Investigating Olanzapine Efficacy Markers Based On The IPSC Model Of Patients With Schizophrenia

Objective: Schizophrenia(schizophrenia,SCZ)is a systemic mental disease characterized with emotional,cognitive and behavioral incoordination.The first and second generation Antipsychotic drugs developed based on the hypothesis of dopaminergic,serotonergic and other neurotransmitter regulation disorders are still the cornerstone of clinical treatment.Olanzapine has become the first-line drug in clinical treatment of SCZ due to its strong binding of dopamine receptor,5-HT receptor and cholinergic receptor,well therapeutic effect on negative and positive symptoms,and less adverse reactions.However,there are obvious individual differences in the efficacy of this drug,and there is no quantitative efficacy-related molecular marker for the precise treatment of olanzapine in SCZ.Genetic polymorphism and abnormal transcriptional expression are one of the important mechanisms of individual differences in SCZ drug effects.The lack of brain tissue in patients makes the progress of basic research on genetic markers of SCZ drug effects slow.Human induced pluripotent stem cells(i PSCs)technology makes it possible to obtain nerve cells from SCZ patients in vitro and creates conditions for sequencing of multi-omics pharmacodynamic markers.Based on this technology,the study will be included three parts: Construct neural stem cell lines derived from i PSCs of SCZ patients and screen genetic markers of olanzapine resistance;Verify candidate genetic markers of olanzapine efficacy in animal model;Explore the molecular mechanism of candidate genetic markers participating in olanzapine resistance.Finally,this study screened candidate molecular markers for accurate use of olanzapine,which laid the foundation for subsequent basic and clinical research.Methods: In the first part of the study,samples of patients with TRS and SCZ were matched according to the latest clinical diagnostic guidelines for SCZ and treatment resistance schizophrenia(TRS),and i PSCs lines were constructed using non-integrated reprogramming techniques;the constructed i PSCs were identified by morphology,immunofluorescence,chromosome karyotype,and three-layer differentiation test;m RNA-seq technology was used to perform high-throughput sequencing on the above NSCs.Olanzapine resistance genetic markers were obtained through GO analysis preliminarily.q PCR and Western Bolting were used to re-screen the primary screening genetic markers to obtain SCZ olanzapine efficacy related candidate genetic markers.The second part of the study,according to the results of the first part,we constructed olanzapine resistance gene transfection mouse model by brain stereotactic microinjection technology.Take diazepine(MK801)acute-induced schizophrenia mouse model as a positive control,then detect the behavior by open field test,prepulse inhibition and elevated plus maze;After giving a therapeutic dose of olanzapine,observe the behavioral changes of the above animal models to verify the phenotypic relationship between candidate genetic markers and the pharmacodynamic changes of olanzapine.The third part of the study,based on the pathological mechanism of neurotransmitter disorder,RNA interference(RNAi)technology was used to construct the model of candidate gene silencing primary cortical neurons.The molecular targets of neurotransmitter balance were detected by q PCR and Western blotting.Through the above research,the possible mechanism of candidate genes participating in SCZ olanzapine resistance was preliminarily demonstrated,which laid the foundation for subsequent large-scale basic and clinical research.Results: In the first part of the study,using non-integrated reprogramming technology,we constructed i PSCs lines from SCZ and TRS patients successfully.The cell morphology showed a typical cloned-like growth state,and the cell nuclear-to-mass ratio,morphology,cell tightness and edge sharpness all met the typical characteristics of i PSCs.Immunofluorescence staining results of showed positive expression of i PSCs characteristic markers Nanog and SOX2.Chromosome number and karyotype analysis are normal,no genetic aberration occurred.Transplanted teratoma has significant differentiation ability in the three germ layers in vivo,which proves that the pluripotent differentiation potential of the i PSCs.Confirm of the pluripotent identity of NSCs generated with this approach by immunostaining for the Nestin and SOX2.The m RNA-seq results showed that 867 genes in TRS group had significant changes compared with SCZ group.GO enrichment analysis showed that nervous system development(GO:0007399)was the signal pathway with the most significant changes in TRS group.q RT-PCR and Western boltting were used to re-screen the five protein coding genes in neurodevelopmental enrichment genes.Deleted in colorectal cancer(DCC)was found for the first time be related to SCZ olanzapine resistance,the deletion of this gene and phenotypic in olanzapine efficacy deserve further study.Based on the results of the first part,our group successfully constructed frontal cortex DCC knock down(KD)animal model using stereotactic injection technology in the second part of the study.q RT-PCR and Western Blotting results showed that DCC expression level in DCC KD mice was significantly lower than WT mice(P<0.05),proved that the model was established successfully.The results of behavioral tests show that,the spontaneous movement,prepulse inhibition rate and behavioral anxiety of frontal cortex DCC knockout mice were significantly different from those of the control group(P<0.05),and there was no significant difference(P>0.05)between the frontal cortex DCC knockout mice and MK801 acute induced SCZ model(P>0.05),indicating that frontal cortex DCC KD induce SCZ-like behavior changes in C57BL/6 mice.Importantly,the therapeutic dose of olanzapine can effectively improve the SCZ behavior abnormality induced by MK801(P<0.05).However,it can not effective improve the behavior abnormality in DCC knockdown mice(P>0.05).The results of behavioral phenotype in animal models proved that DCC gene deletion was one of the important factors for the treatment resistance of olanzapine in SCZ.The third part of the study showed that there was no significant change in the expression of γ-aminobutyric acid transporter(VGAT)of inhibitory neurons(P>0.05),while glutamine synthetase(GLUL),glutamate dehydrogenase(GLUD),and glutamate decarboxylase(GAD)in the glutamate metabolic system of excitatory neurons were all significantly decreased(P<0.05).Its postsynaptic membrane receptor N-methyl-D-aspartic acid receptor(NMDAR)andα-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor(AMPAR)family and Long-term potentiation(LTP)-related postsynaptic density protein95(PSD95)were all significantly decreased(P<0.05),indicating that the deletion of DCC gene affected the glutamate metabolism and function of neurons.In addition,the expression levels of GLUL and PSD95 proteins in frontal cortex DCC KD mice and NSCs from TRS patients were also significantly decreased(P<0.05),and olanzapine was ineffective for the changes,suggesting that glutamatergic dysregulation caused by DCC gene deletion might be one of the important mechanisms of the treatment resistance of olanzapine in SCZ.Conclusion: The deletion of DCC gene in nerve cells is associated with olanzapine resistance in SCZ.The glutamatergic dysregulation characterized by decreased expression of GLUL and PSD95 induced by DCC knockdown is one of the important factors for olanzapine resistance,and the mechanism deserves further study.