M6A Demethylase FTO Regulates Dopaminergic Neurotransmission Deficits Caused By Arsenite

PART ?EFFECTS OF ARSENITE EXPOSURE TO CHRONIC DRINKING WATER ON DOPAMINERGIC NEUROTRANSMISSION IN MOUSE CORTEXObjective: To establish a model of arsenite exposure via drinking water,and observe the effects on neurobehavioral function,cortical histopathology and ultrastructure and the expression of key proteins of dopaminergic neurotransmission in cortex,in order to provide theoretical basis for revealing the cortical dopaminergic nerve conduction disorder induced by arsenite.Methods: Healthy C57BL/6J male mice,aged 7 weeks,were randomly divided into the following 4 groups: control group,0.5 mg/L arsenite group,5 mg/L arsenite group and 50 mg/L arsenite group(n=16per group).After exposure,the Morris water maze,the eight-arm maze,the shuttle box and the open field were employed to evaluate neurobehavioralfunction of the mice.The concentration of arsenite and dopamine in the cortex was detected by Atomic fluorescence spectron-metry and High performance liquid chromatography,respectively.The ultra-structural were observed by transmission electron microscopy and neuronal pathological structure were observed by Nissl staining.The expression of TH,DAT,COMT and DRD proteins in cortex were detected by Western-Blot.Results:(1)After exposure,there was no significant change in body weight between the control and the arsenite-treated group(P>0.05).(2)Morris water maze results showed that the swimming distance and escape latency of arsenite-treated groups were significant longer than those of controls(P<0.05),the time spent in the target quadrant was significantly decreased in arsenite-treated groups(P<0.05),while there was no significant difference in swimming speed among 4 groups(P>0.05);the results of the eight-arm maze showed that all arsenite-treated groups were significant increased in the latency,total entries into the arm,the test time,the number of reference memory errors,and the number of working memory errors(P<0.05);the shuttle box results showed that compared with the control mice,the arsenite-treated mice exhibited significant increases in the active avoidance and passive avoidance times(P<0.05),number of passive avoidance,while the number of active avoidance responses were sharply decreased due to arsenite exposure(P<0.05);the results of open field experiments showed that the number of defecations ofarsenic-exposed groups increased significantly compared with the control(P<0.05),moreover,the central square duration and distance moved in the center were both reduced by arsenite exposure(P<0.05),while there was no significant difference in the number of central square entries(P>0.05).(3)Atomic fluorescence spectroscopy results showed that the concentrations of arsenite were significantly enhanced in arsenite-treated groups compared with control and the dopamine content was significantly decreased in arsenite-treated groups was detected by High performance liquid chromatography(P<0.05).(4)Nissl staining showed that the number of Nissl bodies decreased with the increase of arsenite exposure dose and the bodies showed vacuolar degeneration,the edges were blurred and weakly stained.The transmission electron microscopy revealed that the mean and maximal postsynaptic density(PSD)thickness of arsenic-treated group were much larger than that of the control group(P<0.05),however,there were no significant differences in the lengths of the active zone,the width of the synaptic clefts among 4 groups(P>0.05).(5)Compared with the control group,the expression of TH,DAT,COMT,DRD1,DRD2,DRD4 and DRD5 proteins in dopaminergic neurtransmission were significantly decreased after 6 months of arsenite exposure(P<0.05),and there was no significant change in DRD3(P>0.05).Conclusion: Chronic arsenite exposure via drinking water could affect the synthesis,transport,decomposition and binding process ofdopamine,resulting in a decrease of dopamine content in the cortex of mice,accompanied by neuronal damage and synaptic ultrastructural abnormalities,which induced a series of neurobehavioral dysfunction.PART ?STUDY ON THE MECHANISM OF m6 A DEMETHYLASE FTO REGULATES DOPAMINERGIC NEUROTRANSMISSION DEFICITS CAUSED BY ARSENITEObjective: To further verify whether arsenite could induce dopaminergic neurotransmission disorder using a cell model and explore the role of FTO in arsenic-induced dopaminergic nerve conduction disorder,so as to provide experimental basis for targeting regulation of FTO to prevent or intervene in neurodegenerative diseases caused by arsenic.Methods: The CCK-8 method was used to assess the effect of arsenite on the cell viability of PC-12 cells.The High performance liquid chromatography was used to detect the concentration of dopamine in PC-12 cells.The protein expression of TH,DAT and COMT in PC-12 cells was determined by Western-blot.The m6 A modification level in cortex tissue and PC-12 cells were tested using dot-blot assay and immunofluoresence.Western-blot was used to detect the protein expression level of FTO in tissues and cells,and the co-localization of FTO and THwas observed by immunofluorescence.Finally,the FTO knockdown and overexpression cell model was established by siRNA and CRISPRa transfection technology,respectively;and detected the changes of m6 A levels and key proteins in the dopamine pathway.Results:(1)After 24 h of arsenite exposure,the viability of PC-12 cells was significantly decreased,and there was a significant dose-dependnt relationship.(2)The results of High performance liquid chromatography showed that the concentration of dopamine in PC-12 cells decreased after arsenite exposure(P<0.05).The expression of TH,DAT,COMT proteins in PC-12 cells were significantly decreased in response to arsenite exposure(P<0.05),which were consistent with the animal model.(3)Dot-blot assay and immunofluorescence showed that the level of m6 A modification in tissues and cells increased significantly after arsenite exposure.(4)Compared with control group,the expression of FTO protein in tissues and cells was significantly decreased(P<0.05),as well as FTO and TH co-localization.(5)When the FTO expression was down-regulated,the m6 A modification level of the arsenite exposure group was more obvious than that of the control,and the protein expression of TH,DAT and COMT were significantly lower in the arsenite-treated group(P<0.05);after overexpression of FTO,the level of m6 A modification decreased,as well as the expression of TH,DAT and COMT protein in FTO CRISPRa and arsenite double exposure group increased compared with arsenite exposuregroup alone(P<0.05).Conclusion: Arsenite could cause a decrease in cell viability,an increase in m6 A modification level,a decrease in FTO protein expression.With the change of FTO expression level,the expression level of the key proteins in the dopaminergic neurotransmission pathway will change.The m6 A demethylase FTO did play an important role in the regulation of arsenite-induced dopaminergic neurotransmission disorders to some extent;and suggest that the dynamic regulation of m6 A by FTO may have a certain protective effect on the neurotoxicity caused by arsenite.