Effects And Mechanism Of Specific Knockout Of TPP2 Gene In Excitatory Neurons On Learning And Memory In Mice

BackgroundTripeptidyl peptidase II（TPP2）is a member of the eukaryotic serine peptidase family,which can remove tripeptides from the N-terminal of longer peptides.It plays an important role in cell autoimmunity,aging,and metabolism.Studies have found that patients with abnormal TPP2 gene function cause autoimmune diseases and aseptic encephalitis.Our previous experiments demonstrated that compared with wild-type mice,TPP2 systemic knockout mice developed severe immunodeficiency and impaired learning and memory^[1].It is unknown whether the impairment of learning and memory ability of mice is caused by the impairment of immune system caused by TPP2 gene deletion or directly acting on the nervous system.PurposeIn order to exclude the effect of immune system deficiency on the learning and memory ability of TPP2 gene-deficient mice,we selected mice with specific loss of TPP2function in excitatory neurons as the experimental subjects to study the effect of TPP2 on learning and memory in the nervous system,in order to explore its mechanism and provide reference for the treatment of neurodegenerative diseases.Methods1.Conditional knockout mice were obtained and identified.By mating homozygous TPP2 mice with a flox locus with Cre mice with tissue-specific expression,the flox locus is activated to knock out the TPP2 gene between the two flox sites,and the mice that specifically knock out the TPP2 gene in excitatory neurons are obtained.The genotypes of mice were identified by PCR.2.Animal behavior experiments（water maze test Morris water maze）.Record the experimental data such as the time（latency）that the mouse took to find the platform for the first time during the training period and the test period,speed,and times of crossing the platform,and use statistical software to statistically analyze the data of each group and evaluate the hippocampal dependent learning and memory ability of mice in each group.3.Record EEG signals in vivo.Electrodes were implanted in the brain（CA1 region of the hippocampus）of female mice using an in vivo multi-channel microfilament array electrode implantation method.The local field potential（LFP）signals of neurons in the mouse brain were recorded and analyzed on the 6th postoperative day to explore the effect of TPP2 deletion on learning and memory signals in hippocampal neurons.4.Detect the activity of related proteins in TPP2 knockout cells.Using cell monoclonal technology,Western blotting（WB）and other experimental methods to explore the changes of protein activity related to metabolism in 293T cells after knocking out TPP2gene.Results1.Using hybridization and PCR technology,mice with specific knockout of TPP2gene in excitatory neurons(Cre⁺-TPP2^-/-)were successfully obtained.2.The learning and memory abilities of mice were tested by Morris water maze test.It was found that:（1）About 60 days and 180 days of age,the incubation period of the control group(Cre^--TPP2^flox/flox)and the experimental group(Cre⁺-TPP2^-/-)gradually decreased with the increase of days,and the latency period on the fifth day was significantly lower than the first day（P<0.05）.（2）Compared with Cre⁺-TPP2^-/-mice,the incubation period of Cre^--TPP2^flox/flox mice in the training period increased at about 60 days of age and decreased at about 180 days of age,but there was no statistical significance（P>0.05）;（3）Cre^--TPP2^flox/flox mice,both male and female,had a significantly lower overall latency during the training period at around 180 days of age than mice at around 60 days（P<0.05）;（4）The incubation period of Cre⁺-TPP2^-/-mice during the training period was about180 days old compared with the mice of about 60 days old,and there was no significant difference in females（P>0.05）;Males showed a decrease（P>0.05）,but generally higher than Cre^--TPP2^flox/flox.3.In vivo multi-channel microfilament array electrodes were implanted to detect electrical signals.It was found that theγ-rhythm andθ-rhythm of Cre⁺-TPP2^-/-mice were significantly lower than those of Cre^--TPP2^flox/flox mice（P<0.01）.4.Compared with normal 293T cells,the expression levels of metabolism-related proteins P-AMPK and P-ULK1 were significantly increased in TPP2 gene-deficient cells（P<0.05）,and the expression level of P-Raptor was significantly decreased（P<0.05）.Conclusion1.Specific knockout of TPP2 gene in excitatory neurons causes impaired learning and memory function.2.Specific knockout of TPP2 gene in excitatory neurons can lead to the decrease ofγ-rhythm andθ-rhythm,which are closely related to learning and memory,and the molecular mechanisms may be mediated through the AMPK/ULK1/m TOR signaling pathway.