Proteomic And Transcriptomic Analyses Explore The Mechanism Of Entorhinal Cortex Involved In Alzheimer’s Disease

Background:Alzheimer’s disease(AD)is the most common neurodegenerative disorder.Its clinical features are progressive memory loss and cognitive dysfunction,which brings a heavy economic burden to society and families.The primary pathological changes in AD are deposition of extracellular β-amyloid plaques,neurofibrillary tangles,neuronal loss,synaptic dysfunction and neuroinflammation.Although great progress has been made in basic and clinical research,there is currently no treatment method that can prevent or reverse the progression of AD disease.Therefore,the etiology of the disease still needs to be fully elucidated.The entorhinal cortex(EC)is an important region of the cortical-hippocampal interaction and also the most susceptible region in AD,which play a crucial role in the formation and retrieval of memories.Therapeutic strategies and preventive measures to protect against entorhinal degeneration would be of substantial value in AD.Methods:In this study,we used frozen human brain EC tissues of AD and controls from National Human Brain Bank for Development and Function.We used TMT-based proteomics and RNA-seq-based transcriptomics technology to analyze the expression profiles of genes and proteins in human EC tissue.Immunohistochemistry and label-free quantitative mass spectrometry methods are used to verify protein expression.Through the combined analysis of transcriptome and proteomics data,the shared differential genes and differential proteins are obtained.We further used Western Blot and Quantitative Real-Time PCR methods to explore the function of Hub gene CACNG3 which found in omics research,and the possible mechanism of CACNG3 in AD.Results:Through the comparative analysis of AD and the control group,we found that a total of 534 significantly differently expressed genes(107 upregulated and 427 downregulated)and 1,489 significantly differently expressed proteins(372 upregulated and 1,117 downregulated)were identified.GO enrichment analysis demonstrated the similarity between the composition of the transcriptome and proteome.In cell component,the differently expressed genes/proteins are mainly enriched in the cell membrane and synaptic structure.In biological process,the differently expressed genes/proteins are mainly involved in synaptic plasticity,synaptic vesicle recycling.In molecular function,most important enriched terms were for molecular function of binding,catalytic activity,transporter activity,molecular transducer activity and molecular function regulator.By combined analysis of the transcriptomic and proteomic data,51 proteins matched the corresponding genes.We further analyzed 51 shared differentially expressed genes/proteins and found that the functions of Hub gene(GABRG2,CACNG3,CACNB4,GABRB2,GRIK2,and SLC17A 6)were related to ion transport.Expression levels of mRNA and protein of Hub genes were negatively correlated with the pathological changes of AD.Thus,we speculate that disruption of ion transport in the EC might be a key event that triggers synaptic dysfunction and a critical signaling pathway that may initiate the pathology of AD.In order to further study the mechanism of Hub gene in AD,we first studied whether the changes of Hub gene at the cellular level were consistent with the results of human data.It was found that only the protein expression of Hub gene CACNG3 decreased in N2aAPP cells,which was consistent with the results of omics data and IHC.Then we found that Aβ can reduce the content of CACNG3 mRNA and protein.After knocking down CACNG3 with siRNA,we found that the content of synaptic plasticity related genes such as c-Fos and CaMKIIβ decreased,and the transcription level of NLRP3 and MAPT genes increased.Aβ may cause synaptic plasticity dysfunction by regulating CACNG3,and CACNG3 may affect the pathological changes of AD through NLRP3 signals.Our research results preliminarily explain the mechanism of CACNG3 in synaptic disorders and AD pathological changes,and provide potential new targets and treatment strategies for AD prevention and treatment.Conclusion:1.The expression levels of mRNA and protein of the ion transport related genes(GABRG2,CACNG3,CACNB4,GABRB2,GRIK2,SLC17A6)in entorhinal cortex neurons of AD were significantly down-regulated,which might be the key event that triggers synaptic dysfunction.2.The expression levels of ion transport-related genes were negatively correlated with the pathological changes of AD,which might be involved in a critical signaling pathway that initiates pathology.3.CACNG3 might regulate the plasticity of synapses by affecting the expression of genes related to synaptic plasticity,and might affect the pathological changes of AD through NLRP3 signals.