Construction Of UCP2 Knockout Model Of Zebrafish And The Preliminary Study Of Aluminum Toxicity On This Model

Alzheimer’s disease（AD）is an age-related neurodegenerative disorder characterized by learning and memory disorders.The pathological features include the deposition of senile plaques（SP）,neurofibrillary tangles（NFTs）and the reduction of neurons.With the development of aging in the world,the number of AD patient is increasing.AD has affected human health seriously,which brings a great pain and financial burden to the patients and their families.At present,the pathogenesis of AD is still not clear and needs to further explore.In this study,UCP2 was screened out through the aluminum exposed zebrafish model,and the zebrafish model of UCP2 knockout was constructed by the CRISPR/Cas9technique.Then the model was used to study the role of UCP2 in dementia induced by aluminum.The research contents and results in detail are as following:1.Screen the sensitive genes relateted to mitochondrial through zebrafish exposed aluminum modelThe wild zebrafish were exposed to AlCl₃（pH 5.8,100μg/L AlCl₃）for 40 days.The behavioral changes were tested by T-maze,and the sensitive genes related to mitochondrial were screened by RT-qPCR.Compared with the control group,the latency time of zebrafish exposed to AlCl₃ was prolonged（P<0.01）and the ability of learning and memory decreased.The mRNA expressions of ALDOB and PFKFB4 were decreased significantly（P<0.01）.The mRNA expression of UCP2 and P53 were significantly increased（P<0.01）.2.Construct UCP2 knockout zebrafish by CRISPR/Cas9The specific pT7-UCP2-gRNA plasmid was constructed by designing a appropriate target on the functional domain of zebrafish UCP2 gene through the CRISPR/Cas9 system.The specific gRNA（50 ng/μL）and Cas9-mRNA（300 ng/μL）were mixed and microinjected into the zebrafish embryo of one-cell.The activity test was used to confirme the effectiveness.The zebrafish with mutation were feeded for 2-3 months.The genomic DNA of fish tails was extracted,digested by BsmAI and sequenced to confirm the generation of zebrafish mutants.The zebrafish of F0 generation mutants and wild type were mated,the zebrafish of F1 generation mutants male and female were mated.After being sequenced,the F2 generation zebrafish mutants（homozygotes）on embryonic stage were obtained.3.The effect of UCP2 knockout on the development of AD induced by aluminumZebrafish larvae（Wild and F1 heterozygous）were exposed to AlCl₃（150μg/L,pH 5.8）for 96 h,The locomotor activity of zebrafish larvae were recorded by Noldus Tracking system,the cell apoptosis were observed by acridine orange（AO）staining and scoring under fluorescence microscope,the level of reactive oxygen species（ROS）were determined by the biochemical method,and the mRNA expression of neural-related genes were analyzed by RT-qPCR.Compared with the control group,the individual behavior of the zebrafish exposed to AlCl₃ were abnormal,and the distance,speed and frenquencyof swim for zebrafish significantly decreased（P<0.01）,while the ROS level were significantly increased（P<0.01）.UCP2 knockout zebrafish exposed to AlCl₃ caused more obvious abnormalities,in which the zebrafish brain showed significant neurons apoptosis,and the mRNA expressions of PSEN1,PSEN2,TNF-αand IL-1βwere significantly up-regulated（P<0.01）.In conclusion,aluminum exposure reduce the ATP production and affect Ca²⁺reabsorption,which cause the imbalance of Ca²⁺homeostasis and energy metabolism,then,the UCP2 gene expression is upregulated in order to reduce the injury by decreasing ROS production.UCP2 knockout combination with aluminum exposure increase the production of ROS,and activate the expression of PSEN1,PSEN2,TNF-αand IL-1β,which lead to the accumulation of Aβ,neuroinflammation and neurons apoptosis,eventually lead to the cognitive impairments of AD.Regulation of UCP2 may be a good target for treating environmental aluminum exposure syndrome and AD.