Research On The Mechanism Of Foxg1 Improving Alzheimer’s Disease By Promoting Adult Neurogenesis

Alzheimer’s disease（AD）is an age-related neurodegenerative disease characterized by memory decline and cognitive impairment.Loss of neurons is one of its main pathological characteristics.For this reason,in addition to improving neuronal apoptosis and loss of function,promoting the generation of adult newborn neurons is a new direction of current research.In the adult mammalian hippocampus,neural stem cells and neural progenitor cells continue to divide and produce new neurons,a process known as adult neurogenesis.Adult neurogenesis is a multi-step physiological process,including the whole process of proliferation,differentiation,migration and integration of adult neural stem cells into adult neural networks,resulting in the generation of terminal differentiated mature neurons.Neurogenic functional damage in adult mammals is closely related to neurodegenerative diseases,such as Alzheimer’s disease.Promoting the effective differentiation of neural precursor cells into mature neurons in the dentate gyrus（DG）of hippocampus is helpful to the prevention and repair of neurodegenerative diseases.The Foxg1 is a key transcription factor that connects human cognitive function and brain diseases.It is critical for embryonic development and neurogenesis in infancy.Its absence will lead to the dysplasia of the entire cerebral hemisphere.However,the Foxg1 is not only limited to the embryonic brain,but also can be continuously expressed in the adult neural stem cell aggregation area after birth.Up to now,there are few reports on the role of the Foxg1 in adult hippocampal neurogenesis and whether it can improve Alzheimer’s disease by promoting adult neurogenesis.Objectives（1）To detect the correlation between the Foxg1 and the proliferation and activation of adult neural stem cells;（2）To explore whether the Foxg1 can affect the proliferation and differentiation of adult neural stem cells by regulating cell cycle;（3）To explore whether the Foxg1 can promote the progression of adult neural precursor cell lineage;（4）To explore whether the Foxg1 can improve AD by regulating adult neurogenesis.Methods（1）To detect the correlation between the Foxg1 and the proliferation and activation of adult neural stem cells.（1）In vivo model:FOXG1 was induced to overexpress in the subgranular zone（SGZ）of Foxg1^fl/flmice（4 months old）hippocampus by microinjection of targeted brain regions and Cre-AAV mediated gene recombination.The accuracy of injection site was judged by HE staining;The overexpression of FOXG1 was detected by immunofluorescence technique with EGFP labeled antibody.Effects of FOXG1 overexpression on the proliferation of a NSCs were detected by immunofluorescence,immunohistochemistry and Western Blot;The immunofluorescence double labeling method was used to further confirm the influence of FOXG1 overexpression on a NSCs and detect its influence on the activation of a NSCs.（2）In vitro model:Hippocampal primary nerve cells of Foxg1^fl/fland Foxg1^fl/fl-Cre AAV mice were extracted and cultured in vitro.The growth of mature nerve cells was inhibited by serum free medium culture and vitamin A was removed to inhibit the differentiation of neural stem cells in vitro to obtain primary NSCs.In suspension culture,FOXG1 overexpression was observed under optical microscope to evaluate the effect of FOXG1 overexpression on the formation rate of neural spheres in vitro.（2）To explore whether the Foxg1 can affect the proliferation and differentiation of adult neural stem cells by regulating cell cycle.In vitro model:N₂A cell line was used as the research object,and pECMV/Foxg1 plasmids were used to transfect cells through liposome transfection.The overexpression of FOXG1 induced by plasmid transfection was verified by Western Blot;The effect of FOXG1 overexpression on P21 expression was detected by flow cytometry;Then flow cytometry PI staining was combined with P21 immunostaining to explore whether FOXG1 could affect cell cycle by inhibiting P21^CIP1;Hoechst33342 and Pyronin Y were used to stain the DNA and RNA of N₂A cells.The effect of FOXG1 overexpression on cell DNA and RNA content was detected by flow cytometry to evaluate whether FOXG1 overexpression can promote cell cycle exit.HEK293-T cells were taken as the research object,the fluorescent ubiquitin cell cycle indicator FUCCI was introduced,and the cells were infected with FUCCI virus packaged in AAV vector,and the infected cells were screened with puromycin.FUCCI cells were transfected with pECMV/Foxg1 plasmids by liposome transfection.The overexpression of FOXG1 induced by plasmid transfection was verified by Western Blot;The cell cycle changes induced by FOXG1 overexpression tracked by FUCCI system were observed by fluorescence microscope.（3）To explore whether the Foxg1 can promote the progression of adult neural precursor cell lineage.（1）In vivo model:On the basis of verifying the successful construction of FOXG1overexpression model,the impact of FOXG1 overexpression on the expression of neuron lineage markers was detected by immunofluorescence technology,immunohistochemical technology and Western Blot,so as to evaluate whether FOXG1 overexpression can promote the development of adult a NSCs lineage.（2）In vitro model:N₂A cell line was used as the research object.Cells transfected with pECMV/Foxg1 plasmids were detected by the staining of fluorescent labeled phalloidin on the cytoskeleton of filamentous actin in the hippocampal DG region of adult mice.The synaptic growth was detected with fluorescence microscope to evaluate whether FOXG1overexpression could enhance the synaptic plasticity of post mitotic neurons.（4）To explore whether the Foxg1 can improve AD by regulating adult neurogenesis.In vivo model:Nine months old APP/PS1 mice were introduced as AD in vivo model,and FOXG1 was induced to overexpress in DG area of hippocampus of AD mice by targeted microinjection in brain area and AAV mediated gene recombination technology.The accuracy of injection site was judged by HE staining;The overexpression of FOXG1 was detected by GFP labeled antibody using immunofluorescence technique.The effect of FOXG1 overexpression on the proliferation of a NSCs was detected by immunofluorescence technique;The effect of FOXG1overexpression on the expression of neural lineage markers in AD model mice was detected by immunofluorescence technology,so as to evaluate whether FOXG1 overexpression can promote the lineage progression of AD model mice.Results（1）Cre-AAV microinjection successfully induced FOXG1 overexpression in hippocampal DG region of Foxg1^fl/flmice;In the absence of significant differences in endogenous q NSCs populations,FOXG1 overexpression caused an increase in the number of q NSCs in the DG region of the adult mouse hippocampus;FOXG1 overexpression promotes the proliferation and activation of q NSCs in the hippocampal DG region of adult mice;Overexpression of FOXG1 causes the increase of spherulation rate of neural stem cells cultured in vitro in suspension.It is proved that FOXG1 can promote the proliferation and activation of a NSCs.（2）FOXG1 overexpression can increase the number of cells in G₁-phase and inhibit the expression of P21 in G₁-phase;Overexpression of FOXG1 can promote cells blocked in G₁-phase to exit the cell cycle and enter G₀-phase.It is proved that FOXG1 can make cells stay in the G₁-phase by inhibiting P21,and promote the increased G₁-phase cells to exit the cell cycle.（3）FOXG1 overexpression can promote the proliferation of IPCs in the DG region of adult mouse hippocampus and increase the number of IPCs in the DG region;FOXG1 overexpression can promote the differentiation of neural precursor cells in the DG area of the adult mouse hippocampus to produce nerve cells and mature neurons;FOXG1 overexpression can enhance synaptic plasticity of post mitotic neurons;FOXG1 overexpression could not induce a NSCs to differentiate into oligodendrocytes.（4）APP/PS1 mice were injected with Foxg1-AAV and verified the GFP label,which proved that FOXG1 was successfully induced to overexpress in the pathological brain area of AD model mice;Overexpression of FOXG1 can increase the quantity of q NSCs in hippocampus DG region of AD mice;FOXG1 overexpression can increase the number of IPCs and mature neurons in the hippocampus DG region of AD model mice.ConclusionsFOXG1 has the function of expanding the adult NSC pool and promoting the progression of the adult neural precursor cell lineage.Its mechanism may be achieved by inhibiting P21 to regulate cell cycle.FOXG1 can improve the neuron loss associated with AD by promoting the neurogenesis in adult hippocampus.