Effects Of Paternal Co-Exposure To High-Fat Diet And Cadmium On Offspring Cognitive Ability And Its M6A Modification Mechanism

Background:The incidence of cognitive impairment has been increasing in recent years,and the mainly risk factors of cognitive impairment are environmental factors exposure.Studies showed that single environmental heavy metal cadmium（Cd）or high-fat diet（HFD）could cause contemporary cognitive impairment.However,the effect and mechanism of paternal co-exposure to HFD and Cd on offspring cognitive function remain unclear.Objective:This study explored the effect of paternal co-exposure to HFD and Cd on offspring cognitive function and its m6A modification mechanism.Methods:This study included whole animal experiments and cell researches.The animal experimental scheme was as follows:1.To explore the effect of paternal co-exposure to HFD and Cd on cognitive function and hippocampal aging level in offspring,5-week-old male mice were exposed to standard control diet,Cd Cl₂（100 mg/L）through drinking water,high-fat diet,and high-fat diet plus Cd Cl₂ for 10 weeks.Next,a part of the male mice were euthanized to collect the sperm for methylation sequencing.The remaining male mice mated with normal female mice,and the hippocampi of offspring at GD18 and PND35 were collected.The offspring at PND105 were tested by Morris water maze,and their hippocampi were collected for detecting the neuron development,aging and m6A modification levels.2.To explore the role of paternal sperm m6A reduction in paternal HFD and Cd-caused hippocampal neuronal senescence and cognitive deficits in offspring,the testes of 10-week-old exposed mice were treated with the m6A inhibitor STM2457（50 mg/kg）once a week,a total of 5 times.Some 15-week-old male mice were euthanasia to collect sperm for m6A and Rhoa detection,and the remaining male mice were mated with normal female mice.The offspring were naturally raised to 10 weeks old for Morris water maze testing,and their hippocampi were collected for the detection of hippocampal aging,m6A modification and Rhoa levels.The cell experiment scheme was as follows:1.To establish the mouse hippocampal neuronal senescence model,mouse hippocampal neuronal cells（HT22 cells）were administrated with H₂O₂（200μM）,a common inducer of cellular senescence,for 24 h,and the aging level of HT22 cells was detected after harvest.2.To explore the effect of H₂O₂ treatment on the expression of RHOA and IGF2BP1,HT22 cells were treated with H₂O₂（200μM）for 0,6,12 and 24 h to detect the expression levels of RHOA and IGF2BP1.3.To investigate the role of Rhoa in mouse hippocampal neuronal senescence,HT22 cells were pretreated with Rhoa si R for 24 h,and then incubated with H₂O₂（200μM）for 24 h.The aging level of HT22 cells was detected.4.To explore the role of IGF2BP1 in Rhoa stability in aging mouse hippocampal neuronal cells,HT22 cells were pretreated with Igf2bp1 si R for 30-36 h,then administrated using H₂O₂（200μM）for 12 h,and finally incubated with the pan transcription inhibitor actinomycin D（Act D,5μg/m L）for 0,3 and 6 h to detect the decay rate of Rhoa m RNA.Results:The results of behavioral experiments showed that paternal co-exposure to HFD and Cd significantly prolonged the escape latency and decreased times spent in the target quadrant and the number of platform crossings in offspring,suggesting that the cognitive function of the offspring was impaired.HE and Nissl staining results indicated that paternal co-exposure to HFD and Cd reduced the number of neurons and increased the number of pyknotic nuclei in offspring hippocampi.Golgi-cox staining results also showed that paternal co-exposure to HFD and Cd markedly reduced the dendritic spine density and the number of dendritic branches of hippocampal neurons in offspring.Further results indicated that paternal co-exposure to HFD and Cd significantly elevated the SA-β-Gal positive area percentages and the levels of senescence-related proteins,p16and p21,in offspring hippocampi.The above results suggested that the number and structure of hippocampal neurons in the offspring were damaged.Mechanistically,paternal co-exposure to HFD and Cd obviously promoted the m RNA expression of Mettl3and increased the total RNA m6A level in paternal sperm.Me RIP-seq results showed that the m6A modification and m RNA levels of Rhoa,a cellular senescence inducer,were significantly increased in HFD plus Cd-treated sperm.Interestingly,paternal co-exposure to HFD and Cd markedly elevated Rhoa m RNA,its m6A level and the m6A reader protein IGF2BP1 expression in offspring hippocampi.STM2457,a specific inhibitor of METTL3,markedly mitigated paternal environmental exposure-caused the elevation of hippocampal Rhoa m6A,neuronal senescence and cognitive deficits in offspring,suggesting that METTL3-mediated m6A modification elevation in paternal sperm contributed to paternal co-exposure to HFD and Cd-increased Rhoa m6A and neuronal senescence in offspring hippocampi.The results of the in vitro experiments showed that Rhoa si R significantly reversed mouse hippocampal neuronal senescence,and Igf2bp1si R significantly reduced the level and stability of Rhoa in aging mouse hippocampal neuronal cells.The above results suggested that paternal co-exposure to HFD and Cd could cause offspring hippocampal neuronal senescence via elevating the level of Rhoa m RNA and its m6A in offspring hippocampi.Conclusion:Paternal co-exposure to HFD and Cd induces offspring hippocampal neuronal senescence and cognitive deficits,and the mechanism may be that paternal sperm m6A-mediated Rhoa stabilization involves in paternal co-exposure to HFD and Cd-caused offspring hippocampal neuronal senescence.