Discovery Of MicroRNA As Neuregulin 1 Downstream Targets For The Effect Of Olanzapine On Schizophrenia-related Mitochondrial Dysfunction

Objective: Schizophrenia is a complex brain dysfunction disease with an overall incidence of approximately 1 %.A large number of studies have shown that factors such as disordered regulation of neurotransmitters in central nervous cells,abnormal neural development due to genetic mutations,and disorders of energy metabolism in nerve cells are closely related to the occurrence of schizophrenia.Mitochondria,as important organelles for regulating energy metabolism of nerve cells,play an important role in the synthesis,the release of neurotransmitters and the differentiation and development of neural stem cells.The in-vitro study of this research group found for the first time that olanzapine,the second-generation atypical anti-schizophrenia drug,has a significant therapeutic effect on schizophrenia-like mitochondrial dysfunction mediated by neuregulin 1(NRG1).This study was carried out to verify the relationship between the protective effect of mitochondria above olanzapine and NRG1 from the overall animal level,and to further explore the downstream regulatory mechanism.Methods: First of all,in the first part of the study,the whole knockout(NRG1 KO)mouse model of NRG1 was constructed by CRISPR / Cas 9 technique,and the NRG1 expression level in the brain was respectively verified by qRT-PCR,Western Blot and immunohistochemistry;wild-type group(WT),MK801 group,NRG1 KO group and NRG1 KO+Olanzapine group were set up,using open field experiment to detect spontaneous activity of mice,pre-pulse inhibition(PPI)method to detect sensory gating function,social experiment to test social ability of mice,and the Morris water maze to test their learning and memory ability;A series of neurobehavioral tests were performed on mice,observing the ultrastructure of mitochondria in the brain of mice with transmission electron microscope,and detecting the swelling level of brain mitochondria to evaluate the effect of NRG1 on mitochondria.Through the above studies,the relationship between olanzapine’s anti-schizophrenia-like mitochondrial damage and NRG1 was verified from the overall animal level.Previous studies of the group have shown that NRG1 regulates the changes of downstream mitochondrial function through post-transcriptional regulation.For this reason,in the second part,the research group used miRNA sequencing technology to conduct unbiased sequencing of miRNA molecules with significant changes in brain tissues of NRG1 KO mice,and further adopted bioinformatics related technologies to lock in the molecules with extremely significant changes miR-143-3p,which is closely related to cellular energy metabolism;qRT-PCR was used to verify the expressed relationship between NRG1 and miR-143-3p at three levels of NRG1 KO mouse brain tissue,SH-SY5 Y cell line and rat primary cortical neurons;building miR-143-3p mimic and miR-143-3p inhibitor models were established at SH-SY5 Y cell line and rat primary cortical neurons,and MitoSOX Red fluorescent probe method was used to detect mitochondria ROS,JC-1 fluorescent probe method was used to detect the mitochondrial membrane potential,and Western blot was used to detect the expression levels of cell-related proteins,which further clarified the regulatory effect of miR-143-3p on mitochondrial function;Finally,the group also verified the regulatory effect of olanzapine on miR-143-3p,and clarified the relationship between this effect and NRG1.From the above studies,it was clear that mir-143-3p was an important target for NRG1 downstream regulation of olanzapine anti-SCZ-like mitochondrial dysfunction at the in vitro and overall levels.In order to further clarify the direct target of miR-143-3p in regulating mitochondrial function,in the third part,the group performed mRNA sequencing on miR-143-3p overexpressing nerve cell;combined with literature preliminary screening,highly significant down regulation of target genes with mitochondrial regulation was observed and verified by qRT-PCR;dual luciferase reporter probe method was used to lock miR-143-3p target gene Mitochondrial Creatine Kinase 1B(CKMT1B);Western Blot and immunostaining were used to further verify the regulatory relationship between miR-143-3p and CKMT1 B.Through the above research,the target genes of miR-143-3p regulating mitochondrial function were identified,which laid a foundation for the subsequent research on direct targets of olanzapine anti-schizophrenia-like mitochondrial function.Results: In the first part,compared with the WT group,NRG1 KO mice showed significant schizophrenia-like behavioral changes similar to the MK801 group,including pre-pulse inhibition(p<0.05),and increased spontaneous activity(p<0.05 0.05)and social dysfunction(p<0.05),but did not have a significant effect on learning and memory function(P>0.05);consistent with the previous in vitro test results,the ultrastructure of brain mitochondria in NRG1 KO mice was significantly changed,the double-layer membrane structure gap was widened,and the electron cloud density of contents was significantly reduced.Compared with the WT group,the isolated brain mitochondria of NRG1 KO mice were not sensitive to calcium-induced mitochondrial swelling changes(p>0.05);olanzapine significantly improved the neurobehavioral and mitochondrial morphological and functional changes induced by MK801 in mice(P<0.05),but had no effect on the schizophrenia-like behavior abnormalities and mitochondrial damage induced by NRG1 KO.The above results prove that NRG1 is a key target for regulating olanzapine’s anti-sperm-like neuronal mitochondrial dysfunction from the overall animal level.In the second part,miRNA-seq results showed that compared to WT,46 miRNAs were up-regulated in NRG1 KO mouse brain tissue,and 143 miRNAs were down-regulated(P<0.05).After NRG1 KO mouse brain tissue and SH-SY5 Y cells and rat primary cortical neurons NRG1 KD model verified by qRT-PCR,miR-143-3p was initially identified as a key target for potential mitochondrial regulation downstream of NRG1(p<0.05);results of miR-143 mimic and miR-143-3p inhibitor model showed that,compared with the control group,the cells with low miR-143-3p expression showed significant mitochondrial functional damage,including increased mitochondrial ROS level and decreased mitochondrial membrane potential(P<0.05).it is important that 1,10 μM Austria Niazapine NRG1 dependently increases the expression level of miR-143-3p,and can effectively inhibit the increase of mitochondrial ROS level and the decrease of mitochondrial membrane potential caused by low expression of miR-143-3p(P<0.05).The above results indicate that miR-143-3p is one of the key targets of olanzapine’s anti-schizophrenia-like mitochondrial damage downstream of the NRG1 pathway.In the third part,miR-143-3p overexpression neuronal mRNA sequencing GO enrichment analysis results showed that significant changes in important signaling pathways appeared such as the negative regulation of ATPase activity(GO: 0032780),contraction and signal transduction of cardiomyocytes(GO: 0086002)and synaptic tissue function(GO: 0050808)and other important signal pathways(P<0.05),the results further corroborate the second part related to the regulation of miR-143-3p in regulating energy metabolism of nerve cells;the mitochondrial creatine kinase 1B(CKMT1B)and PAPPA candidate mRNAs were obtained by preliminary screening in the genes with significant changes combined with literature investigation,and through SH-SY5 Y cells and rat primary cortical neurons miR-143-3p mimic and miR-143-3p inhibitor model qRT-PCR verification,mitochondrial creatine kinase 1B(CKMT1B)was obtained from the target gene with significant down-regulation of miR-143-3p overexpression;compared with the control group,the low expression of miR-143-3p can make the CKMT1 B protein level significantly increased(P<0.05);the results of the dual luciferase reporter probe test showed that compared with CKMT1 B / miR NC,the fluorescence intensity of the CKMT1 B / miR-143-3p mimic group was significantly reduced(P<0.01),while CKMT1 B / The fluorescence intensity of the miR-143-3p inhibitor group increased significantly(P<0.01).This result proves that CKMT1 B is the target gene of miR-143-3p;the results of Western Blot and immunostaining tests further verify the above relationship.Importantly,it was found that olanzapine can significantly reduce the protein level of CKMT1 B cells caused by miR-143-3p inhibitor(P<0.05).The above results indicate that CKMT1 B is the target gene of miR-143-3p,which may mediate the effect of olanzapine against sperm-like mitochondrial dysfunction.Conclusion: The miR-143-3p downstream of the NRG1 pathway is one of the key targets for regulating the mitochondrial dysfunction of olanzapine against schizophrenia-like neurons.Its mitochondrial regulatory target gene is CKMT1 B.The mechanism of action still needs to be further verified.This study further expands our understanding of the molecular mechanism of mitochondrial function damage in seminal neurons,and discovered miR-143-3p,as a potential molecular marker associated with the efficacy of olanzapine,provides a theoretical research foundation for the subsequent clinical genotyping of patients,and individualized precision treatment.