Role Of NEGR1 On High Fat Diet-induced Cognitive Impairment In Obese Mice

BACKGROUND:In recent years,the number of overweight and obese people worldwide has increased dramatically;meanwhile,the incidence of various cognitive dysfunctional diseases including Alzheimer’s disease,is increasing with the aging of the population.Cognitive decline can seriously affect the quality of life of patients and their families and bring heavy financial burden.However,there is no effective treatment available yet.Several studies have shown that the increase of saturated fatty acids associated with high fat diet contribute to the occurrence of cognitive impairment through directly acting on the hippocampal tissue or indirectly promoting hippocampal inflammation.Neurofibrillary tangles caused by hyperphosphorylation of hippocampal neuronal tau protein is one of the major pathological changes of cognitive dysfunction.Neuronal growth regulator 1(Negr1),is an adhesion molecule on the surface of cell membranes,and its involvement in neurodevelopmental disorders and psychiatric disorders such as depression has been previously reported in the literature.The role of the obesity susceptibility gene negrl in obesity-related cognitive dysfunction is unclear.The aim of this study was to investigate the effect of NEGR1 on tau protein phosphorylation in hippocampal neurons of mice with high-fat diet-induced obesity cognitive dysfunction,thereby providing new ideas for an in-depth understanding of the mechanisms of obesity-related cognitive dysfunction.OBJECTIVE:(1)To identify the NEGR1 expression in mice with cognitive dysfunction induced by high-fat diet(HFD).(2)To clarify whether the knockdown of hippocampal neuron Negr1 in HT22 cells improves tau protein hyperphosphorylation.(3)To investigate the influence of NEGR1 on kinase phosphorylation and its involvement in the hyperphosphorylation of neuronal tau proteins.(4)To explore whether inflammatory cytokines are involved in NEGR1-related tau protein hyperphosphorylation.METHOD:(1)Male C57BL/6J mice(4 weeks old)were assigned to either a fed with regular diet(RD)(n=8)or HFD(n=12),respectively.The DIO and the control mice were fed for 16 weeks later to evaluate the establishment of obesity-related cognitive dysfunction through the cognitive-related behavioral tests and the measurements of phosphorylation level of tau protein in hippocampus.(2)Three hippocampal tissue samples each from RD and HFD fed mice for 16 weeks were selected for RNA sequencing(RNAseq).Gene set enrichment analysis(GSEA)was employed to conduct intersection analysis of different gene sets.Real Time Quantitative PCR(RT-qPCR),Western blotting(WB)and immunohistochemistry were used to detect the expression of NEGR1 in hippocampus of RD and HFD mice.(3)First,a cellular model of tau protein hyperphosphorylation was induced in HT22 cells using palmitic acid(PA).Detection of the effect of knockdown of NEGR1 on PA-induced tau protein phosphorylation in HT22 cells.Male C57BL/6J mice were fed with RD(n=16)or HFD(n=24),in which neuronal labeled(hSyn)AAV-shNegrl or AVV-shNT was performed at week 16 by hippocampal stereotaxic injection.The mice were fed with RD and HFD until 24 weeks for cognitive behavior and detection of tau phosphorylation.(4)The scramble and OE-Negr1 HT22 cells were subjected to proteomics and phosphomics sequencing,and followed by the application of bioinformatics methods to identify for differential signaling pathways.Protein-protein interaction networks(PPI)were used to identify kinases that exhibited altered expression between the two groups,and tau protein phosphorylation kinases Mapk1 and Mapk3 were found to be significantly upregulated in the OE-Negr1 group.Subsequently,tau phosphorylation was detected again after MAPK inhibitor treatment in scramble and OENegrl HT22 cells.Detection of the effect of knockdown of NEGR1 on MAPK signaling pathway in hippocampal neurons of HFD mice.(5)First,RT-qPCR was used to detect the mRNA levels of proinflammatory cytokines in hippocampus of RD and HFD mice as well as HT22 cells treated with PA.Secondly,tau protein phosphorylation and NEGR1 expression levels in HT22 cells treated with IL1β were detected by WB,which was the most obvious change in HT22 cells.Then,NEGR1 antibody was used to block NEGR1 on the surface of HT22 cells,and the levels of tau protein phosphorylation and ERK phosphorylation in HT22 cells were once again detected.RESULT:(1)Compared with the RD group,C57BL/6J mice fed with HFD for 16 weeks showed impaired recognition memory,and the phosphorylation of tau protein in the hippocampus of the HFD group was significantly increased.(2)GSEA analysis of RNA sequencing results in hippocampus of RD and HFD groups showed that Negr1 was enriched in hippocampal tissue of HFD group.(3)Negr1 knockdown in HT22 cells attenuated PA-induced tau protein phosphorylation in HT22 cells.After knockdown Negr1 in hippocampal neurons of mice fed with HFD for 16 weeks,cognitive dysfunction was improved despite continued high-fat diet.(4)The OE-Negr1 group of HT22 cells,compared with the Scrambled group,showed increased levels of tau protein phosphorylation.By proteomics and phosphomics analysis of scrambled and OE-Negrl HT22 cells,we found that elevated phosphorylation levels of tau protein phosphorylation kinases ERK 1/2,p38 MAPK in OE-Negr1 HT22 cells,and the use of ERK inhibitors ameliorated NEGR1 induced tau protein hyperphosphorylation.The phosphorylation levels of ERK1/2,p38 MAPK were reduced after knockdown of hippocampal neuron Negr1 in HFD group.(5)The mRNA levels of pro-inflammatory cytokines in the hippocampus of HFD mice were increased.Similarly,PA can increase the IL1β mRNA levels in HT22 cells.NEGR1 antibody attenuated the IL1βinduced increase in ERK phosphorylation and tau phosphorylation in HT22 cells.CONCLUSION:The expression of NEGR1 increased in the hippocampus of HFDinduced obese mice,which activates the ERK/MAPK pathway,and subsequently caused tau protein hyperphosphorylation and is involved in the development of cognitive decline in obese mice.