FoxG1 As A Potential Therapeutic Target For Alzheimer’s Disease With A Particular Focus On Neuronal Cell Cycle Regulation

Alzheimer’s Disease（AD）is a neurodegenerative disease with progressive memory and cognitive impairment as its primary clinical features.β-amyloid（Aβ）deposition and neuronal apoptosis are considered to be crucial factors in the pathogenesis of it.Studies have shown that there are neuronal cell cycle re-entry and cell cycle pathological abnormalities in the AD brain.FoxG1 is an important transcription factor in the development of the brain,its role and mechanism in AD are rarely reported at home and abroad.In this study,we found that 1)the occurrence of AD is accompanied by down-regulation of FoxG1 expression and up-regulation of P21 expression;2)FoxG1 can reduce Aβdeposition and neuronal apoptosis in AD mice,and improve behavioral defects in AD mice;3)FoxG1 improves the occurrence of AD by targeting neuron cell cycles,which in turn reduces AD neuron cell cycle re-entry and G₂/M blocking.As a result,FoxG1 is likely to be a new target for AD treatment and intervention.Objectives（1）We aimed to explore the role of FoxG1 and P21 in the pathogenesis of AD.（2）We aimed to further demonstrate whether FoxG1 mediates the down-regulation of P21 and whether there is a correlation between FoxG1 and P21.（3）To explore whether FoxG1 can improve the occurrence of AD.（4）To investigate whether FoxG1 can improve the occurrence of AD by regulating the abnormal neuronal cell cycle entry and cell cycle progression.Methods（1）We aimed to explore the role of FoxG1 and P21 in the pathogenesis of AD:（1）Construction and verification of AD cell model and AD transgenic mouse model:Choosing（2 months old and 6 months old）APP/PS1 mice as model animals:through molecular biology technology and behavior technologies（NOR,NPR,PPI and MWM）to verify the apparent effectiveness（memory,cognition,sensory information gating ability）and mechanism effectiveness of AD model mice;Evaluation of Aβdeposition and apoptosis-related parameters;Transfecting APPswe plasmid in N₂A cell line to construct an AD vitro model and then to detect the expression of Aβby Western blotting.（2）Using immunoblotting and immunohistochemistry techniques to check the protein of P21 and FoxG1 in AD in vivo and vitro models.（3）Flow cytometry and Immunoblotting were used to check the apoptosis of N₂A cell line after P21overexpression.（2）We aimed to further demonstrate whether FoxG1 mediates the down-regulation of P21 and whether there is a correlation between FoxG1 and P21:CAG-loxp-stop-loxp-FoxG1-IRES-EGFP(FoxG1^fl/fl)transgenic mice were introduced and microinjection of r AAV2/9-CMV-Cre-WPRE-p A（Cre-AAV）into cortical brain region.FoxG1 overexpression mice(FoxG1^fl/fl+Cre-AAV)were obtained by Cre/Lox P gene recombinant technique.Then the expression of P21 was detected.FoxG1 and P21plasmids were overexpressed by liposome transfection in HEK293T cells line,and whether there was a direct correlation between FoxG1 and P21 was verified by CO-IP technology.（3）To explore whether FoxG1 can improve the occurrence of AD:Construction of FoxG1 overexpression mice(AD/FoxG1^fl/fl+Cre-AAV)on the basis of AD:AD mice and FoxG1^fl/flwere crossed to obtain progeny AD/FoxG1^fl/fl.Using Cre/Lox P gene recombination technology,Cre-AAV was microinjected into the cortex of AD/FoxG1^fl/flto obtain AD/FoxG1^fl/fl+Cre-AAV mice.Evaluating whether the overexpression of FoxG1 could reduce the Aβplaque deposit by immunohistochemical staining.Using behavioral experiments including NOR,PPI and MWM experiments to evaluate whether the abnormal behaviour of AD mice after FoxG1 overexpression was improved,and to show the effectiveness of overexpression of FoxG1 in the brain to improve AD-like behavio.（4）To investigate whether FoxG1 improve the occurrence of AD by regulating the abnormal neuronal cell cycle entry and cell cycle progression:（1）Assessing the expression of P21 and P21 kinase-PAK3,and detecting the expression of marker proteins in each phase of cell cycle G₁,G₁/S,G₂ and G₂/M by immunohistochemical technique,and checking the abnormal cell cycle marker proteins of AD neurons.Bcl2and BAX were used to check the apoptosis of neurons after overexpression of FoxG1.（2）We separately transfected FoxG1,APPswe and APPswe+FoxG1 plasmids in N₂A cells line,and then explored the cell distribution in G_0/1,S and G₂/M phases with a cell cycle test kit.Results（1）Compared with 2-month-old AD mice,Aβdeposition in 6-month-old APP/PS1mice was more significant.NOR and NPR experiments showed that 6-month-old AD mice exhibit deficits in short-term habituation.MWM experiment showed that AD mice had insufficient spatial memory and learning abilities,and PPI experiment demonstrated low prepulse inhibition.Therefore,6-month-old APP/PS1 mice were selected as the experimental model（AD mice）.FoxG1 expression decreased and P21expression was up-regulated and accompanied by apoptosis in AD mice and N₂A cells line transfected with APPswe plasmid model.In addition,P21 overexpression induced increasing in apoptosis rate by flow cytometry.（2）FoxG1^fl/fl+Cre-AAV transgenic mice showed down-regulation of P21,and in vitro experiments confirmed that FoxG1 has a direct relationship with P21 by Co-IP technology.（3）FoxG1 reduced the expression of Aβdeposition.NOR experiment showed that FoxG1 overexpression improved the deficits in short-term habituation of AD mice,and MWM experiment showed that FoxG1 overexpression could improve the lack of spatial memory in AD mice and obtained slightly higher prepulse inhibition in PPI experiment.（4）AD vitro model showed that the cell cycle passed the G₁/S checkpoint,but blocked in the G₂/M phase.Overexpression of FoxG1 reduced the blockage of AD cells and thus reduced cell apoptosis.FoxG1 overexpression could antagonize the expression of P21 and its kinase PAK3,and negatively regulated the expression of cell cycle ectopic marker protein.Conclusions:The occurrence of AD is accompanied by the down-regulation of FoxG1expression;FoxG1 can alleviate Aβdeposition and neuronal apoptosis in AD mice,and improve behavioral defects in AD mice;FoxG1 improves the occurrence of AD by reducing AD neuronal cell cycle re-entry and G₂/M-phase block.Therefore,FoxG1 is likely to become a new target for AD treatment and intervention.