The Role Of RNA Demethylase FTO In Manganese-induced Hippocampal Learning And Memory Disfunction

Objective:Manganese（Mn）is an essential trace element in the human body,but long-term close contact with Mn compounds and lack of protection can cause manganism.The early clinical manifestations of patients with Mn poisoning are dizziness,headache,and memory loss.After excessive Mn enters the body,it can accumulate in the hippocampus,an important organ of learning and memory.Interfering with the learning and memory signal pathways and inducing learning and memory disorders.Recent studies have found that N6-methyladenosine（m⁶A）has potential neurobiological functions in the nervous system,and messenger ribonucleic acid（mRNA）m⁶A methylation can regulate local translation during memory formation.Fat mass and obesity-associated protein（FTO）is an m⁶A demethylase and plays an important role in the formation of mouse hippocampal-dependent memory.The purpose of this study was to investigate the role of FTO in Mn-induced hippocampal learning and memory disfunction,and to provide an experimental basis for further research on the mechanism of Mn toxicity and prevention strategies.Methods:Clean（CL）adult wild-type C57BL/6 mice were selected,half is female and half is male,the body weight is 20±2 g.It was conducted in 2 stages,divided into 11groups,10 mice in each group,with a total number of 110.In vivo experiments using brain stereotactic methods for Mn exposure and intervention in C57BL/6 mice.MnCl₂was injected to construct the Mn exposure models,AAV5-FTO was injected to construct the FTO overexpression models.After the infection,recover for 1 week,and the body weight of the mice was weighed weekly.Small animal body composition analysis,morris water maze,T maze and radial arm maze behavior experiments were performed.4 mice in each group were perfused with 4%paraformaldehyde,2 were embedded the whole brain,and 2 were removed hippocampus for transmission electron microscope experiments.The whole brain of 6 mice was taken from each group,bilateral hippocampus was separated,and the whole brain and hippocampus were weighed.Take one side hippocampus of each mouse,labeled it in an EP tube,and freezed it for protein extraction.Put the other side hippocampus into an enzyme-free EP tube and add RNAiso to extract RNA.Brain slices were cut with the vibrating slicer,exposed and intervened using artificial cerebrospinal fluid perfusion with drugs.Brain slice Mn exposure models,MA2FTO inhibition models.After exposure and intervention,electrophysiological examination of brain slices was performed using the MED64 system.SH-SY5Y cells were used for in vitro experiments.Cells were exposed to MnCl₂ for24 h to establish a cell Mn exposure models.Intervention with MA2 for 2 h to construct FTO inhibition models.After exposure and intervention,CCK-8 method was used to detect cytotoxicity.The morphology of the cells was observed with an inverted microscope,and proteins and RNA were extracted for detection.Result:Behavioral tests found that Mn exposure can impair the spatial memory,alternating memory,working memory,and reference memory of mice.After weighing,it was found that Mn exposure did not affect whole brain organ coefficients and hippocampus organ coefficients in mice.After HE staining and Nissl staining,it was found that Mn can damage the hippocampus morphology of mice.TEM observations revealed that Mn can cause damage to the hippocampal ultrastructure.The MED64system test showed that the electrophysiological condition of the hippocampus was impaired.Western blotting and RT-PCR result showed that Mn exposure inhibits FTO protein and mRNA expression.AAV-5 intervention overexpressed FTO,MA2intervention inhibited FTO function,which further proved that FTO plays an important role in Mn-induced learning and memory,hippocampal morphology injury,SH-SY5Y cell damage and electrophysiological injury in mice.Conclusion:Mn can affect the ability of learning and memory in mice and damage the hippocampal morphology.Mn can reduce FTO expression in hippocampus and SH-SY5Y cells.Mn can down-regulates FTO expression in the hippocampus and leading to learning and memory impairment.