M6A Methylation Modifications In Astrocytes Effects On Alzheimer’s Disease And Its Mechanism

Objective Alzheimer’s disease（AD）is a neurodegenerative disease characterised by the deposition of Aβplaques and phosphorylation of tau proteins,and the aberrant activation of astrocytes is closely associated with the development and progression of AD.m6A modification is one of the most abundant chemical modifications on eukaryotic m RNAs,and is involved in the regulation of biological functions.It is involved in the regulation of biological functions by modulating the variable shear,translation and degradation of m RNAs.In recent years,it has been shown that enzymes responsible for m6A modification are significantly reduced in the postmortem brain tissue of AD patients and are directly related to neuronal survival and function.However,the regulatory role of m6A modification on astrocytes in AD has not been reported.Based on that,the aim of this study was to investigate the effects of m6A methylation modification in astrocytes on astrocyte activity and its relevance to the pathological features of AD disease by using the AD mouse model.Method The matched APP/PS1 transgenic mice and wild type mice of different months of age were used to detect changes in the expression of m6A methylation-related proteins（METTL3,METTL14,FTO,WTAP,etc.）and the total m6A levels of RNA in the hippocampus and cortex by western blot and dot blot methods.APP/PS1-Mettl14^f/fmice were obtained by crossbreeding Mettl14-loxp(Mettl14^f/f)mice and APP/PS1 double transgenic mice,and Mettl14 was specifically knocked out from astrocytes based on the Cre-loxp recombinase system and adeno-associated virus（AAV）lateral ventricular stereotaxic injection technique（Mettl14 knockout group,AAV-GFAP-Cre）.Firstly,the effects of AAV-GFAP-Cre on learning and memory functions in mice were examined using water maze and open field experiments.Subsequently,changes in the number and area of Aβplaques in the brain tissue of APP/PS1-Mettl14^f/f/AAV-GFAP-Cre mice and control mice were detected using immunofluorescence and other methods,and the expression levels of proinflammatory cytokines in neural tissue were measured by ELISA.In vitro experiments,we constructed Mettl14 knockdown astrocyte lines and further validated the effect of Mettl14 knockdown on astrocyte inflammatory factor expression（IL-6,TNF-α,CCL2,etc.）by PCR and ELISA.Finally,based on Me RIP and RNA-seq techniques,we further analyzed and validated the effects of astrocyte Mettl14 specific knockdown on gene expression and related signaling pathways,and explored the underlying molecular mechanisms.Results Compared to wild-type mice Mettl14 expression and the level of total m6A on RNA were decreased in APP/PS1 double transgenic mice.2.Behavioral results indicated that Mettl14 depletion in astrocytes lead to the improvement of spatial memory ability.3.Immunofluorescence results show that conditional knockdown of Mettl14 in astrocytes has no significant change in the effect on Aβplaque deposition in the brain.4.In vivo/in vitro inflammatory index assays revealed that Mettl14 deletion in astrocytes effectively inhibited the expression of proinflammatory cytokines such as IL-6,TNF-αand the chemokine CCL2 compared to controls.5.Based on analysis of sequencing results,it was found that the reduction in inflammation levels caused by knockdown of Mettl14may be mediated by altering the expression of inflammatory negative regulators such as Dusp1 and Socs3.Conclusions Mettl14-mediated m6A methylation in astrocytes plays an important role in regulating inflammation during the development of AD.Down-regulating m6A levels in astrocytes by inhibiting Mettl14 expression can effectively inhibit of astrocyte-mediated neuroinflammation,thereby altering the inflammatory environment and slowing down the pathological development of AD.