Study On The Relationship Between Repetin And Schizophrenia

Repetin(RPTN) protein is a member of S100 calcium-binding protein family, with two EF-hand domains in the N terminal that can reversely bind calcium and internal tandem repeats.RPTN is known to be expressed in the normal epidermis, but is also high in the acrosyringium, the inner hair root sheath and in the filiform papilli of the tongue. S100B, another member of S100 family, is abundant in central nervous system(CNS) and has been suggested to be a susceptibility gene for bipolar disorder, schizophrenia and cognitive dysfunction. Neurons have highly developed Ca2+ signalling systems responsible for regulating many neural functions such as the generation of brain rhythms, information processing and the changes in synaptic plasticity that underpins learning and memory. Our previous study for the first time found that RPTN is expressed in CNS, with high expression in prefrontal cortex and hippocampus where is closely associated with schizophrenia and depressive disorder. The expression of RPTN in hippocampus and prefrontal cortex of mouse subjected to chronic unpredictable mild stress is decreased, suggestting its potentional function in mental disorders. But the functions and molecular mechanisms in nervous system is unknown. In this study, the underlying molecular mechanism of RPTN in CNS is explored.Research aims1) SerumRPTN levels in normal human of different ages and gendersCollecting serum of normal human of different ages and genders,examine RPTN levels,using statistical method to analyze serum RPTN levels with the correlation between age and gender.2) Serum RPTN levels in normal human subjects, patients with schizophrenia and bipolar disorder and drug usersCollecting serum from normal human subjects, patients with schizophrenia, bipolar disorder and drug users, examine RPTN levels respectively. Then using statistical method analyze serum RPTN levels among normal human, schizophrenia, bipolar disorder and drug users.3) Serum RPTN levels in mouse treated with atypical antipsychoticsSince the majority of the patients enrolled in the study were under atypical antipsychotic therapy, to rule out the possibility that the reduction of serum RPTN was due to antipsychotics, we tested the effect of three widely used drugs (Olanzapine, Quetiapine and Aripiprazole) serum RPTN levels in mouse and using statistical methods to analyze.4) Platelet concertrations of RPTN from healthy and schizophreniaDue to mental disorder patients with high S100B protein in platelet, the origin of serum RPTN is currently unclear. We measure platelet concentrations of RPTN from healthy and schizophrenia and using statistical methods to analyze.5) Research on the RPTN promoter transcriptional sctivityThe RPTN promoter contains the sequence TTNCNNNAA (N for any base),this sequence is a target for JAK-STAT signaling. We made a pGL2-RPTN promoter luciferase report vetor,then determine the effect of 5-HT2A receptor antagonist Olanzapine, Ca2+ and 5-HT2A receptor agonist DOI on RPTN promoter transcr-iptional activity by measuring luciferase activity.6) Structure and bioinformatics prediction of RPTNUsing bioinformatics method,examing sequence space of the RPTN and α-synuclein, β-amyoid, tau, Disrupted-in-Schizophrenia-l,then we computered the Z-score.7) Research on the functions of RPTN in nervous systemTo investigate the expression pattern of RPTN in nervous system, immunohisto-chemitry was used to examine the RPTN expression in neural stem cell of mouse brain.One of the pathogenesis of schizophrenia is the decline of hippocampal autophagy. Under physiological and pathological conditions,many factors can induce selective autophagy. Rapamycin, an inhibitor of mTOR signaling and an inducer of autophgay, was used to induce HEK293T cell autophagy, then we analyzed the effect of overexpression RPTN rapamycin induced autophagy.Patients, materials and methods1) Research on serum RPTN levels in normal human subjects, schizophrenia, bipolar disorder and drug usersCollection the serum of normal human and patients, ELISA Kit was used to examine the serum RPTN levels. One-way ANOVA was run in comparisons among multiple groups of humans and different ages and genders of normal human subjects.2) Research on serum RPTN levels of normal mice and injection of antipsychotic drugs in mice3) 36 mice were divided into four groups and administered intraperitoneally with atypical antipsychotics,Olanzapine (n=9),Quetiapine (n=7), Aripiprazole(n=9) or PBS as control (n=11) for 24 days.27 mice were divided into two groups,11 mice were maintained as control group,16 mice were housed in individual cages and assigned as CUMS group. Collection the serum of these mice,ELISA Kit was used to examine the serum RPTN levels. One-way ANOVA was run in comparisons among multiple groups of mice.4) Research on platelet concentrations of RPTN from healthy and schizophrenia5) Isolation of healthy and schizophrenia platelets from whole blood, ELISA Kit was used to examine the platelet RPTN levels,t-test was used to compare the two groups.6) Research on the effect of promoter transcriptional activation by Olanzapine, Ca2+ and DOIConstruction of report gene vector pGL2-RPTN promoter,transient transfected pGL2-RPTN promoter vetor with HEK293T cell, treat the cells with Olanzapine 100uM, Ca2+ 2mM and DOI 10uM after 6 h.Measure the luciferase after 48 h. One-way ANOVA was run in comparisons among these groups.7) Structure and bioinformatics prediction of RPTNComparative analysis of the sequence of RPTN protein,prediction the LIR domain of RPTN.COTH was used to predict the dimers of RPTN and α-synuclein, β-amyoid, tau, DISC1.And the Z-score was computered.8) Reaearch on the functions of RPTN in nervous systemIsolation and cultivation neural stem cell of mice brain, immunohistochemitry was used to examine the RPTN expression in neural stem cell.Grow cells on coverslips 12 h. Transient transfected with pCDNA-3.1 Control vetor and pCDNA-3.1-RPTN ORF vetor for 12 h.Treat the cells with rapamycin 24 h,then the Autophagy Detection Kit was used to assay.Results1) Compared with the normal, significantly diminished serum RPTN levels in patients with schizophrenia and bipolar disorder (p<0.005). Compared with the normal, significantly diminished serum RPTN levels in drug users (p<00001).2) Olanzapine treated mice significantly increase serum RPTN levels (p<0.0001), Quetiapine treated mice significantly increase serum RPTN levels (p<0.05), Aripiprazole treated mice serum RPTN levels are no significant difference to normal mice.3) Platelet concentrations of RPTN are significantly diminished in schizophrenia (p<0.05).4) Transcriptional activity of RPTN promoter is significantly increased by treated with Olanzapine (p<0.05),transcriptional activity of RPTN promoter is significantly increased by treated with Ca2+(p<0.01), transcriptional activity of RPTN promoter is no significant difference between control and DOI treated cells.5) Comparative analysis of the sequence of RPTN protein,the result showed that F LIR and Y LIR in the N-terminal 44-47 and 74-77 amino acid. RPTN and α-synuclein, β-amyoid, tau, DISCI can construct protein-protein dimers and the Z-score was greater 2.5.6) Immunohistochemitry showed that RPTN punctiform expression in neural stem cell of mouse brain. The autophagy of HEK 293 T cell treated with rapamycin was significantly increased by overexpression RPTN.Conclusions1) Compared with the healthy subjects, serum RPTN levels in patients with schizophrenia and bipolar disorder were significantly diminished.2) Significantly increased serum RPTN level in mice treated with Olanzapine.3) Platelet concentrations of RPTN are significantly diminished in schizophrenia.4) Transcriptional activity of RPTN promoter is significantly increased by Olanzapine and Ca2+.5) F_LIR and Y_LIR motif are present in the N-terminal 44-47 and 74-77 of RPTN protein.6) RPTN can potentially form protein-protein dimers with α-synuclein, β-amyoid, tau, and DISCI.7) RPTN expressed in mouse brain neural stem cells as cellular puncta.8) The autophagy of HEK 293T cells treated with rapamycin was significantly enhanced by RPTN overexpression.