Effects Of Transcranial Direct Current Stimulation On Synaptic Plasticity In The Hippocampus Of APP/PS1 Mice With Alzheimer's Disease By Exploring Hippo/Yap Activatio

Transcranial direct current stimulation(tDCS)is a non-invasive brain nerve regulation technique,which regulates the transmembrane potential of nerve cells and changes the excitability of cerebral neurons by applying anode(excitation)or cathode(inhibition)dependent low-intensity subthreshold direct current to specific brain regions.Clinical studies have shown that tDCS has a good application prospect in the prevention and treatment of Alzheimer’s disease,especially in early intervention.AD is characterized by progressive neuronal loss and cognitive decline,which in a sense is called synaptic disease.On the one hand,synaptic plasticity is the basis of normal learning and memory,cognitive function and behavior,and synaptic loss and dysfunction are the key features of many neurodegenerative diseases,which occur in the ultra-early stage of AD or even in the early stage of mild cognitive impairment(MCI),prior to the deposition of β-amyloid protein,hyperphosphorylation of Tau protein and neurofibrillary tangles(NFT).On the other hand,the current research and development of drugs targeting A β has failed,and the efficacy of existing therapeutic drugs is very little.Scientists hope to explore more effective intervention methods and targets,and move the intervention window forward.It has not been reported whether tDCS has a protective effect when synaptic plasticity is impaired in the early stage of AD.Therefore,revealing its effect and molecular mechanism is helpful to clarify whether tDCS can target synaptic plasticity and its mechanism in the early stage of AD,and promote the clinical application and promotion of tDCS.In this study,4-month-old APP/PS1 mice were used as early AD mouse model.The right frontal cortex of mice was anodic stimulated for 30 minutes per day.After continuous intervention for 4 weeks:①Evaluate the effects of tDCS on spatial memory of Morris Water Maze and learning ability of mice,A β plaque deposition and the number of mature neurons in intervention area and hippocampal subarea by behavioral and histopathological examination.②Evaluate the effect of tDCS on the expression and localization of presynaptic membrane protein Synapsin I and postsynaptic protein PSD95 in the intervention region and hippocampal subregion,on the synaptic ultrastructure of the above brain regions,and on the LTP of hippocampal CA1 neurons by histopathological and electrophysiological examination and electron microscopic examination.③Analyze the regulatory effect of tDCS anodic stimulation on the number of excitatory/inhibitory synapses in the stimulation area from the point of view of the theory of yin-yang balance in traditional Chinese medicine.④The significant differential expression of Hippo/Yap pathway in the pathological process of AD was confirmed by bioinformatics analysis,and the activation of tDCS to the key molecule Yap in different brain region Hippo/Yap pathway was further observed by histopathology.⑤In vitro experiments,direct current(DC)stimulation was applied to PC 12 differentiated neurons and primary cortical neurons induced by NGF to observe the effect of DC on axonal growth and synaptic formation,as well as the inhibitory effect on A βsynaptic connection loss;and further verify the activation effect of DC on Yap by blocking Yap signal transduction.This study will help to clarify whether tDCS can reeduce Aβ deposition and neuron loss in early AD model mice,partially explain the possible mechanism and TCM theoretical basis of tDCS regulating synaptic plasticity,and provide scientific basis for the application of tDCS intervention targeting synaptic plasticity in AD.Part Ⅰ The effects of transcranial direct current stimulation on spatial learning and memory ability of APP/PS1 mice in early and middle stage and its mechanismOBJECTIVE:To study the effects of tDCS on the pathological and spatial learning and memory ability of APP/PS1 mice in early and middle stage.METHODS:Thirty 4-month-old APP/PS1 mice were randomly divided into control group(AD group)and transcranial direct current stimulation intervention group(TDCS group),and 15 wildtype mice were used as negative control group.The electrode cap was fixed on the right frontal cortex of mice with medical tissue glue and dental cement as an anode stimulation electrode device,and a protective collar was worn to prevent the mice from scratching off.The cathode stimulation electrode was placed on the chest and abdomen of mice.Conductive gel was injected into the electrode cap at each intervention,and a 3.14mm2 stimulation electrode was inserted into the electrode cap.After the power was turned on,200uA(output density 6.37mA/cm2)stimulation intensity was applied for 30min.After continuous stimulation for 30 days(no stimulation was applied on the last day of the week),control group(AD group)mice underwent the same operation,but no power supply was connected to apply direct current stimulation.After perfusionfixation,brain tissues were collected for immunofluorescence detection to evaluate the pathological results in the coronal section of brain tissues of mice in each group after intervention(n=3).Meanwhile,Morris water maze test was conducted to evaluate the spatial learning and memory ability of mice in each group(n=6).RESULTS:Histological results showed that 5-month-old APP/PS1 mice showed a decreasing trend in mature neuron marker NeuN positive(NeuN+)expression in prefrontal cortex and hippocampus CA3 region,but there was no statistical difference compared with Control group.However,Aβ had been deposited and accumulated in each brain region,showing as scattered Aβ plaque deposition,which was significantly different from that in the Control group.After 4 weeks of tDCS,the number of NeuN+cells in hippocampal CA1 and CA3 regions increased,and the difference was statistically significant compared with that in AD group.The amount of Aβ plaques decreased in DG and prefrontal cortex region,which was significantly different from that in AD group.Morris water maze results showed that during the training stage,all the three groups showed a certain degree of learning ability,but there was no significant difference in the latency results.In the test phase of space exploration,there were no significant differences between the three groups of animals in the number of platform crossing and the residence time in each quadrant.However,tDCS increased the residence time of the model animals in the quadrant of the target platform.CONCLUSIONS:1.There was no significant spatial learning and memory impairment in 5-month-old APP/PS1 mice,and tDCS did not imrove the learning ability of 4-month-old APP/PS1 mice in the morris water maze training stage after 30 days of intervention,but only increased the residence time of the animals in the quadrant of the target platform in the test stage.2.tDCS can increase the expression of mature neuron marker Neun in hippocampal CA1 and CA3 regions of APP/PS1 mice,and reduce the amount of Aβ plaque deposition in prefrontal cortex and hippocampal DG region of APP/PS1 mice.Part Ⅱ The effects of transcranial direct current stimulation on synaptic structure and plasticity of AD miceOBJECTIVE:To study the effect of tDCS on synaptic plasticity of APP/PS1 mice in early and middle stages.METHODS:Grouping and intervention methods were the same as described in the first part.At the end of the intervention,the paraffin sections of the brain tissues of mice were separated after perfusion with 4%paraformaldehyde,and then immunofluorescence double-labeled staining was performed(n=3)to evaluate the expression of synaptic protein marker molecules PSD95 and Synapsin in different brain regions of mice in each group after intervention.The effect of tDCS on synaptic protein expression and synaptic loss was evaluated by co-localization of two key proteins.The right frontal cortex and the right hippocampus of each group were separated and fixed with glutaraldehyde.The ultrastructural changes of synapses in these brain regions were observed by transmission electron microscopy.The brain tissues of mice in each group were separated for field potential recording,and coronal slices with a thickness of about 300um containing the hippocampus were prepared.Stimulation electrodes were placed in the hippocampus CA3 area for high-frequency stimulation to induce long-term potentiation(LTP).The recording electrodes were placed in the radiation layer of the hippocampus CA1 area.LTP was recorded for 60 minutes after LTP induction,and the changes of LTP in each group were compared.RESULTS:Immunofluorescence results of synaptic protein in different brain regions showed that synaptic protein loss in 5-month old APP/PS1 mice mainly occurred in hippocampal CA3 and DG regions,and the expression intensity of PSD95 and Synapsin in these two regions was decreased compared with negative control group and there is statistical difference between each group.The ultrastructure of synapses showed that the number of synapses decreased in the right prefrontal cortex and right hippocampus of the APP/PS1 mice.The results of field potential recording of hippocampal CA1 showed that the FEPSP of 5month-old APP/PS1 mice was significantly lower than that of control group,suggesting that LTP was damaged,which proved that the synaptic plasticity of APP/PS1 mice was damaged too.After 30 days of stem prognosis,tDCS can improve the loss of synaptic protein in CA3 and DG region of hippocampus of APP/PS1 mice,increase the number of synaptic connections in right prefrontal cortex and right hippocampus of APP/PS1 mice,and significantly restore the expression levels of hippocampal FEPSP and LTP to the expression levels of wild-type control group.CONCLUSIONS:1.Although 5-month-old APP/PS1 mice did not show significant spatial learning and memory impairment,they showed different degree of decreased synaptic protein expression intensity and loss of synaptic connections in prefrontal cortex and hippocampus,and did not show significant LTP impairment.2.TDCS can inhibit the loss of synaptic proteins in prefrontal cortex and hippocampus of 5-month-old APP/PS1 mice,improve the synaptic connection structure,and enhance the expression level of damaged LTP.Part III The effects of transcranial direct current stimulation on Hippo/Yap pathway of APP/PS1 miceOBJECTIVE:Apply bioinformatics analysis toinvestigate the differential expression of HIPPO/Yap pathway in the development of AD disease,and to investigate the activation of HIPPO/Yap pathway by tDCS.METHODS:Bioinformatics analysis was used to analyze two datasets,GSE1297 and GSE36980.0.05 and Fold change(FC)>2 were screened out for up-regulated and down-regulated genes.After the intersection of these differentially expressed genes,Venn plot was used to obtain the genes with low expression in both data sets.Based on the expression of GSE1297,KEGG pathway enrichment analysis was performed by GSEA to analyze the enrichment of differential pathways in Alzheimer’s disease.Finally,by detecting the expression of core protein Yap of Hippo/Yap pathway in different brain regions of mice in each group,the expression of Yap and the effect of tDCS on Yap expression were evaluated in 5month-old APP/PS1 mice.RESULTS:Through bioinformatics analysis,249 up-regulated and 812 down-regulated differential genes were screened in GSE1297 and GSE36980 dataset.Further KEGG pathway enrichment analysis showed that HIPPO/Yap pathway was significantly enriched in APP/PS1 group.The results of Yap immunofluorescence staining in different brain regions of APP/PS1 mice showed that the expression of Yap in hippocampus CA3 and DG region was significantly decreased compared with control group.After tDCS intervention,the expression levels of Yap in these two brain regions was significantly increased compared with that in AD group.In prefrontal cortex and hippocampus CA1 region,tDCS tended to promote the expression of Yap,but the difference was not statistically significant compared with AD group.CONCLUSIONS:1.The results of bioinformatics analysis showed that HIPPO/Yap pathway was significantly enriched in AD group,which might be involved in the pathological process of AD.Pathological results also showed that the expression of Yap which is the core molecule of the Hippo/Yap pathway was decreased in the CA3 and DG regions of the hippocampus in early and middle age AD mice,suggesting that the activation of the Hippo/Yap pathway was inhibited in early and middle age AD mice.2.TDCS can activate Yap expression in CA3 and DG region of APP/PS1 mice.Part Ⅳ The promotion effects of direct electrical stimulation on axonal growth and synaptic formation of cultured neurons in vitroOBJECTIVE:To observe the effects of Direct current(DC)on neuron growth and synaptic connection ultrastructure in vitro.METHODS:The primary cortical neurons of neonatal mice and suckled mice were isolated in vitro by stimulating PC 12 cell to differentiate into neurons by NGF.The primary cortical neurons of neonatal mice were cultured in 3D using AD cell model indμced by Aβ.The effects of different intensity(5 μA/20 μA/50 μA/100 μA/200 μA)direct current on neuronal differentiation and axon growth length and quantity were detected by ordinary bright field microscopy.The appropriate intensity of external direct current stimulation was decidedby western blot of synaptic proteins PSD95/Synapsin,Yap and P-Yap.The effect of 50μA direct current stimulation on the formation of synaptic connections between neurons in vitro was evaluated by transmission electron microscopy.RESULTS:In the experiment to evaluate the effects of different current intensity on the growth and number of neuronal axons,a control experiment with NGF stimulation and no stimulation was applied to confirm that 50μA DC could promote the growth of neuronal axons and increase the number of neuronal axons more effective.Western blot results showed that 50μA DC could increase the expression of PSD95 and Synapsin.Through transmission electron microscope observation,50μA DC couldincrease of the connection structure between neurons.In the primary neuron validation experiment,it was observed that the density of neural network was higher than that of the control group after the extension of 50μA DC for 7 days,and the expressions of NeuN/Yap and synaptic protein PSD95/Synapsin was improved.In the Aβ-induced AD cell model,50μA DC stimulation suppress the decrease in Aβ-induced network connection density.Electron microscopy results suggested that 50μA DC inhibited the formation of synaptic connections in the in vitro 3D culture environment.CONCLUSIONS:1.50μA direct current can promote the expression of synaptic protein in PC12differentiated neurons and primary cortical neurons induced by NGF and increase the expression level of Yap in cells.50μA direct current can promote the synaptic connection structure between PC12-differentiated neurons induced by NGF.2.DC suppressed the decrease in density of neuronal connection network caused by Aβin 3D culture environment,and increased the synaptic connection structure between neurons.