Role And Mechanism Of RNA Demethylase FTO Regulating GRIN1 Expression In Manganese Induced Learning And Memory Dysfunction

Objective:Manganese（Mn）is a neurotoxic substance that exceeds the standard in drinking water sources in many parts of China.It has the ability to penetrate the blood-brain barrier and accumulate in the hippocampus,leading to symptoms such as memory loss and emotional instability.The demethylase fat mass and obesity-associated protein（FTO）is highly expressed in the brain and plays a significant role in brain development,learning,memory,as well as Alzheimer’s disease and other brain disorders.The hippocampus is rich in the expression of N-methyl-D-aspartate（NMDA）receptors.Among them,N-methyl-D-aspartate ionic glutamate receptor 1（GRIN1）is an essential subunit and a key subunit for learning and memory formation.The upstream and downstream regulatory mechanisms of the key molecule GRIN1 in FTO regulation of learning and memory have not yet been elucidated.SRY-box containing gene 2（SOX2）is a common transcription factor that can provide potential effector points for screening FTO abnormalities caused by manganese poisoning in the population.YTH N6methyladenosine RNA binding protein 3（YTHDF3）is an RNA methyl recognition protein（readers）that can collaborate with other recognition proteins.By binding to m RNA containing m⁶A and promoting the degradation of target m RNAs.In this study,we utilized human neuroblastoma cell line SH-SY5Y,mouse hippocampal neuron cell line HT22,and rat adrenal pheochromocytoma cell line PC12for in vitro experiments.For in vivo experiments,C57BL/6 mice were employed.And mouse hippocampal slices were utilized for electrophysiological experiments.Exploring the role and mechanism of demethylase FTO in the modification of GRIN1 m⁶A under manganese-induced learning and memory impairment is of great practical significance.We employed various experimental methods,including behavioral assessments,molecular biology techniques,and bioinformatics analyses,to uncover new targets and strategies for the prevention and control of manganese poisoning.These efforts aim to safeguard the health of individuals exposed to environmental manganese in China.Methods:1.In the first part of the animal experiment,C57BL/6 mice（half male and half female）were selected and grouped according to the principle of weight balance（n=16）.The manganese stained mice were divided into four groups and intraperitoneally injected with 0.9%Na Cl and Mn Cl₂（12.5,25,50 mg/kg）.The FTO inhibitory components of mice were divided into 5 groups,and FTO inhibitor MA2（20 mg/kg）was subcutaneously injected.The adenovirus overexpression components of FTO in mice were divided into 6 groups,with stereotactic injection of AAV5 virus(virus titer was1×10¹² Vg/m L,1μL for each side,total injection 2μL virus).The intervention agonist overexpression components of FTO in mice were divided into 5 groups,subcutaneous injection of FTO agonist VPA（4 mg/kg）.The treatment concentration is 5 mg/kg.Poison once a day for two consecutive weeks.Before and after exposure and intervention,record the weight of mice,conduct body composition analysis,and conduct behavioral experiments.Perform ICP-MS detection,observe hippocampal morphology,and detect protein and m RNA expression.The electrophysiological experiment of hippocampal slices was conducted using a vibration slicer to cut hippocampal slices from the whole brain samples of wild-type C57BL/6 mice.The slices were perfused with artificial cerebrospinal fluid containing drugs for 5 minutes for toxicity and intervention.The manganese stained brain slices were divided into four groups,and Mn Cl₂（25,100,400μmol/L）was used for manganese staining.The FTO inhibitory component of brain slices was divided into 5 groups,using the FTO inhibitor MA2（100μmol/L）intervention.The overexpression components of FTO in brain slices were divided into 5 groups and intervened with FTO agonist VPA（2 mmol/L）.After exposure and intervention,the MED64 system was used for electrophysiological testing of brain slices.Record the f EPSP waveform,long-term potentiation（LTP）induction success rate,%f EPSP amplitude before and after LTP induction,and paired pulse facilitation（PPF）ratio.Cell experiments were conducted using human neuroblastoma cell line SH-SY5Y cells,mouse hippocampal neuron cell line HT22 cells,and rat adrenal medullary chromaffin tumor cell line PC12 cells（highly differentiated）.The manganese stained cells were divided into four groups,and Mn Cl₂（125,250,500μmol/L）was used for manganese stainingμmol/L.Cell FTO inhibitory components were divided into 5 groups,using FTO inhibitor MA2（100μmol/L）intervention.Cell FTO overexpression was divided into 5groups and intervened with FTO agonist VPA（2 mmol/L）.The exposure time was 24 h,and the intervention time was 48 h.After exposure and intervention,the cell viability was detected using the CCK-8 method.Observe cell morphology using an inverted microscope,extract proteins and m RNA for detection.2.In the second part of the bioinformatics analysis used SH-SY5Y cells for RIP-seq of FTO.Using SH-SY5Y cell Lentivirus transfection to construct FTO overexpression group,RNA seq transcriptome sequencing was performed.Apply GO enrichment analysis and Pathway analysis to process sequencing results.C57BL/6 mice（half male and half female）were selected for animal experiments and grouped according to the principle of weight balance（n=16）.The GRIN1 inhibitory component of mice was divided into 10 groups,and the GRIN1 inhibitor MK-801（0.6 mg/kg）was subcutaneously injected.The overexpression group of GRIN1 in mice was divided into10 groups,and the GRIN1 agonist NMDA（0.6 mg/kg）was subcutaneously injected.The treatment concentration is 5 mg/kg.Poison once a day for two consecutive weeks.Record the weight of mice,conduct body composition analysis,and conduct behavioral experiments.Perform ICP-MS detection and detect protein and m RNA expression.The hippocampal slices experiment used a vibration slicer to cut hippocampal slices from the whole brain samples of wild-type C57BL/6 mice.The slices were perfused with artificial cerebrospinal fluid containing drugs for exposure and intervention,with a perfusion intervention time of 5 minutes.The brain slices were divided into 8 groups using the GRIN1 inhibitor MK-801（100μmol/L）.The overexpression components of GRIN1 in brain slices were divided into 8 groups,using the GRIN1 agonist NMDA（25μmol/L）.After exposure and intervention,the MED64 system was used for electrophysiological testing of brain slices.Record the f EPSP waveform,LTP induction success rate,%f EPSP amplitude before and after LTP induction,and PPF ratio.Cell experiments were conducted using SH-SY5Y cells,HT22 cells,and PC12 cells（highly differentiated）.The manganese stained cells were divided into four groups,and Mn Cl₂（125,250,500μmol/L）was used for manganese staining.Cell FTO inhibitory components were divided into 5 groups,using FTO inhibitor MA2（100μmol/L）intervention.Cell FTO overexpression was divided into 5 groups and intervened with FTO agonist VPA（2mmol/L）.SH-SY5Y cells transfected with Lentivirus were divided into 4 groups.The GRIN1 inhibitory component of cells was divided into 8 groups,using the GRIN1inhibitor MK-801（10μmol/L）.The overexpression component of GRIN1 in cells was divided into 8 groups,using the GRIN1 agonist NMDA（10μmol/L）.After exposure and intervention,CCK-8 method was used to detect cytotoxicity,cell morphology was observed under an inverted microscope,protein and m RNA were extracted for detection.3.In the third part of cell experiments,the manganese stained SH-SY5Y cells were divided into four groups,and Mn Cl₂（125,250,500μmol/L）was used for manganese staining.The SH-SY5Y cell transient si YTHDF3 group was divided into four groups,and the SH-SY5Y cell Lentivirus transfected YTHDF2 overexpression group was divided into four groups.Detect the expression levels of YTHDF1 and YTHDF2 proteins in SH-SY5Y cells after manganese exposure.After using SH-SY5Y cells si YTHDF3 and overexpressing YTHDF2,the expression level of GRIN1 was detected.The bioinformatics analysis,four databases including NCBI,UCSC,EPD,and Ensemble were selected to predict FTO promoters,and the JASPAR 2022 database was selected to predict FTO transcription factors.293T cells were used for double Luciferase experiment to verify the FTO transcription factor SOX2.The cell SOX2 intervention group was divided into 7 groups,using the SOX2 inhibitor propranolol（10μmol/L）and agonist O4I2（20μmol/L）intervention in SH-SY5Y cells to further validate the impact of transcription factor SOX2 on the expression of FTO.Results:1.Manganese can accumulate in the hippocampus of mice,and its content increases with the increase of manganese exposure dose.The results of Morris water maze,T-maze,and radial arm maze staining showed that manganese exposure can reduce the learning and memory abilities of mice.The MED64 planar microelectrode array recording system detected that manganese exposure can cause electrophysiological damage to the hippocampal slices of C57BL/6 mice.HE staining and Nissl staining revealed damage to the nucleus of the hippocampus and a decrease in Nissl bodies,but no changes were observed in the whole brain coefficient and hippocampal coefficient.Manganese exposure can reduce the activity and damage the morphology of human Neuroblastoma cell line SH-SY5Y,mouse hippocampal neuron cell line HT22 and rat adrenal Pheochromocytoma cell line PC12.The expression of FTO in mouse hippocampus and three kinds of cells decreased significantly.The intervention of FTO inhibitor MA2 cannot significantly affect the expression of FTO,but can exacerbate manganese induced learning and memory dysfunction in mice,exacerbating electrophysiological damage to the hippocampal slices.Cell viability and morphological damage of three types of cells after intervention with FTO inhibitor MA2.The intervention of FTO agonist VPA can significantly increase the expression of FTO.In the animal experiment section,the AAV-5 overexpression FTO group was added,indicating that the intervention effect of VPA was the same as that of overexpression FTO,further verifying the function of the FTO agonist VPA.2.We used SH-SY5Y cells to carry out RIP seq experiment on RNA demethylase FTO.We used Lentivirus to transfect SH-SY5Y cells to construct FTO overexpression group and transfect them with manganese,and then sequenced the cells with RNA seq transcriptome.After intersection of RIP-seq and RNA-seq genes,6 genes were selected to be related to the nervous system.After verification,GRIN1 was selected as the key gene for learning and memory in the study.The expression of GRIN1 in the hippocampus and three types of cells of mice significantly decreased after manganese exposure.After MA2 intervention,the expression of GRIN1 decreased.After VPA intervention,the expression of GRIN1 significantly recovered.After the intervention of NMDA receptor inhibitor MK-801,the electrophysiological damage to mouse hippocampal slices intensified,and the learning and memory abilities of mice decreased.After the intervention of NMDA receptor agonist NMDA,the electrophysiological damage to the mouse hippocampal slices was restored,and the learning and memory abilities of the mice were significantly improved.3.The m RNA expression levels of m⁶A methylation related recognition proteins YTHDF1,YTHDF2 and YTHDF3 remained unchanged in SH-SY5Y cells after manganese exposure.After using SH-SY5Y cells si YTHDF3,it was observed that the expression level of GRIN1 increased,but after overexpressing YTHDF2,the expression level of GRIN1 decreased.After manganese exposure,the expression levels of YTHDF1and YTHDF2 proteins in SH-SY5Y cells were almost the same.FTO promoters were obtained using four databases:NCBI,UCSC,EPD and Ensemble.Then,the JASPAR2022 database was used to predict FTO transcription factors,and 20 transcription factors that can affect both humans and mice were screened.With the increase of manganese exposure dose,the m RNA expression of transcription factor SOX2 decreases,and its predicted score is the highest and all are on the positive chain of the promoter.The double Luciferase reporter gene experiment was used to verify the binding of transcription factor SOX2 with the FTO promoter.SOX2 has the function of up regulating the expression of FTO.To further validate the effect of transcription factor SOX2 on the expression of FTO,the agonist O4I2 of SOX2 and the inhibitor propranolol were used.Conclusion:1.Manganese can lead to a decrease in learning and memory abilities in the hippocampus of mice by reducing FTO expression.2.Manganese exposure can down regulate the expression of FTO through the transcription factor SOX2,leading to the degradation of the m RNA of NMDA receptor GRIN1 in a YTHDF3 dependent manner,resulting in learning and memory impairment in the hippocampus of mice.