Dissecting The Role Of Paternal Methyl Donor-rich Diet On Hippocampal Dependent Learning And Memory In Offspring Mice

DNA methylation is a major epigenetic modification involving the addition of a methyl group to the 5 position of cytosine by DNA methyltransferase?DNMT?to form 5-methylcytosine?5-mC?.This chemical modification alters the physical structure of the DNA,preventing DNA-binding proteins access during transcription processes,ultimately silencing the affected gene.DNA methylation patterns are particularly sensitive to diet.Food-derived nutrients such as folic acid?FA?,vitamin B₁₂,betaine,methionine and choline are prominent sources of methyl donors.Methyl groups for DNA methylation are provided through C₁ metabolism.There is growing evidence that methyl donors are critical during pregnancy and that dietary deficiency or excess may have an impact on epigenetic programming in human subjects as in animals.It is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring,it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation.Given the emerging appreciation that sperm contribute epigenetic information to offspring,we performed a study to test the hypothesis.Young adult C57BL/6J males were randomly assigned to two groups.The control diet?CD?group was fed a standard Teklad global 18%protein rodent-breeding diet while the methyl donor-rich diet?MD?group received a methyl donor-enriched diet(enriched for FA,L-methionine,choline,zinc,betaine and vitamine B₁₂)based on the same formula.After 6 weeks of feeding,males were individually mated with young adult129S6/SvEv female mice to generate the F1 offspring mice.At first,we assessed behavioral and cognitive functions?such as the spatial learning and memory,the motor coordination,grip strength,emotion and so on?of the F1 mice by Morris water maze,context fear conditioning,accelerating rotarod,a grip strength meter system,open field and startle reflex.To explore the underlying cellular mechanisms,we employed deep,intrahippocampal CA1 electroence phalogram recordings to measure local field potentials in awake and freely behaving F1 offspring of CD and MD fathers,and in further step we investigated the intrinsic and synaptic properties of CA1 pyramidal neurons of the F1 offspring mice by field potential recording and whole-cell patch.We performed hippocampal gene expression analyses by gene expression microarray analyses,targeted DNA methylation analysis and MeDIP-chip to identify differentially expressed genes in MD F1 mice.The results of our first part of the research are as follows:1.Morris water maze test:the probe trial data revealed that there was a significant interaction between paternal diet?between-subjects factor?and quadrant?within-subjects factor?with regards to quadrant occupancy time and target crossing measures.We also examined hippocampus-dependent learning and memory in CD F1and MD F1 mice using an associative contextual fear-conditioning paradigm,and the data revealed reduced freezing levels in MD F1 mice.Moreover,additional behavioral assessment revealed normal exploratory behavior,anxiety-related behaviors,motor skills and sensory processing in the F1 offspring of MD fathers.2.In vivo hippocampal EEG recording:analyses of oscillatory activity in the theta range revealed reduced overall theta activity in the F1 offspring of fathers on the MD relative to control.3.Field potential recording:these experiments revealed reduced levels of LTP in the offspring of MD fathers relative to controls,however basal synaptic transmission was normal in MD offspring slices.4.Patch clamp recording:compared to that of CD F1 animals,CA1 neurons of MD F1 mice displayed reduced AP half-width,larger fAHP,an increased first spike latency and decreased firing numbers.However,there were no significant differences in resting potential,input resistance,spike amplitude and the AP threshold of CA1pyramidal neurons between the two groups.IPSC recordings revealed decreased sIPSC frequency,and unaltered sIPSC amplitude in CA1 pyramidal neurons of MD F1 offspring relative to CD F1 controls,however no significant changes in both sEPSCs and mEPSCs,indicating overall excitatory synaptic transmission onto CA1pyramidal neurons was unaltered in MD F1 mice.5.Hippocampal gene expression analyses identified two notably differentially expressed genes in MD F1 mice,Mat2a and Kcnmb2.And targeted and genome-wide DNA methylation analyses identified differential methylation of Kcnmb2 regulatory regions MD F1 offspring.The BK channel?₂,encoded by Kcnmb2,has a role in the inactivation of BK currents.BK channels play significant roles in regulating a range of physiological processes,including AP firing,neurotransmitter release and also take part in the regulation of learning and memory processes.Considering the function of the BK channels and our above research findings,we speculate that Kcnmb2 may be the target gene,which could contribute to alterations in synaptic plasticity,theta oscillations and behavior observed in MD F1 mice.So we addressed whether overexpression AAV-mediated of Kcnmb2 in the CA1 region of the dorsal hippocampus can rescue the neurophysiological alterations observed in MD F1offspring mice.High titers of AAV virus engineered to overexpress Kcnmb2?AAV₁-hSyn1-Kcnmb2-IRES-GFP;Kcnmb2-AAV?or control virus?AAV₁-hSyn1-GFP?were stereotaxically injected into the CA1 region of the dorsal hippocampus of CD F1 mice,as well as MD F1 mice.The viral infection in the CA1 region was confirmed by GFP fluorescence.Relative expression of Kcnmb2 in hippocampus was measured by real-time qRT-PCR.Behavior and electrophysiological experiments were performed 4–6 weeks following virus injection.The results showed that:1.Confocal fluorescence microscopy showed that successful AAV transduction in hippocampal area CA1 based on GFP-associated fluorescence,as well as Kcnmb2expression measured by immunofluorescence staining.Kcnmb2 expression in the hippocampus was quantified by qPCR:AAV-Kcnmb2 increased Kcnmb2 expression in the hippocampus of both CD and MD F1 mice compared to AAV-control virus.2.A probe trial test of morris water maze revealed that CD F1 control-AAV mice,CD F1 Kcnmb2-AAV mice and MD F1 Kcnmb2-AAV mice showed significantly higher occupancy values in the target quadrant than in all the other quadrants,indicating highly targeted searching of the mice in these groups,while this was not the case in MD F1 control-AAV mice.3.We tested whether AAV-mediated overexpression of Kcnmb2 in CA1 of dorsal hippocampus rescued LTP deficits in MD F1 mice,our experiments did reveal reduced early LTP measured at 0–10 min post-tetanus in the SC-CA1 synapses of the MD F1 mice compared to the CD F1 mice that received same control virus injection.In contrast,the early LTP in the SC-CA1 synapses of MD F1 mice receiving AAV-Kcnmb2 injection?MD-Kcnmb2?was comparable to that recorded in both CD-Kcnmb2 and CD-control mice.4.Our whole-cell current-clamp recordings revealed that the MD F1 neurons infected by control virus?MD-control?displayed reduced AP half-width,larger fAHP,and decreased firing numbers.In contrast,MD F1 pyramidal neurons infected by AAV-Kcnmb2?MD-Kcnmb2?displayed similar firing properties as infected CD F1neurons?CD-Kcnmb2?,including AP half-width,fAHP and firing numbers.Based on the above experimental results,we arrive at an opinion that transient exposure of male mice to a methyl donor-rich diet before mating exerts intergenerational effects on learning and behavior in offspring mice.Excessive paternal methyl donor intake results in Kcnmb2 promoter hypermethylation associated with reduced Kcnmb2 expression which may contribute to the impairments in MD F1offspring mice.Taken together,our study identified adverse effects of a paternal methyl donor-rich die on cognitive and neural functions in offspring animals,raising the possibility that paternal dietary factors may be relevant causal factors for mental health issues in the subsequent generation.Our study also provides a targeting gene,Kcnmb2,for treatment of BK channel related mental disorders,including autism,Alzheimer's disease and schizophrenia.In the long run,these studies will not only help enrich our understanding of the potential effects of dietary supplementation on epigenetic processes,but also will ultimately help instruct and guide public health policies in the future.