Differentially Expressed Genes In Aging Mice With Cognitive Alteration And Depression-and Anxiety-like Behaviors

Background:With rapid socioeconomic development,the life expectancy is increasing.However,age-related disorders such as Alzheimer ’s disease(AD)and dementia afflict the vast majority of aged patients,which become the most significant public health issues and considerable challenges around the world.Indeed,the aging process may involve a series of behavioral and psychological dysfunction(such as depression,anxiety and abnormal pain response affect)due to neurodegenerative changes in cell homeostasis and biological pathways[1].Therefore,understanding molecular mechanisms underlying these age-related symptoms may open a new avenue in prevention and treatment of age-related disorders.Animal models are the key tools for studying the behavioral and pathological changes in aging.Animal behavioral abnormalities during aging are important parameters,which help us to understand pathological mechanism of age-related disorders and its treatment.Previous studies shown that the ACC(anterior cingulate cortex),amygdala played a key role in cognitive dysfunction,depression and anxiety-like behaviors.As part of the limbic system of the brain,the hippocampus was closely related to short-term memory,long-term memory and spatial localization.Therefore,exploring changes in gene expression in the ACC,amygdala and hippocampus during aging might identify new targets for age-related symptoms.Research purposes:In this experiment,we detected change in cognitive dysfunction,depression and anxiety-like behaviors and pain-related behaviors.Further studies by RNA sequencing of ACC,hippocampus and amygdala may provide a new target for research on abnormal behavior related to aging.Research methods:1.Detecting cognitive function,depression-and anxiety-like behavior and pain-related behavior with agingFirst,Novel-object recognition and Y-maze spontaneous alternation tests were used to evaluate the recognition memory and working memory of 2-,12-and 24-month old mice,respectively.Morris water maze test was then performed on 2-and 12-month old mice.Depression-like behaviors of three groups of mice with different ages were studied by forced swimming and tail suspension tests.Anxiety-like behavior in aging mice was evaluated by open field test and elevated plus maze test.Finally,the Von Frey method and hot plate method were used to detect the mechanical and thermal pain of the mice,respectively.2.Analyze the differentially expressed genes in the brain regions and verify the sequencing results by RT-PCR experiments.The anterior cingulated cortex,amygdala and hippocampus of 2-,12-and 24-month old mice were used in this experiment.Differentially expressed genes were then screened in ACC,amygdala and hippocampus compared with 2-month old mice(| Foldchange |>1,P<0.05).Then,four genes(Defb1,Ostn,Igfbpll and Klk6)with significant changes were selected for RT-PCR experiment to verify RNA sequencing.3.Using bioinformatics analysis to analyze the differentially expressed genesVenn analysis was performed with depression-and anxiety-related genes and DEGs in ACC and amygdala of 12-and 24-month old mice.Then,Venn analyses were also used to analyze DEGs associated with cognitive impairment in the hippocampus of 12-and 24-month old mice.Finally,the molecular functions,cellular pathways,KEGG pathway involved and the interaction between DEGs were analyzed though GO enrichment,KEGG pathway and PPI network analysis.Results:1.Changes of cognitive dysfunction,depression-and anxiety-like behaviors and pain related behavior with aging.First,we tested cognitive function in 2-,12-and 24-month old mice.Novel-object recognition test,Y-maze spontaneous alternation test and Morris water maze test showed that the recognition memory,working memory and spatial memory of 12-and 24-month old mice all suffered varying degrees of impairment.In the forced swimming test and the tail suspension test,the immobility time of the old mice was significantly prolonged,which indicated that the old mice had depression-like behavior.Both open field test and the elevated-plus maze test showed that aging mice exhibited apparent anxiety-like behavior.Finally,the results of pain-related behavioral testing showed that no significant difference was observed in aging mice,whether in the mechanical or thermal pain threshold.2.Screening differentially expressed genes in ACC,amygdala and hippocampus and verifying sequencing results by RT-PCR.A large number of DEGs were screened from ACC,hippocampus and amygdala of 12-and 24-month old mice,respectively.Subsequent experiment showed that the results of RT-PCR experiments and RNA sequencing analysis were consistent.3.Using bioinformatics analysis to analyze the differentially expressed genes.A certain number of depression or anxiety-related DEGs were found in ACC,amygdala of 12-and 24-month old mice through Venn analysis.Similarly,DEGs related with cognitive dysfunction have also been found in the hippocampus.GO enrichment analysis showed that the number of DEGs involved in immune response,cell proliferation and differentiation significantly changed.Subsequent Venn analysis showed that there were genes related to inflammation and apoptosis in different brain regions,and the number of DEGs increased with aging.KEGG analysis also showed that inflammation and apoptosis-related DEGs were involved in most of the pathways,and PPI interaction analysis showed that inflammation and apoptosis-related genes played a vital role.Conclusion:1.There were age-related cognitive function,depression and anxiety-like behavior changes in mice,but there was no pain-related behavioral changes occurred.2.RNA sequencing analysis shows that inflammation and apoptosis-related genes play important roles in aging-induced behavioral abnormalities.