| Asprosin is the 140-residue,C-terminal product of the fibrillin 1 protein(encoded by FBN1).The peptides have various physiologic functions,including the release of hepatic glucose and elevation of glucose.Considerable evidence has already shown that Asprosin is a high risk factor for type 2diabetes mellitus.Therefore,exploring the role of Asprosin in diabetic-associated cognitive dysfunction(DACD)is of great significance.N6-methyladenosine(m~6A)is the most prevalent modification in the mRNA of eukaryotes.Numerous studies show that METTL3-m~6A-YTHDF1axis in hippocampus positively regulates learning and memory.m~6A modification catalyzed by METTL3(methyltransferase like 3,also known as MTA70)can facilitate learning and memory,which is dependent on m~6A readers(YTH domain-containing family member 1,YTHDF1).We therefore undertake the pathogenesis of DACD from the perspective of hippocampal METTL3-m~6A-YTHDF1 axis regulating.To sum up,to investigate whether asprosin contributes to the development of DACD via regulating hippocampal METTL3-m~6A-YTHDF1axis,we performed researches in the following three aspects:1)To confirm whether the Asprosin level is elevated and the METTL3-m~6A-YTHDF1 axis is disturbed in the hippocampus of diabetic rats.2)To clarify whether Asprosin overexpression in hippocampus leads to cognitive impairment as well as the disorder of hippocampal METTL3-m~6A-YTHDF1 axis.3)To comfirm whether knock-down of Asprosin attenuates the the development of DACD and normalises the hippocampal METTL3-m~6A-YTHDF1 axis.Part Ⅰ There are dysfunction of cognitive,elevation of hippocampal Asprosin,and disorder of hippoampal METTL3-m~6A-YTHDF1 axis in diabetic ratsObjective:To comfirm the development of DACD is associated with the elevation of Asprosin and the disorder of METTL3-m~6A-YTHDF1 axis.Methods:After fed with high-fat diet(HFD)for four weeks,rats were intraperitoneal injected with streptozotocin(STZ,30 mg/kg).Eight weeks after administration,the novel object recognition(NOR)test and Morris water maze(MWM)test were used to evaluate the cognitive function of rats.Then rat hippocampus tissues were collected and analysed:Asprosin mRNA levels were analyzed by RT-qPCR;Expression levels of the expected proteins were confirmed by western blot analysis,including Asprosin,the synapse-related proteins(PSD95 and SYN1),glutamate receptors(NMDAR1,NMDAR2A,NMDAR2B,GluA1,and GluR2),METTL3,and YTHDF1.The abundance of m~6A in hippocampus was measured by m~6A RNA Methylation Quantification Kit(Colorimetric).The changes of m~6A modification and gene expression were analysed respectively by m~6A-seq and transcriptome sequencing.Results:1)Diabetic rats presented cognitive impairment:The diabetic rats showed a significantly decreased discrimination index by NOR test;In MWM test,the escape latency to the platform of the diabetic rats was markedly prolonged,while the percentage of time in target quadrant and the times of crossing platform were remarkably reduced,indicating impairment in cognition of diabetic rats.2)Hippocampal synaptic plasticity of diabetic rats was impaired:The levels of synapse-related proteins(PSD95 and SYN1)expression were significantly lower in the hippocampus of diabetic rats,while the glutamate receptors proteins(NMDAR1,NMDAR2A,NMDAR2B,GluA1,and GluR2)expression were remarkably increased.These datas suggested that hippocampal synaptic plasticity of diabetic rats was impaired.3)The level of Asprosin in hippocampus of diabetic rats was elevated:Both mRNA and protein levels of Asprosin were increased in the hippocampus of diabetic rats.4)The METTL3-m~6A-YTHDF1 axis in the hippocampus of diabetic rats was disordered:The abundance of m~6A modification as well as the expressions of METTL3 and YTHDF1 were all decreased in the hippocampus of diabetic rats,suggesting that there is disorder of METTL3-m~6A-YTHDF1axis in the hippocampus of diabetic rats.5)The changes of m~6A modification and gene expression in the hippocampus of diabetic rats:In the hippocampus of diabetic rats,m~6A-seq data identified 692 differential peaks with up-regulated m~6A modifications and1210 differential peaks with down-regulated m~6A modifications,functional enrichment analysis showed that the hub genes were mainly enriched in dendritic spine development and axon regeneration;Transcriptome sequencing identified 140 up-regulated differential genes and 99 down-regulated genes,which functions were mainly enriched in synapse,Parkinsons disease,oxidative phosphorylation,and Alzheimers disease;Combined analysis of transcriptome sequencing and m~6A-seq identified 28 differential genes,which were associated with Parkinsons disease and Alzheimers disease.Part Ⅱ Overexpression of hippocampal Asprosin induces the cognitive impairment and the disorder of hippocampal METTL3-m~6A-YTHDF1 axis in normal ratsObjective:To determine that the overexpression of hippocampal Asprosin leads to the cognitive dysfunction as well as the impairment of synaptic plasticity and the disorder of METTL3-m~6A-YTHDF1 axis in the hippocampus.Methods:After adaptation of one week,rats were respectively treated with AAV-Asprosin or AAV-Control(10E×10 vg,i.c.v.)for once.After four weeks,the cognitive function of rats was evaluated by NOR test and MWM test.The expressions of synapse-related proteins,glutamate receptors,METTL3,and YTHDF1 were measured by Western blot.The ultrastructure of synapses in hippocampus was detected by transmission electron microscopy(TEM).The level of glutamate in the hippocampus was measured by the high-performance liquid chromatography-tandem mass spectrometry(LC-MS).The abundance of m~6A in hippocampus was measured by m~6A RNA Methylation Quantification Kit(Colorimetric).The changes of m~6A modification and gene expression in hippocampus were detected by m~6A-seq and transcriptome sequencing,respectively.Results:1)Overexpression of hippocampal Asprosin impairs the cognition of normal rats:Overexpression of hippocampal Asprosin significantly decreased the discrimination index of normal rats in the NOR test.Overexpression of hippocampal Asprosin increased the escape latency to the hidden platform and decreased the percentage of time in the target quadrant as well as the times of crossing platform of normal rats in MWM test.These results suggested that overexpression of hippocampal Asprosin impairs the cognition of normal rats.2)Overexpression of hippocampal Asprosin leads to synaptic plasticity disorders in normal rats:Overexpression of hippocampal Asprosin decreased the expression of PSD95 and SYN1,increased the expression of NMDAR1,NMDAR2A,NMDAR2B,GluA1,and GluR2,and did not change the concentration of glutamate in the hippocampus of normal rats.Overexpression of hippocampal Asprosin obviously reduced the synaptic density,length of synaptic active zone,PSD thickness,as well as increased the synaptic cleft width in the hippocampus of normal rats.Thus,these results suggested that Asprosin induces the disorders of synaptic plasticity in the hippocampus of normal rats.3)Overexpression of hippocampal Asprosin leads to the hippocampal METTL3-m~6A-YTHDF1 axis disorder:Overexpression of hippocampal Asprosin decreased the expression of METTL3 and YTHDF1,as well as reduced the abundance of hippocampal m~6A,which suggests that overexpression of hippocampal Asprosin leads to the hippocampal METTL3-m~6A-YTHDF1 axis disorder.4)Effects of hippocampal Asprosin overexpression on m~6A modification and gene expression in hippocampal of normal rats:In the hippocampus of AAV-Asprosin treated group,the m~6A-seq data identified 1014 differential peaks with up-regulated m~6A modifications and 759 differential peaks with down-regulated m~6A modifications,the functional enrichment analysis showed that the hub genes were mainly enriched in G protein-coupled receptor signaling pathway,immune response,NOD-like receptor signaling pathway,and neuroactive ligand-receptor interaction;Transcriptome sequencing identified 79 up-regulated differential genes and 83 down-regulated differential genes,which mainly enriched in ionotropic glutamate receptor complex,regulation of AMPA receptor activity,postsynaptic density assembly,dendritic spine morphogenesis,and PI3K-Akt signaling pathway;The combined analysis of m~6A-seq and transcriptome sequencing identified 10differential genes,which enriched in Hippo signaling pathway.Part Ⅲ Knock-down of hippocampal Asprosin ameliorates DACD and reverses the disorder of the hippocampal METTL3-m~6A-YTHDF1 axis Objective:To determine that knock-down of hippocampal Asprosin ameliorates DACD and redresses the disorder of METTL3-m~6A-YTHDF1axis in the hippocampus.Methods:After four weeks of HFD feeding,rats in the model group were treated with AAV-Asprosin-sh RNA(10E×10 vg,i.c.v.),and then injected with STZ(30 mg/kg,i.p.)for once.Eight weeks after administration,the cognitive function of rats was evaluated by NOR test and MWM test.The expressions of synapse-related proteins,glutamate receptors,METTL3,and YTHDF1were measured by Western blot.The ultrastructure of synapses in hippocampus was detected by TEM.The level of glutamate in the hippocampus was measured using the LC-MS.The abundance of m~6A in hippocampus was measured by m~6A RNA Methylation Quantification Kit(Colorimetric).The changes of m~6A modification and gene expression in hippocampus were detected by m~6A-seq and transcriptome sequencing,respectively.Results:1)Knock-down of hippocampal Asprosin ameliorates cognitive function of diabetic rats:Knock-down of hippocampal Asprosin significantly increased the discrimination index of diabetic rats in the NOR test.Knock-down of hippocampal Asprosin decreased the escape latency to the hidden platform as well as increased the percentage of time in the target quadrant and the times of crossing platform of diabetic rats in MWM test.These results suggested that knock-down of hippocampal Asprosin ameliorates the cognitive dysfunction in diabetic rats.2)Knock-down of hippocampal Asprosin improves synaptic plasticity disorders in diabetic rats:Knock-down of hippocampal Asprosin enhanced the expression of PSD95 and SYN1,reduced the expression of NMDAR1,NMDAR2A,NMDAR2B,GluA1,and GluR2,as well as decreased the concentration of glutamate in the hippocampus of diabetic rats.Knock-down of hippocampal Asprosin obviously increased the synaptic density,length of synaptic active zone and PSD thickness,as well as decreased the synaptic cleft width in the hippocampus of SD rats.These results suggested that knock-down of hippocampal Asprosin improves the synaptic plasticity disoders in the hippocampus of diabetic rats.3)Knock-down of hippocampal Asprosin redresses the disorder of METTL3-m~6A-YTHDF1 axis in the hippocampus of diabetic rats:Knock-down of hippocampal Asprosin increased the expression of METTL3and YTHDF1,and up-regulated the abundance of hippocampal m~6A,which suggested that knock-down of hippocampal Asprosin reverses the disorder of METTL3-m~6A-YTHDF1 axis in the hippocampus of diabetic rats.4)Effects of knock-down of hippocampal Asprosin on m~6A modification and gene expression in diabetic rats:In the hippocampus of AAV-Asprosin-sh RNA treated diabetic rats,the m~6A-seq data identified 845differential peaks with up-regulated m~6A modifications and 736 differential peaks with down-regulated m~6A modifications,the functional enrichment analysis showed that the hub genes were mainly enriched in glycolysis/gluconeogenesis,NOD-like receptor signaling pathway,immune response and calcium signaling pathway;Transcriptome sequencing identified140 up-regulated differential genes and 99 down-regulated differential genes,which mainly enriched in innate immune response,Parkinson disease,oxidative phosphorylation,and PI3K-Akt signaling pathway;The combined analysis of m~6A-seq and transcriptome sequencing identified 29 differential genes,which enriched in inflammatory response,neuroactive ligand-receptor interactive,PI3K-Akt signaling pathway.Conclusion:Asprosin contributes to the development of DACD via negatively regulating the hippocampal METTL3-m~6A-YTHDF1 axis. |
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