The Regulation Of Demethylase FTO On VGLUT1 In Manganese-induced Learning And Memory Dysfunction In Mice

Objective:Manganese(Mn)is a metal element necessary to maintain the body’s normal growth,development and cell homeostasis,and as a cofactor for neurotransmitter synthesis and metabolic enzymes,it plays an important role in human and animal brain development and function.Excessive Mn can accumulate in the hippocampus through the blood brain barrier(BBB)and cause neurotoxicity,affecting the brain’s learning and memory capabilities.Studies have found that N6-methyladenosine(m~6A)is most distributed in the brain and plays an important role in learning and memory,dopamine transmitter transmission and other neurological functions.Fat and obesity-related protein(FTO),one of the demethylases of m6A,plays a very important role in the learning and memory functions of the mouse hippocampus.Studies have reported that Type 1 vesicle glutamate transporter(VGLUT1)is involved in learning and memory as a specific marker of glutamate(Glu)terminal in the central nervous system.It is confirmed that the long-term potentiation(LTP)of the hippocampal CA1 region of VGLUT1 gene-deficient mice is significantly reduced,which leads to the decline of learning and memory ability.Methods:In this study,90 adult clean-level wild-type C57BL/6 mice(half of male and female,weight 22-26 g)were divided into 9 groups according to their body weight,with10 mice in each group.The first group was the control group and was given 0.9%normal saline,Groups 2-4 are low,medium,and high-dose manganese-exposed groups;manganese chloride was given at doses of 12.5,25,and 50 mg/kg respectively.Groups5-9 are AAV5-Blank Control,AAV5-FTO Control,AAV5-Blank Mn(50),AAV5-FTO Mn(50),AAV5-FTO Mn(50)+CSB6B groups.The Mn infection method is intraperitoneal injection,while the recombinant adeno-associated virus vector(AAV5)and the VGLUT1inhibitor Chicago sky blue(CSB6B)injection method are stereotactic brain.After the intervention,Morris water maze and T maze behavior experiments were performed to test the learning and memory ability of mice in each group.The content of Mn in the hippocampus of the brain was detected by inductively coupled plasma-mass spectroscopy(ICP-MS).The HE staining method was used to observe the pathological changes of the hippocampus in each group.Western blotting experiment detected the protein expression of FTO and VGLUT1 in the hippocampus of each group.q RT-PCR experiment detected the m RNA expression levels of FTO and VGLUT1 in the hippocampus of each group of mice.The co-expression of FTO and VGLUT1 proteins in each group was observed by immunofluorescence double staining.The m~6A level of VGLUT1 m RNA in the hippocampus of each group of mice was detected by Me RIP-q PCR experiment.Results:Behavioral Morris water maze and T maze results found that Mn exposure can lead to the loss of spatial learning and working memory in mice,resulting in learning and memory dysfunction.ICP-MS detection found that the Mn content in the hippocampus of mice gradually increased with the increase of Mn dose,and the high manganese group was significantly higher than the control group.The HE results showed that compared with the control group,the hippocampal tissue morphology of the mice was significantly changed accompanied by nerve cell deformation disorders and other injuries.The protein and m RNA expression levels of FTO and VGLUT1 in the hippocampus of mice in the manganese-exposed groups changed.The expression levels of FTO protein and m RNA in the middle manganese group and high manganese group showed a downward trend compared with the control group.At the same time,compared with the control group’s hippocampal VGLUT1 protein and m RNA expression levels,both the medium manganese group and the high manganese group decreased.Immunofluorescence staining revealed that FTO and VGLUT1 were co-expressed on nerve cells in hippocampal CA1,CA3,and DG regions,and the fluorescence intensity of both decreased with the increase of Mn dose.The Me RIP-q PCR experiment detected the m~6A enrichment content of VGLUT1 in the mouse hippocampus and found that the high manganese group was significantly enriched compared with the control group.After overexpression of FTO in the hippocampus of mice,the results of ICP-MS found that the overexpression of FTO and VGLUT1 inhibitors did not affect the accumulation of manganese in the hippocampus of mice.The results of q RT-PCR and Western blotting showed that the transcription and protein expression levels of FTO and VGLUT1 in the hippocampus of mice after overexpression of FTO were significantly higher than those of the blank control group.The transcription and protein expression levels of FTO and VGLUT1 in the hippocampus of mice overexpressing FTO and stained with Mn were decreased.Immunofluorescence staining found that similar fluorescence intensity levels appeared in the CA1,CA3 and DG regions of the mouse hippocampus.After overexpression of FTO,the fluorescence intensity of FTO and VGLUT1 in the hippocampus of mice increased significantly,while the fluorescence intensity of FTO and VGLUT1 in the hippocampus of mice overexpression also decreased after the intervention of Mn.Me RIP-q PCR experiments showed that the m~6A methylation modification of hippocampal VGLUT1 m RNA was reduced after overexpression of FTO,and the m~6A methylation modification of VGLUT1 m RNA in the hippocampus tissue of mice that overexpressed FTO and stained Mn increased.The morphology of the hippocampus in the overexpression group did not change,while the hippocampus of the mice overexpressing FTO and inhibited by both Mn and VGLUT1 showed histopathological changes.Behavioral results showed that overexpression of FTO did not significantly change the learning and memory of mice,while overexpression of FTO and simultaneous exposure to Mn decreased the learning and memory ability of mice.The inhibition of VGLUT1 caused mice learning and memory impairment.Conclusion:1.Excessive Mn exposure can accumulate in the hippocampus and cause tissue pathological damage,resulting in learning and memory dysfunction.2.Mn can increase the methylation modification of VGLUT1 m RNA m~6A in the hippocampus by reducing the expression of FTO.3.Manganese reduces the expression of VGLUT1through the increased VGLUT1 m RNA m~6A methylation modification in the hippocampus,which in turn leads to learning and memory dysfunction.