Effect Of Loss Of Function About Lanosterol Synthase(LSS) On Learning And Memory Ability Of C57BL/6J Mice

Background and purpose:Learning and memory is a kind of advanced brain electrical activity commonly found in mammals.It is an adaptive process in which individuals integrate and consolidate the received information through contact with the environment.Its main three processes are the process of receiving,merging and storing external information.According to the length of time the brain takes to complete the corresponding molecular events,it is divided into short-term memory(STM)and long-term memory(LTM).Learning and memory are multiple brain functions.The brain electrical activity that is coordinated by the regions,the most clearly studied is the hippocampus,which is located in the deep outer part of the temporal lobe of mammals.The cooperative information processing of the hippocampal dentate gyrus(DG)-CA1-CA3 neural circuit is formed by memory.Key anatomical areas.It is currently known that there is no new gene expression during short-term memory.The main molecular event is the post-transcriptional modification of related genes,while long-term memory is related to new protein synthesis.A disease closely related to learning and memory is Alzheimer's disease,which is a neurodegenerative disease.The pathogenesis is currently unknown and effective treatments are lacking.Therefore,in-depth study of animal learning and memory processes and related molecular mechanisms is one of the main goals of this research.This study uses the LSS+/-gene knockout mouse model to test its memory ability and combine multi-omics research,which aims to reveal the role of lanosterol synthase in neurometabolism;this work is cholesterol metabolism and learning and memory Provides a link between them,and sets LSS as a potential drug treatment target.Methods:(1)General situation of mouse brain:Take 8 weeks old mature C57BL/6J mice born in the same litter,use 2%pentobarbital sodium at a dose of1ml/kg to anesthetize the mice,and draw the heart The blood was processed and sacrificed,and the serum was separated and used for lipid index detection.The mouse brain was completely isolated,weighed,and then the hippocampus was isolated for detection of related indicators.(2)Behavioral experiments:The mature C57BL/6J mouse strains of 8 weeks old were used for animal behavioral experiments.All animal experiments were approved by the Animal Ethics Committee of Anhui Medical University.100 SPF-level female 8-week-old wild type(Wide type,WT)and LSS knockout mice(LSS+/-)born in the same litter were divided into several groups for behavioral testing.Before the start of the behavioral test,all experimental animals were raised in the SPF standard environment to 8 weeks of age and then brought into the behavioral laboratory to adapt for at least one week to eliminate the interference of the environment on the animals.In order to rule out the influence of sleep on animal behavior,we started behavioral tests in mice at 18:00 every day.All tests are carried out in the light cycle behavior laboratory from 08:00 to 20:00.In order to exclude the influence of different behaviors on animals,only one behavior test was performed for each batch of animals.All videos were analyzed by behavioral software,and all behavioral experiments were carried out using random double-blind experimental methods to eliminate the influence of experimenters'subjective factors on behavioral experiments.(3)Use hematoxylin-eosin staining(HE),Golgi staining,transmission electron microscope imaging and other methods to observe whether there are changes in the microstructure and ultrastructure of the hippocampus of different groups of mice;immunohistochemical staining and immunofluorescence to observe specific proteins Express differences.Use HPLC-MS for non-targeted lipid metabolomics to detect differences in lipids in each group of hippocampus;use transcriptome sequencing to detect differentially expressed genes in each group of hippocampus and screen differential genes related to learning and memory;real-time fluorescent quantitative PCR(Real time-q PCR)to verify the differential gene expression screened by sequencing.Western blotting(Western blotting,WB)method detects the expression of related proteins related to learning and memory,and simultaneously detects the expression of SREBP1,SREBP2,and ERK and AKT signaling pathway related proteins.Results:(1)General situation of mice:Among the same-sex mice born in the same litter,there was no significant difference in body weight,appearance,brain weight and brain appearance between wild group and gene knockout group.The hippocampus of gene knockout group The expression of LSS gene was significantly down-regulated.There was no significant difference between the hippocampal HE staining groups.(2)Behavioral test of mice:Compared with wild group mice,LSS knockout mice showed more obvious anti-anxiety-like behaviors(p<0.05)and higher mobility(p<0.05).),it also showed better memory ability in the spatial positioning memory test(p<0.05).(3)Detection of serum lipids:There was no significant difference in TG and CHO between wild group and gene knockout group,and there was no significant difference in LDL-C and HDL-C.(4)The mouse hippocampal transcriptome sequencing results suggest that the WT group and LSS+/-group are in energy metabolism,ribosome binding protein,nervous system development,long-term potentiation(LTP),long-term inhibition(LTD),Alzheimer's disease,glycine neuron pathway,glutamate neuron pathway,dopamine neuron pathway,nicotine addiction,positive fear memory regulation and other processes are different.(5)Real-time PCR analysis revealed that the expression levels of 14 genes related to neurotransmitter receptors in the hippocampus of the LSS^(+/-)group were significantly changed compared with those in the WT group;ribosomes in the hippocampus of the LSS^(+/-)group Compared with the WT group,the expression levels of 20 genes related to binding protein indicated down-regulation,3 genes indicated up-regulation,and 8 genes were not significantly different.(6)The results of the hippocampal non-targeted lipid metabolome analysis showed that compared with the WT group,the PS of the LSS^(+/-)group was significantly higher,the PC was higher,and the PE was lower,while the changes of other phospholipids were not obvious.(7)Hippocampal transmission electron microscopy imaging results show that the mitochondria,endoplasmic reticulum and myelin structure of the hippocampus of LSS^(+/-)mice are more complete,and the number of ribosomes is more abundant than that of WT mice.(8)(8)Western-blotting results showed that compared with the same group of wild-type mice,neuromodulator 3(NGN3)and neurotrophin 4(NXPH4)were significantly up-regulated in LSS knockout mice.Zinc finger family receptor 268(Zif268)and calmodulin-dependent protein kinase II(Ca MKII)were decreased in LSS(+/-)mice before and after Morris water maze training.Conclusion:Unconditional heterozygous knockout mice of LSS gene show better spatial learning and memory capabilities and more pronounced anxiolytic-like behaviors.The molecular mechanism is partly related to the metabolism of phospholipids.