The Role And Mechanism Of Prkcaa In Regulating The Social Behavior Of Zebrafish

Protein kinase C(PKC)is a class of protein kinases that phosphorylate serine and threonine residues of proteins and are classified into three isoforms,classical PKC,novel PKC and non-classical PKC,according to their structural characteristics.Protein kinase Cα(PKCα/PRKCA)belongs to the classical PKC family and has been shown to be an important regulator of circadian rhythms,while mutations in the PRKCA gene are strongly associated with human psychiatric disorders such as autism spectrum disorders and schizophrenia.However,the role of PRKCA in regulating social behaviour in animals and its underlying mechanisms remains to be explored,and further study are needed to construct knockout models.The doubling of the prkca gene in the genome of zebrafish produced two paralogous homologs,prkcaa and prkcab,located on chromosomes 6 and 3,respectively.Zebrafish Prkcaa and Prkcab contain 670 and 671 amino acids,respectively,with 87% sequence similarity.Both genes were expressed in all tissues except the liver,but the expression patterns differed,with the highest expression levels of prkcaa in ovarian tissue,followed by the eye and brain,and prkcab in brain tissue,followed by the eye and gill.Exogenous expression in 293 T cells revealed that prkcaa was localised in the cytoplasm;prkcab was found to be localised not only in the cytoplasm but also in the cell membrane.To further investigate the role of the prkcaa gene in regulating animal social behaviour,we constructed a zebrafish mutant model with deletion of the prkcaa gene using CRISPR/Cas9 technology and obtained "-2 bp" and "-11 bp " The mutation of the prkcaa gene did not affect the development,growth and reproduction of zebrafish under normal conditions.The prkcaa knockout mutant was used as a model for behavioural experiments,including novel tank experiments,mirror biting experiments,social preference experiments and shoaling experiments.The results of the novel water tank experiment showed that the latency(P<0.001),total number of times(P<0.001)and duration of stay(P<0.001)of prkcaa pure mutants entering the upper layer of the novel water tank were significantly lower than those of WT,showing anxiety-like behavioural phenotypes;the results of the mirror bite experiment showed that the number of times(P<0.001)that the mutants entered the Approach Area(AA)was significantly lower than that of WT.AA(P<0.01)was significantly lower,and the duration(P<0.01)and frequency(P<0.001)of pre-mirror bites were significantly lower,implying a significant reduction in the tendency/behaviour of the mutant to attack in front of the mirror;there was a highly significant difference in preference values for homogeneous groups between mutants and WT in the social preference experiment(P<0.001);mutant fish in the clustering experiment showed a more looser and larger schools,as reflected by increases in mean inter-fish distance(P<0.001),mean near-neighbour distance(P<0.001),and group area(P<0.001),in addition to a significant increase in outlier frequency(P<0.001)and a significant decrease in polarisation values representing the consistency of swimming direction in the fish,suggesting that dysfunction in Prkcaa leads to deficits in the social competence of zebrafish.The above results suggest that zebrafish prkcaa has an important role in the regulation of emotional behaviour in zebrafish.To investigate the mechanism of Prkcaa’s role in regulating fish behavior,we used RNA-seq to analyze the differences in gene expression patterns in WT and 3145 A mutant brains in the morning(9:00 AM)and evening(9:00 PM),respectively.The sequencing results showed a significant effect of both genotype(WT vs.3145A)and sampling time(night vs.morning)on gene expression in zebrafish.Of these,175 circadian genes were affected by prkcaa mutations,including 44 night-preferred genes(higher expression at night,GS1)and 131 morning-preferred genes(higher expression in the morning,GS2)prkcaa mutations had a significant effect on the expression of morning-preferred genes(P<0.001),but not on night-preferred genes(P=0.1045).The representatives are the immediate early genes including egr2a(early growth responsegene 2a),egr4,fosaa(V-fos FBJ murine osteosarcoma viral oncogene homolog Aa),fosab and npas4a(neuronal PAS domain protein 4a).Down-regulation of these genes at night was attenuated by Prkcaa dysfunction.These genes are also significantly associated with neurological development and neural activity.Consistent with this,prkcaa mutants exhibited reversed circadian motor rhythms,with significantly higher activity at night than in the morning,mainly in the form of significantly higher motor distance at night than during the day(P<0.05),and in addition we divided the zebrafish swimming speed into four motor states based on large,medium,small and stationary movements,and found that compared to WT fish a significantly higher proportion of large movements in the morning and higher proportion of moderate locomotion,the zko3145 A mutant had more small locomotion in the morning and more moderate locomotion in the evening.Our data confirm the role of PRKCA in regulating social interactions in animals and link social behavior deficits to disordered circadian rhythms,providing new insights into the molecular mechanisms underlying the function of Prkcaa.