The Role And Mechanism Of METTL16 Regulating MAT2A MRNA M~6A Methylation Modification In Alzheimer’s Disease Pathology And Learning Memory

N6-methyladenosine（m⁶A）is the most prevalent RNA modification that contributes to normal embryonic brain development and memory formation,and also plays an important role in synaptic function.Changes in synaptic function are major mechanisms in Alzheimer’s disease（AD）.Recent studies have shown that m⁶A modification and its related proteins play key roles in the development of AD,but the exact mechanism is not yet clear.Our group has found that methyltransferase-like protein 16（METTL16）enhanced the overall m⁶A by promoting the stability of methionine adenosyltransferase 2A（MAT2A）m RNA.Based on this,we further demonstrated the mechanism of METTL16in AD model mice.Objective:This experiment investigated the role and mechanism of METTL16 in regulating MAT2A m RNA m⁶A methylation in 5x FAD mice,and deeply investigated the physiopathological function of METTL16 in AD pathology and learning memory.These data provides new research avenues and directions for further study of AD.Methods:1.Effect of METTL16 on learning memory,postsynaptic dense protein 95（PSD95）and synaptic vesicle protein（Syp）and dendritic spine density,and Aβ_1-42 expression in 5x FAD mice.The mouse hippocampal tissues of 6-month-old 5x FAD male mice and 6-month-old C57BL/6J male mice were peeled and m RNA extracted,and m⁶A colorimetry was used to detect the overall level of m⁶A methylation in the hippocampal region of 5x FAD mice.Western blot was used to detecte the expression levels of METTL16 proteins in mouse hippocampal tissues.Using the brain stereotaxic injection technique,AAV9-oe-METTL16 adeno-associated virus was performed in the CA1 region of the hippocampus bilaterally.These mice were divided into AAV-oe-control group and AAV-oe-METTL16 group,10 mice in each group.After successful overexpression of METTL16,the m⁶A methylation level in the hippocampal region of 5x FAD mice was detected by m⁶A colorimetry.The learning and memory abilities of mice were examined by new object recognition,Y-maze and Morris water maze assay.After the behavioral assay,the changes in the expression level of synaptic function-related proteins in the hippocampus were detected by Western blot and q RT-PCR,the dendritic spine density of neurons in the CA1 region of the hippocampus was detected by Golgi staining,the expression level of Aβ_1-42 in the hippocampus was detected by ELISA.2.Effect of MAT2A on learning memory,synaptic protein PSD95,Syp and dendritic spine density,and Aβ_1-42 expression in 5x FAD mice.The mouse hippocampal tissues of 6-month-old 5x FAD male mice and 6-month-old C57BL/6J male mice were peeled and proteins extracted,and Western blot was used to detecte the expression levels of MAT2A proteins in mouse hippocampal tissues.Using the brain stereotaxic injection technique,AAV9-oe-MAT2A adeno-associated virus was performed in the CA1 region of the hippocampus bilaterally.These mice were divided into AAV-oe-control group and AAV-oe-MAT2A group,10 mice in each group.After successful overexpression of MAT2A,the learning and memory abilities of mice were examined by new object recognition,Y-maze and Morris water maze assay.After the behavioral assay,the changes in the expression level of synaptic function-related proteins in the hippocampus were detected by Western blot and q RT-PCR,the dendritic spine density of neurons in the CA1 region of the hippocampus was detected by Golgi staining,the expression level of Aβ_1-42 in the hippocampus was detected by ELISA.3.Effects and mechanisms of METTL16 regulation of MAT2A on learning memory capacity,synaptic protein PSD95,Syp and dendritic spine density and Aβ_1-42expression in 5x FAD mice.Using the brain stereotaxic injection technique,AAV9-oe-METTL16adeno-associated virus was performed in the CA1 region of the hippocampus bilaterally.These mice were divided into AAV-oe-control group and AAV-oe-METTL16 group,5 mice in each group.After successful overexpression of METTL16,the hippocampal MAT2A expression level in mice was detected by Western blot.The m⁶A enrichment of the fragmented RNA was performed by Anti-m⁶A-RIP assay using antibody against m⁶A methylation to make adsorbed magnetic beads to detect changes in the m⁶A methylation level of MAT2A m RNA in mouse hippocampal tissue after overexpression of METTL16.Using the brain stereotactic injection technology,bilateral hippocampal CA1 region AAV9-oe-M16+sh-MAT2A adeno-associated virus was performed.These mice were divided into AAV9-oe-M16+sh-control group,10 mice in each group.New object recognition,Y-maze and Morris water maze assay were used to detect the learning and memory ability of mice after overexpressing METTL16 while knocking down MAT2A,and after the behavioral detection,Western blot and q RT-PCR were used to detect the changes in the expression level of hippocampal synaptic function-related proteins in mice after overexpressing METTL16 while knocking down MAT2A.The dendritic spine density of neurons in the CA1 region of the hippocampus of mice after overexpressing METTL16 and knocking down MAT2A was detected by Golgi staining,and the level of Aβ_1-42 in mouse hippocampal tissues was detected by ELISA.Results:1.M⁶A colorimetric experimental results showed that the overall level of m⁶A in hippocampal tissues of 5x FAD mice was reduced compared to WT mice.Western blot results showed that METTL16 expression levels were reduced in hippocampal tissues of 5x FAD mice compared with WT mice.The results of Western blot and q RT-PCR experiments showed that the overexpression efficiency was significant.The results of the m⁶A colorimetric method showed that the overall level of m⁶A in hippocampal tissues of 5x FAD mice increased after overexpression of METTL16 compared with control mice.The results of the new object recognition experiment showed that,after overexpressing METTL16,the preference degree of mice for new objects was significantly enhanced in the object recognition test experiment with an interval of 2 h and 24 h compared with the control group.The results of the Y maze experiment showed that,after overexpressing METTL16,the proportion of exploration time and exploration distance of mice in new hetero arms increased significantly compared with the control group.The results of the Morris water maze experiment showed that in the positioning cruise experiment,the AAV-oe-METTL16 group had reduced escape latency on day 5 compared to the AAV-oe-control group;in the exploration experiment,mice in the AAV-oe-METTL16group traversed the platform significantly more often than the AAV-oe-control group and took longer to explore in the target quadrant than the AAV-oe-control group.The results of Western blot experiments showed that the expression of PSD95 and Syp in hippocampal tissues of METTL16 mice was elevated compared with the control group.The results of q RT-PCR experiments showed that PSD95 and Syp m RNA levels were elevated in hippocampal tissues of mice overexpressing METTL16compared with the control group.The results of the experimental results of Golgi staining showed that the dendritic spine density of hippocampal neurons in the CA1 region of overexpressing METTL16 mice was significantly increased compared with the control group.The results of ELISA detection showed that the expression level of Aβ_1-42in mouse hippocampal tissues overexpressing METTL16 was reduced compared with the control group.2.Western blot results showed that MAT2A expression levels were reduced in hippocampal tissues of 5x FAD mice compared with WT mice.Western blot experimental results showed that the expression level of MAT2A protein in mouse hippocampal tissues increased after overexpression of METTL16 compared with the control group.Western blot and q RT-PCR experimental results showed that the MAT2A overexpression efficiency was significant.The results of the new object recognition experiment showed that,after overexpressing MAT2A,the preference degree of mice for new objects in the object recognition test experiment with an interval of 2 h and 24 h was significantly enhanced compared with the control group.The results of the Y maze experiment showed that,the proportion of exploration time and exploration distance of mice in the new arm increased significantly after overexpressing MAT2A compared with the control group.The results of the Morris water maze experiment showed that in the positioning cruise experiment,the AAV-oe-MAT2A group had reduced escape latency on day 5 compared to the AAV-oe-control group;in the exploration experiment,mice in the AAV-oe-MAT2A group traversed the platform significantly more often than the AAV-oe-control group and took longer to explore in the target quadrant than the AAV-oe-control group.Western blot results showed that the expression of PSD95 protein and Syp protein in mouse hippocampal tissues was increased after overexpression of MAT2A compared with the control group.The results of q RT-PCR experiments showed that PSD95 and Syp m RNA levels were elevated in hippocampal tissues of mice overexpressing MAT2A compared with the control group.The results of the experimental results of Golgi staining showed that the dendritic spine density in the CA1 region of the hippocampus of mice overexpressing MAT2A was significantly increased compared with the control group.The results of ELISA detection showed that the level of Aβ_1-42 in mouse hippocampal tissues overexpressing MAT2A was reduced compared with the control group.3.Western blot and q RT-PCR experimental results show that the knockdown efficiency is significant.The results of the new object recognition experiment showed that compared with the control group,after overexpressing METTL16 and knocking down MAT2A,the preference for new objects in the object recognition test experiment with an interval of 2h and 24h was significantly reduced.The results of the Y maze experiment showed that compared with the control group,after overexpressing METTL16 and knocking down MAT2A,the proportion of exploration time and exploration distance of mice in the new hetero arms were significantly reduced.The results of the Morris water maze experiment showed that in the positioning cruise experiment,the AAV-oe-M16+sh-MAT2A group had increased escape latency on day 5 compared to the AAV-oe-M16+sh-control group;in the exploration experiment,mice in the AAV-oe-M16+sh-MAT2A group traversed the platform significantly less often than the AAV-oe-M16+sh-control group and took less time to explore in the target quadrant than the AAV-oe-M16+sh-control group.Western blot results showed that the expression of PSD95 protein and Syp protein in mouse hippocampal tissues after overexpressing METTL16 while knocking down MAT2A was reduced compared with the control group.The results of q RT-PCR experiments showed that compared with the control group,overexpression of METTL16 and knockdown MAT2A at the same time reduced PSD95 and Syp m RNA levels in mouse hippocampal tissues.The results of the Golgi staining assay showed that compared with the control group,the dendritic spine density of hippocampal neurons in the CA1region of mice overexpressing METTL16 and knocking down MAT2A group was significantly reduced.The results of ELISA detection showed that the expression levels of Aβ_1-42in mouse hippocampal tissues that overexpressed METTL16 while knocking down MAT2A were elevated compared with the control group.Anti-m⁶A-RIP experimental results showed that after overexpression of METTL16,the level of MAT2A m RNA m⁶A methylation in hippocampal tissues of 5x FAD mice was increased compared to control mice.Conclusions:1.M⁶A methylation modification levels and METTL16 expression were reduced in the hippocampus of 5x FAD mice.Overexpression of METTL16increased the overall level of m⁶A methylation,improved learning memory,increased PSD95 and Syp expression levels and dendritic spine density,and decreased Aβ_1-42 accumulation in mice.2.5x FAD mice showed reduced MAT2A expression in hippocampal tissue.Overexpression of MAT2A improved learning memory,increased PSD95 and Syp expression levels and dendritic spine density,and reduced Aβ_1-42accumulation in mice.3.Overexpression of METTL16 increased MAT2A expression in mouse hippocampal tissues and improved learning memory,increased PSD95 and Syp expression levels and dendritic spine density,and decreased Aβ_1-42accumulation by regulating MAT2A m RNA m⁶A methylation modifications.