Study On Molecular Mechanisms Of VPA Promoting Neural Stem Cell Differentiation And Improving Alzheimer’s Phenotypes

ObjectiveEpigenetic regulations involved in multiple aspects of neuronal function and development,including occurrence and development of Alzheimer’s disease(AD)and other neurodegenerative diseases.Sodium valproate(VPA),nonspecifically inhibits the function of Class Ⅰ and Class Ⅱ HDACs,promotes histone hyperacetylation.In our previous study,we found that VPA selectively increased the levels of histone H4K16 ac and H3K9 ac in the process of promoting the differentiation of embryonal carcinoma cells.However,genes and pathways that regulated by VPA orchestrated histone acetylation during the differentiation process are still need to be investigated.On the other hand,it is reported that VPA alleviates memory deficits and pathological characteristics,such as senile plaques(SP)and intracellular neurofibrillary tangles(NFTs),in transgenic mouse model of Alzheimer’s disease by inhibiting the activity of GSK-3β.It is not yet clear whether VPA reduces β-amyloid production via HDAC inhibition.Recently,it is reported that the abnormal differentiation of neural stem cells is closely related to AD.However,whether VPA is involved in the differentiation of neural stem cells,thereby improving the behavior and pathological characteristics in AD mouse models,needs further research.Therefore,we first studied the genes and pathways that affected by histone acetylation during the differentiation process of neural stem cells promoted by VPA.Next,we studied whether VPA could improve behavior and memory functions in APP/PS1 mouse by reducing Aβ production via HDAC inhibition,and by promoting the differentiation of hippocampal neurons.Our research results will provide new information for further revealing the pathogenesis and developing new treatments for AD.Methods1.In our research,NE-4C neural stem cells were selected to study the effect of VPA on neuronal lineage differentiation.The expression levels of stem markers including Nestin,Oct4,and Sox2,and differentiation markers such as Vimentin,Neu N,and GFAP in NE-4C cells were detected by Western Blots and immunofluorescence assays.Acetylation levels of histone H4,H4K16 and H3K9 in NE-4C cells were detected by Western Blot.2.The whole transcriptome of NE-4C cells was sequenced by RNA-Seq.The chromatin accessibility of NE-4C cells was captured and sequenced by ATAC-Seq.The chromatin binding or distribution of histone H4K16 ac modifications in NE-4C cells were detected by CUT&TAG.A combined analysis of RNA-Seq,ATACSeq,and CUT&TAG was used to identify genes and pathways that affected by VPA promoted histone acetylation in the process of neurological lineage differentiation.3.The results of RNA-Seq,ATAC-Seq,and CUT&TAG suggested that VPA could affect the chromatin accessibility and transcription changes of genes in the hippo signaling pathway through H4K16 ac hyperacetylation.The protein expression level of YAP1,a key factor in the hippo signaling pathway,was detected by Western blotting while nuclear localization of YAP1 was detected by immunofluorescence assay.4.Swedish double mutant APP overexpressed N2a(N2a-APPswe)cells,SH-SY5Y(SH-SY5Y-APPswe)cells,and double transgenic AD mouse model expressing mutant human presenilin 1(PS1-d E9)and Swedish mutant human amyloid precursor protein(APPswe)were selected to study the effect of VPA on APP expressions and to investigate related molecular mechanisms.5.Effects of VPA,WT161(a specific histone deacetylase 6 inhibitor)and Santacruzamate A(a specific histone deacetylase 2 inhibitor)on expressions of HDACs in N2a-APPswe and SH-SY5Y-APPswe cells,and in APP/PS1 mouse cortex and hippocampus were detected by Western Blot.6.Effects of HDAC6 on expressions of APP metabolism-related proteins and JNK/c-Jun were observed by knocking down or promoting expressions of HDAC6 in N2a-APPswe cells.7.Effects of VPA,WT161 and Santacruzamate A on expressions of APP metabolism related proteins(APP,ADAM10,BACE1,PS-1 and Aβ42)in N2a-APPswe and SH-SY5Y-APPswe cells,and in APP/PS1 mouse cortex and hippocampus were detected by Western Blot and ELISA.8.Effects of VPA and WT161 on expressions of p-JNK,JNK3,and c-Jun in N2 aAPPswe cells were detected by Western Blot.9.Aβ plaque depositions in cerebral cortex and hippocampus of APP/PS1 mouse were detected by immunohistochemistry and thioflavin-S staining.We also detected the effects of VPA and WT161 on daily behavior,short-term learning,spatial localization,and long-term memory in APP/PS1 mouse by nest building test,novel object recognition,and Morris water maze.10.Effects of VPA and WT161 on expressions of p-JNK,JNK3,and c-Jun in APP/PS1 mouse cerebral cortex were detected by Western Blot.11.The expression levels of stem marker Oct4,and differentiation markers Neu N and GFAP in hippocampus of APP/PS1 mice were detected by Western Blot.Protein expression levels of YAP1 in hippocampus of APP/PS1 mice was detected by Western blotting.ResultVPA may mediate the loss of pluripotency and promotes differentiation of NE-4C cells by inhibiting YAP1 expression.1.VPA promotes the differentiation of NE-4C cells into mature neurons by increasing the acetylation levels of total histone H4 in NE-4C cells,especially at H4K16 ac and H3K9 ac sites.2.The integrated KEGG enrichment analyses of RNA-Seq,ATAC-Seq and CUT&TAG indicated that the chromatin accessibility and transcription levels of key regulatory genes in the hippo signaling pathway were significantly altered by VPA in NE-4C cells,the expression of YAP1 was significantly inhibited in the process of VPA promoted NE-4C cells differentiation.These results suggested that VPA increases the acetylation level of histone H4K16,causing changes in chromatin accessibility,leading to down-regulation of YAP1 expression in the hippo signaling pathway during the differentiation of NE-4C cells into mature neurons.These results suggested that VPA may mediate the loss of pluripotency and promote differentiation of NE-4C cells by inhibiting the YAP1 expression.VPA could reduce the levels of soluble Aβ1-42 by inhibiting HDAC6 in N2 aAPPswe and SH-SY5Y-APPswe cells.1.VPA could significantly inhibit expressions of HDAC6 in N2a-APPswe and SHSY5Y-APPswe cells.Further analysis of the relationship between HDAC6 and APP metabolism-related proteins showed that HDAC6 was positively correlated with BACE1 and PS-1 protein expressions in N2a-APPswe cells,but negatively correlated with the ADAM10 protein expression.2.VPA and WT161(a specific HDAC6 inhibitor)significantly inhibited BACE1-mediated amyloid proteolysis,thereby reducing the level of Aβ1-42 in N2 aAPPswe cells.While Santacruzamate A,a specific HDAC2 inhibitor,had no significant effect on BACE1-mediated amyloid proteolysis.These results suggested that VPA may reduce the levels of soluble Aβ1-42 by inhibiting HDAC6 in N2a-APPswe and SH-SY5Y-APPswe cells.3.VPA and WT161 could inhibit the expression of p-JNK,JNK3,and c-Jun in N2 aAPPswe cells.After knocking down of HDAC6 expression,we found that expressions of JNK3 and c-Jun in N2a-APPswe cells decreased significantly.These results suggested that VPA may inhibit the activation of JNK/Jun pathway by inhibiting HDAC6 in N2a-APPswe cells.4.SP600125(a specific JNK inhibitor)significantly inhibited the activation of JNK/Jun pathway.At the same time,it could inhibit the expression of BACE1 and PS-1 and promote the expression of ADAM10.These results suggested that inhibition of JNK signaling pathway could inhibit BACE1-mediated amyloid proteolysis.VPA could inhibit Aβ production and behavioral deficits by inhibiting HDAC6 and promoting the differentiation of neural stem cells in APP/PS1 mouse.1.VPA and WT161 could improve daily behavior,short-term learning,spatial localization,and long-term memory in APP/PS1 mice.2.VPA and WT161 could improve the deposition of Aβ amyloid in cerebral cortex,hippocampus,and in entorhinal cortex of APP/PS1 mice,decrease the level of plasma soluble Aβ1-42.These results suggested that VPA could inhibit Aβ production and behavioral deficits in APP/PS1 mouse.3.VPA and WT161 significantly inhibited BACE1-mediated amyloid proteolysis in cerebral cortex and hippocampus of APP/PS1 mice.VPA and WT161 could inhibit expressions of p-JNK,JNK3,and c-Jun in the cerebral cortex of APP/PS1 mice.These results suggested that VPA may regulates the expression of BACE1 by inhibiting HDAC6 and the JNK/c-Jun signal pathway,thereby inhibits BACE1-mediated amyloid proteolysis,reduces the Aβ deposition in the brain of APP/PS1 mouse.4.VPA could significantly increase the expression of the neuron marker Neu N while inhibit the expression of Oct4 and GFAP in the hippocampus of APP/PS1 mice.The VPA promoted neuron generations in the hippocampus of APP/PS1 mice,which might relate to the inhibition of YAP1 expression by VPA.These results suggested that VPA may promote the differentiation of neural stem cells in the hippocampus by inhibition of YAP1 expression by VPA,and finally improve the cognitive memory and behavior of AD mice.Conclusion1.In summary,VPA could affect the expressions of genes that related to the hippo pathway through histone acetylation modification at specific sites,thereby promoting the differentiation process of neural stem cells.2.In our study,there could be two main reasons for the improvement of cognition and memory in APP/PS1 mice that treated by VPA: One was to reduce the Aβ amyloid deposition by inhibiting HDAC6,while the other was to promote the differentiation of neural stem cells in the hippocampus.Our study provides new information for understanding the molecular mechanism of VPA treatment of ADrelated phenotypes,as well as in searching new targets for AD therapy.