The Role Of TREM2 Gene In Learning And Memory Changes Of Zebrafish Induced By Nano-alumina

Objective:Aluminum oxide nanoparticles（Al NPs）and aluminum（Al）are used in various areas of production and life,and their toxic effects can damage the nervous system.Studies have found that exposure to toxic substances early in life may be a potential risk factor for neurodegenerative diseases.This study mainly explores the role of Triggering receptor expressed on myeloid cells（TREM2）in the induction of neurodevelopmental disorders in juvenile years and the learning and memory changes in zebrafish in adulthood,and observes the neurotoxicity differences between Al and Al NPs.Methods:The study was divided into two phases,juvenile and adulthood.First,zebrafish embryos were divided into control group,negative control group,TREM2 knockdown group,Al Cl₃group,TRME2 knockdown+Al Cl₃ group,Al NPs group,TREM2 knockdown+Al NPs group.Lentiviral（TREM2 inhibitors）were injected into the yolk sac of the embryo by microinjection within 1 hour of fertilization to knock down the TREM2 gene and infected for 144 hours at a concentration of 50 mg/L.After the end of infection,the developmental toxicity of each group was observed;Neurobehavior of juvenile zebrafish（mean speed,distance traveled,percentage time spent in the outer circle,absolute turn,panic response）was recorded with a motion trajectory tracker;Detect m RNA levels of the TREM2 gene,the neurodevelopmental geneα1-tubulin,mbp,and syn2a;The ELISA method detects the activity of the neurotransmitter acetylcholinesterase（ACh E）and superoxide dismutase（SOD）,an indicator of oxidative stress.In addition,the neural behavior and T-maze experiments of adult zebrafish were detected at 9 months of age to observe adult zebrafish learning and memory changes;Detect m RNA levels of TREM2 gene,AD-related gene appb,psen1,mapta;ELISA detection of AD-associated proteins Aβ_1-42,APP,p-tau;The neurotransmitters acetylcholinesterase（ACh E）,dopamine（DA）,and superoxide dismutase（SOD）,an indicator of oxidative stress.Pathological changes in the brain of zebrafish are observed by electron microscopy and silver staining.Results:（1）Developmental toxicity:There are different degrees of deformity.Among them,the mortality rate,malformation rate and hatchability of Al Cl₃ group increased.After the knockdown of the TREM2 gene,mortality and malformation rates are further increased.The malformation rate of Al Cl₃ group was greater than that of Al NPs group,and the malformation rate of TREM2 knockdown+Al Cl₃ group was greater than that of TREM2knockdown+Al NPs group.（2）Juvenile neurobehavioral injury:the average speed,distance traveled,percentage residence time in the outer ring decreased,absolute angle increased,and panic response ability was weakened in the TREM2 knockdown group,Al Cl₃ group and Al NPs group.After the knockdown of the TREM2 gene,neurobehavioral impairment is further exacerbated.The neurobehavioral damage in the Al Cl₃ group was slightly greater than that in the Al NPs group.（3）Juvenile-related genes were suppressed:the expression of TREM2 gene,neurodevelopmental geneα1-tubulin,mbp and syn2a in zebrafish juveniles decreased significantly in the TREM2 knockdown group,Al Cl₃ group and Al NPs group.After the TREM2 gene knockdown,the TREM2 gene and neurodevelopmental genes of the juveniles are further inhibited.（4）Neurobehavioral impairment in adulthood,decreased learning and memory ability:the average speed,distance traveled,percentage of time spent in the outer ring,and activity of the TREM2 knockdown,Al Cl₃ and Al NPs groups decreased,and the panic response ability was reduced.Neurobehavioral impairment is exacerbated after TREM2 gene knockdown.The incubation period of the Al Cl₃ and Al NPs groups increased,the cumulative residence time decreased,and the learning and ability decreased.After the TREM2 gene knockdown,the learning and memory ability of zebrafish is further impaired.The neural behavior loss of the Al Cl₃ group was greater than that of the Al NPs group,and the learning ability was lower than that of the Al NPs group.（5）Adult zebrafish neurodevelopmental genes were inhibited:the expression of zebrafish neurodevelopmental genesα1-tubulin,mbp and syn2a in the TREM2 knockdown group,Al Cl₃ group and Al NPs group decreased,and after the knockdown of TREM2 gene,the TREM2 gene and neurodevelopmental genes of juveniles were further inhibited.The mbp gene of the TREM2 knockdown+Al Cl₃ group was significantly lower than that in the TREM2 knockdown+Al NPs group.（6）Increased expression of AD-related genes and proteins in adulthood:TREM2 gene remains low in adult treatment groups.The expression of appb and mapta genes in the Al Cl₃group and the Al NPs group were significantly increased.APP,Aβ_1-42,p-tau protein expression is elevated.After knockdown of TREM2 gene,the expression of appb,psen1,mapta gene and APP,Aβ_1-42,and p-tau protein were further increased in each treatment group.The expression of genes and proteins in the Al Cl₃ group was higher than that in the Al NPs group.（7）Neurotransmitters:decreased acetylcholinesterase activity in the juvenile TREM2knockdown group,Al Cl₃ and Al NPs groups.After the TREM2 gene knockdown,neurotransmitter levels decrease further.Acetylcholinesterase activity and dopamine activity decreased in the Al Cl₃ and Al NPs groups in adulthood.After the knockdown of the TREM2gene,acetylcholinesterase activity was further increased,and there was no significant change in dopamine.（8）Oxidative stress:SOD activity decreased in the juvenile TREM2 knockdown group,Al Cl₃ group and Al NPs group.After TREM2 gene knockdown,the SOD activity of TREM2knockdown+Al Cl₃ group and TREM2 knockdown+Al NPs group further decreased.SOD activity decreased in the Al Cl₃ and Al NPs groups in adulthood,and SOD activity further decreased after knockdown of TREM2 gene.（9）Zebrafish brain tissue damage:electron microscopy showed that microglia in the Al Cl₃ group and Al NPs group had hyperplasia,and microglia in the infected group had decreased after the TREM2 gene knockdown.After the Al Cl3 group and Al NPs group and TREM2 gene knockdown,zebrafish nerve cells were damaged,organelles were damaged,swelling,fragmentation,vacuoles,etc.appeared,and autophagosome phagocytosis impaired organelles.Silver staining showed that the Al Cl₃ group and the Al NPs group had black stained nerve fibers,and after the TREM2 gene was knocked down,the number of diseased nerve fibers increased,leading to the phenomenon of fiber entanglement.Conclusions:Al Cl₃ and Al NPs have neurotoxicity,leading to low expression of TREM2 gene,neurodevelopmental disorders,damage to the cholinergic system,and oxidative stress reactions in juvenile zebrafish;The neural damage caused by exposure to Al Cl₃ and Al NPs in juvenile zebrafish larvae can continue into adulthood,resulting in impaired learning and memory abilities.After knockdown of the TREM2 gene,the neurodevelopmental damage caused by Al and Al NPs to juvenile zebrafish was aggravated.The nerve damage continued into adulthood,exhibiting neurotransmitter disorders and oxidative stress damage,further reducing learning and memory abilities,and exhibiting changes similar to Alzheimer’s disease.In addition,Al Cl₃ has higher developmental toxicity and is characterized by higher teratogenicity,and Al Cl₃ and Al NPs have similar neurotoxicity in terms of neurodevelopment and learning memory.The results suggested that the involvement of TREM2 gene in the neurodevelopment and learning and memory changes of zebrafish was related to Alzheimer’s disease-like changes.