, acetylated histones and learning- and memory-related genes.Methods:Primary cultured neurons were cultured and the purity of the neurons was identified with the neural marker neuron-specific enolase (NSE) on the sixth day. Four groups (①FP, ②RFP+RA, ③RA+siRARa, ④RA+Ad-RARa) of the neurons were set on the sixth day. And these neurons were intervented with RA or infected with adenoviruses encoding RFP, siRARa, Ad-RARa, respectively. On the seventh day, the mRNA levels of RARa, CBP and the learning and memory genes were analyzed; and the enrichments levels of RARa on the CBP promoters were examined with CHIP-qPCR. Protein expressions of RARa, CBP, histone acetylation and HAT activity of the neurons were investigated on the ninth day.Results:(1) The primary hippocampal neurons were in good growth condition with full cell bodies, and the synaptics were gradually extended to form mutual connections. The primary cultured cells were identified correctly and the purity of neurons was achieved greater than 90%, which met the requirement of our experiment; (2) The levels of RARa mRNA (p<0.001/0.01) and protein expressions were both up-regulated after RA treatment. And the mRNA and protein levels of RARa were significantly reduced or enhanced in neurons by infection with siRARa and Ad-RARa, respectively; (3) The global HAT activity was also significantly induced by RA compared with the RFP group, and it was significantly repressed by siRARa and enhanced by Ad-RARa in comparison to the RFP group (p<0.001/0.01/0.05). Additionally, CBP mRNA (p<0.001/0.01/0.05) and protein expressions, Ac-H3/H4 protein expressions, and the expressions of the three memory genes (p<0.001/0.01/0.05) were also basically consistent with the changes in RARa levels among the four groups. (4) An enrichment of RARa was clearly detected on the CBP promoter with the results of ChIP-qPCR. Furthermore, the ability of RARa to bind the CBP promoter was also significantly affected by the RARa levels (p<0.001/0.01/0.05).Conclusions:(1) We have successfully cultured and identified the primary hippocampal neurons, and satisfied the requirement of our experiment; (2) We have obtained the corresponding up- or down-regulated RARa-related signals to meet the experiment demands; (3) RARa can regulate CBP, HAT activity, acetylated histone and the downstream memory-related gene expressions; (4) RARa can not only recruit CBP to become a coactivater, but also can directly regulate the expression levels of CBP, involving in the expressions of learning and memory genes regulated by RA signaling pathways.Chapter Ⅱ The mechanism of CBP-dependent histone acetylation modification in RARa-mediated RA signaling pathway regulating learning- and memory-related signalsObjective:To study the specificity of acetylated histone modification regulated by the CBP signal, as well as its mechanism in RARa-mediated RA signaling pathway regulating learning- and memory- related signals.Methods:Primary cultured neurons were cultured. Three parts ((1)① NC,②siCBP, (2)③Ad-RARa+NC,④ Ad-RARa+siCBP/C646, (3)⑤ RFP+NC,⑥RFP+RA+NC,⑦RFP+NC+siCBP) with different groups of the neurons were set on the sixth day. At first, four siCBP and control (NC) lentiviruses were used to infect the primary cultured hippocampal neurons in the first two groups, and the siCBP lentivirus with the most effective inhibition efficacy and function was selected out and used in the following experiments. And then neurons of the other groups were intervented with RA or infected with adenoviruses encoding RFP, siRARα, Ad-RARa or the NC and siCBP lentivirus/inhibitor C646, respectively. On the seventh day, the mRNA levels of RARa, CBP and the learning and memory genes were analyzed; Protein expressions of RARa, CBP, histone acetylation and HAT activity of the neurons were investigated on the ninth day.Results:(1) CBP mRNA and protein expressions in neurons were found to be with varying degrees of decreases after infections of four kinds of lentivirus siCBP. Furthermore, the most effective lentivirus GR806 was found to effectively inhibit the HAT activity and the protein expressions of Ac-H3, Ac-H4 in neurons (p<0.001/0.01/0.05); (2) Based on the agitated changes in HAT activity, histone acetylation, and the learning and memory genes, infection with siCBP in neurons caused significant downregulations of CBP mRNA (p<0.01) and protein expressions, followed by parallel reductions in HAT activity (p<0.05), H3/H4 acetylation, and mRNA levels of Zif268, cFos, FosB (p<0.001/0.05); (3) CBP mRNA expression was reduced significantly (p<0.05) while RARa mRNA was not obviously affected after treatment with siCBP lentivirus (p=ns). And the expressions of RARa protein levels were showed no significant changes after inhibition of CBP with C646. (4) The CBP mRNA (p<0.05) and protein were significantly induced by RA compared to the RFP group, and they were significantly repressed by siCBP in comparison to the NC group (p<0.05). Additionally, the expressions of Ac-H3/H4 protein and the three memory genes (p<0.001/0.05) were also basically consistent with the changes in CBP levels among the three groups.Conclusions:(1) We have successfully selected out and identified the siCBP lentivirus with the most effective inhibition efficacy and function; (2) We have obtained the corresponding changes of RARa and CBP signals to meet the demand of the experiment; (3) CBP specifically regulates the expressions of HAT activity, acetylated histone Ac-H3, Ac-H4 and the downstream memory- related genes in primary hippocampal neurons; (4) RARa and CBP are in the same signaling pathway and RARa is in the upstream. RA signaling pathway regulates the learning and memory function through the CBP-dependent histone acetylation mediated by RARa.