Molecular Mechanism Of MiR-592 Targeting MeCP2 To Regulate NSCs Differentiation And Its Effect In Brain Development And Autism-Like Behavior

Background:Autism spectrum disorder（ASD）is a neurodevelopmental disorder（NDD）marked by increased stereotypic behaviors,impaired communication abilities,and restricted interests.ASD pathophysiology is still unknown owing to genetic and environmental factors.Epidemiological studies have indicated that the prevalence of ASDs has been growing year by year in recent years,suggesting that the disease has been evolved into a public health issue that demands immediate attention.Most of the ASD patients need lifelong care,which brings a serious burden to the society and the family,and is highly concerned by governments,experts and patient families all over the world.Finding demonstrates that neural stem cell（NSC）proliferation,neuronal differentiation and migration,synapse formation,and myelin production are all associated with the development of ASD.MicroRNAs（miRNAs）are small RNA molecules with tissue-and time-specific expression properties.Genome-wide expression profiling has shown that miRNAs can regulate ASD risk gene networks.Several studies have revealed that miRNAs perform regulatory roles in central nervous system（CNS）development,NSC proliferation and differentiation,and neuronal synaptic plasticity.Among them,miR-592 is found in non-coding exon region of the flanking sequence of the autism-related Glutamate metabotropic receptor 8（Grm8）gene.Previous studies have shown that miR-592 plays a key role in the maintenance of proliferation and differentiation in NSCs,neuronal differentiation in vitro,and neuronal damage in ischemic and hypoxic environments.However,the above studies were performed in vitro and in vivo studies were limited.The X-linked methylated CpG-binding protein 2（MeCP2）,a member of the methyl-CpG-binding domain（MBD）protein family,is essential for early embryonic development,neuronal maturation and downstream neural circuit.Studies have shown that MeCP2 is involved in the pathophysiology of ASD,Rett syndrome（RTT）,MeCP2duplication syndrome（MDS）and other diseases.This study aims to investigate the molecular mechanisms of miR-592 targeting Mecp2 to regulate NSCs differentiation and its effect in brain development and autism-like behavior.Objective:To examine the biological functions of miR-592 in mouse CNS development,we generated miR-592 knockout(miR-592^-/-)mice to investigate the mechanistic mechanism through which miR-592 influences brain development via its target gene MeCP2.Furthermore,we aim to explore whether miR-592 can ameliorate the autism-like phenotype in MeCP2 over expressing mice（TG（MECP2）mice）and provide a new clue for autism treatment.Methods:（1）The aberrant neurodevelopmental phenotype of miR-592^-/-mice is investigated.（1）The miR-592^-/-mice were constructed based on CRISPR-Cas9technology.（2）Gel electrophoresis,Sanger sequencing,in situ hybridization（ISH）,and Western blotting were used to identify the knockout results.（3）The effect of miR-592deletion on its host gene Grm8 was evaluated by real-time quantitative PCR（qRT-PCR）and full-length transcriptome sequencing（Isoform Sequencing,Iso-Seq）.（4）The birth rate,sex ratio,body weight,blood glucose,blood lipid levels of miR-592^-/-mice using H&E and Nis staining were detected.（5）Immunofluorescence staining was used to detect the expression of neural differentiation markers and axonal development markers in miR-592^-/-mice.Fluorescence in situ hybridization was utilized to analyze the distribution of miR-592^-/-brain sub regions and sub cellular distribution.（6）Behavioral assays were utilized to assess the neurobehavioral capacities of miR-592^-/-mice,including autonomous activity,anxiety,social interaction,learning,and memory.（2）Combining RNA-seq and proteomic analysis indicates the mechanism through which miR-592 regulates NSC/NPC differentiation by targeting Mecp2:（1）Bulk RNA-seq and Tandem Mass Tags（TMT）technology were used to screen miR-592 target genes and molecular signaling pathways.（2）Double luciferase assay,qRT-PCR,WB,and immunohistochemistry were used to validate the results.（3）The essential biological mechanisms of the miR-592/MeCP2 axis regulating neurogenesis were revealed by observing neuronal morphology and neural stem cell sphere-forming ability in miR-592^-/-mice and TG（MECP2）mice primary cell culture.（4）Based on temporal dynamic transcriptome landscape of miR-592^-/-mice,the growth and developmental changes of miR-592 in two crucial time periods（embryonic and neonatal）were predicted.（5）The spatiotemporal expression profile of miR-592 during CNS development was examined by ISH.（3）miR-592 rescues the autism-like phenotype in TG（MECP2）mice.（1）Immunofluorescence was used to detect MeCP2 expression in the brain of TG（MECP2）mice following intrauterine microinjection of miR-592 mimic at E14.5.（2）After miR-592 mimic microinjection,immunofluorescence staining was performed to detect neural differentiation markers.（3）Whether the injection of miR-592 to TG（MECP2）mice ameliorated the autism-like behavior was testes by open field test（OFT）and new object recognition test（NOP）.Results:（1）miR-592^-/-mice shows developmental abnormalities.（1）Three sg RNAs for miR-592 precursor sequences were constructed,and 12 F0 mice were finally obtained.Founder animals were identified as the mice with the longest knockout fragment（157bp）.（2）The abortion rate of miR-592^-/-mice were 12.9%,whereas female miR-592^-/-littermates had a higher birth rate.Aborted miR-592^-/-mice displayed halted brain development,while knockout mice that developed normally to maturity had aberrant weight increase.No pathological variations were observed in the heart,liver,spleen,lung,kidney,and testes.（3）In situ hybridization revealed that miR-592 was mainly distributed in the cortical region of the brain（ctx）,the sub granular zone of the hippocampal dentate gyrus（SGZ）,and the cytoplasm of neurons in the cerebellum of wild-type mice.In NSCs,miR-592 knockdown reduced the expression of neuronal differentiation markers while increasing the expression of synaptic development indicators.（4）miR-592^-/-mice had reduced anxiety and social behavior,but no significant alterations in learning and memory abilities,suggesting that they have autistic-like behavior.（2）miR-592 can target Mecp2,which affects NSC/NPCs differentiation:（1）Using Bulk RNA-seq and TMT mass spectrometry,we analyzed differentially expressed genes in adult WT mice and miR-592^-/-mice.A total of 132 gene networks were linked to autism susceptibility,all of which were involved in the calcium signaling pathway and axonal development-related biological processes.（2）Double luciferase assay revealed that MeCP2 is an important target gene of miR-592 that regulates brain development.（3）miR-592 knockout NSCs formed spheroids quickly but had a high rate of cell death.Neuronal primary culture demonstrated that neurons lacking miR-592 had substantial axonal branching and a high level of cell death,which is identical to the TG（MECP2）animal phenotype.By blocking the NF-κB/IκBαand increasing the protein expression of the post-synaptic membrane marker PSD95,miR-592/MeCP2promotes synaptogenesis.（4）Transcriptome landscape analysis revealed that miR-592had a significant influence on brain development in the early stage.GO analysis showed that miR-592 had the highest correlation with synaptogenesis.（5）Analysis of miR-592expression profiles peak expression of miR-592 occurred during neurogenesis in mice.The cortical site may be a crucial brain subregion where miR-592 performs its biological function.（3）miR-592 ameliorates the autistic-like phenotype in TG（MECP2）mice:（1）For a long time,miR-592 mimic decreases aberrant MeCP2 expression in the cerebral cortex of TG（MECP2）mice.（2）The total time of object contact in TG（MECP2）mice was significantly increased after miR-592 mimic microinjection,which could partially rescue their autism-like behavior.Conclusions:According to the findings,the critical period for miR-592’s biological function is E14.5.The deletion of miR-592 causes an abnormal increase in MeCP2 expression,which further inhibits the differentiation process of intermediate progenitor populations（TBR2⁺）in the mouse cerebral cortex region via the NF-κB signaling pathway.It causes an autism-like phenotype that is abnormally socially disordered.Our findings further show that intrauterine microinjections of a miR-592mimic can ameliorate autism-like in TG（MECP2）mice.This study provides a new treatment strategy as well as a theoretical foundation for clinical research in autism.