A Critical Role Of RBM8a In Proliferation And Differentiation Of Embryonic Neural Progenitors

Intellectual disability(ID) is commonly found in neurological and psychiatric diseases, such as Alzheimer’s disease and autism. Patients with ID normally have lower Intellectual disability(IQ) score which compare to the general population. Children with ID learn more slowly than a normal child in terms of language, social skills, and the ability to take care of personal needs. Autism spectrum disorder(ASD) is a neurodevelopmental disorder characterized by impaired social and communication skills, repetitive, stereotyped behaviors, and often intellectual and cognitive impairments.Nonsense mediated m RNA decay(NMD) is an RNA surveillance mechanism that controls RNA stability and ensures the speedy degradation of erroneous and unnecessary transcripts. This mechanism depends on several core factors in the exon junction complex(EJC), e IF4A3, RBM8 a, Magoh, and BTZ, as well as peripheral factors to distinguish premature stop codons(PTCs) from normal stop codons in transcripts. Recently, emerging evidence has indicated that NMD factors are associated with neurodevelopmental disorders such asautism spectrum disorder(ASD), Schizophrenia(SCZ) and intellectual disability(ID). However, the mechanism in which these factors control embryonic brain development is not clear. RNA-binding motif protein 8A(RBM8a), an EJC factor, is a ribonucleoprotein with an RNA binding motif that preferably binds to m RNAs during splicing. It is still unclear how RBM8a-mediated specific functions on the proliferation and differentiation of neural progenitors and the neural development of embryonic. In this study, we aim to examin the proliferation and differentiation of function of RBM8 a in the embryonic NPC and make clear the critcal role of RBM8 a in regulate of brain development and finally provide new theoretical to reveal etiology, pathophysiology of the neurological diseases.Our study is included two chapters:Chapter 1: The role of the RBM8 a in proliferation and differentiation of embryonic neural progenitorsObjective To test the expression of RBM8 a in mouse brains from E9 to adult and the proliferation and differentiation of neural progenitor cells in vitro and vivo. We aim to understand the critical role of the RBM8 a in proliferation and differentiation of embryonic neural progenitors.Methods Embryonic brain from different stages and adult mouse brain cortex tissue were desected, lysis and collect total protein. Using Western blot to detect the expression of RBM8 a. We desected the neural progenitor cells from 14 days’ embryonic mice brain and differentiated them in vitro. Then collected their protein and used Western blot to test the expression of RBM8 a. We collected 16 days embryonic mice brain, fixed and cut them and esd the expression of RBM8 a by immunohistochemistry. To test the critical role of RBM8 a in brain development, we construted overexpression RBM8 a and sh RNA plasmids,transfected them to the CAD cell line in vitro, and made sure they can work. In vitro, we transfected overexpression RBM8 a and sh RNA plasmids to CAD cell line and labeled them by Brd U. In vivo, we injected the the plasmids into the embryonic mice brain by using the in utero electroporation technology. On E16 day, we killed the mice, collected the embryonic mice brain, fixed and cut them. After that, we use differents antibodies to do the Brd U labeling and immunohistochemistry.Result1. In Early embryonic brains(E9-E14) express a significantly higher level of RBM8 a when NPCs are actively proliferating(E10-E13), and then have decreased expression at E14 when NPCs begin to differentiate. 2. In the embryonic cerebral cortex, RBM8 a is robustly expressed in nestin positive neural progenitors residing in the ventricular zone(VZ)/subventricular zone(SVZ)(Fig. 1c), but reduced in doublecortin(DCX) positive progenitors or immature neurons in the intermediate zone(IZ) and the cortical plate(CP). 3. In vitro, when knocked down RBM8 a in CAD cells, Brd U labeling and mitotic index were dramatically decreased. Remarkably, when overexpressed of full length RBM8 a in CAD cell, it increased in cell proliferation and increased in both Brd U incorporation and mitoticindex. 4. In vivo, knockdowned the RBM8 a from E13 to E16 in utero decreased the cell number at the VZ/SVZ, and increased the number of cells migrating into the CP, where differentiated neurons are localized. In contrast, RBM8 a overexpression led to a decrease in the cell number migrating into the CP but a increase in the cell number at the VZ/SVZ. In addition, we observed a 32 % increase in cell cycle index in RBM8 a sh RNA transfected embryonic brains. The result suggests that loss of RBM8 a expression causes premature neuronaldifferentiation at the expense of the progenitor pool. Conversly, we observed about 30 % decrease of cell cycle exit when we upregulated RBM8 a levelby overexpression RBM8 a. And upregulation of RBM8 a significantly increases the NPCs’ differentiation and suppresses neuronal differentiation.Conclusion RBM8 a played a critical role in regulation of early neurodevelopment. RBM8 a stimulates embryonic neural progenitor proliferation and suppresses neuronal differentiation. And an optimal level of RBM8 a is essential for proper cell cycle control and NPC’s proliferation and differentiation.Chapter 2: Analysis the genes and signaling pathways regulation by RBM8 a based on RNA-seqObjective Based on RNA-seq Transcriptome analysis, identify the neurological disease’s downstream genes and signaling pathways which may be regulated by RBM8 a in the brain. Then investigated the mechanism of RBM8 a genes regulation of neurodevelopment.Methods We contructed two overexpression RBM8 a and control plasmids: p TRIPZ-m Cherry-RBM8 a and p TRIPZ-m Cherry, then packaged them to lentiviruses. We established a stable neural SH-SY5 Y cell line overexpressing RBM8 a in vitro. Using doxycycline to induce overexpression of RBM8 a in cells and total RNAs was extracted by using TRIzol reagent. Then using Ribo Zero kit to remove the r RNA. RNA were sequenced using the Illumina Hi Seq 2500 system. The RNASeq platform is capable of identifying relative expression,alternative splicing, novel transcripts and isoforms, RNA editing, and allelespecific expression. And then analysis the transcripts which are differentially expressed when RBM8 a is overexpressed. We compared RBM8a-regulated genes with existing databases for psychiatric risk genes to determine whether there were significant gene overlaps. Using the hypergeometric analysis we determined that risk genes in other neurological and psychotic disorders. Then using western blot to examine the protein level of the RNAseq results in mouse CAD cells.We utilized the DAVID bioinformatics tool to analyze the underlying risk genes and signaling pathways that are regulated by RBM8 a. Since EJC proteins have been implicated in alternative splicingwe investigated whether RBM8 a overexpression leads to change of differential alternative splicing. As RBM8 a is involved in NMD, we assessed our list of differentially expressed RNAs to determine how many exhibit characteristics that would make the RNA prone to degradation via NMD.Result 1. We finally constructed the overexpression RBM8 a and control plasmids and package them to lentiviruses. We also established the stable neural SH-SY5 Y cell lines overexpressing RBM8 a and control. Unbiased RNA-Seq generates 50 million high-quality pairedreads per sample and over 95 % of the reads are mapped back to the reference genome. 2. We have found that 7.08 % of transcripts are differentially expressed when RBM8 a is overexpressed. we found many RBM8 a down-regulated genes were significant gene overlaps compared with existing databases for ID, AD, neurodegenerative and neuropsychiatric diseases. To examine the protein level of the RNAseq results in mouse CAD cells, we tested genes involved in autism risk(NLGN1), neurodegeneration(ATXN1), neurogenesis(REST), embryonicdevelopment(TLE4), and neuronal migration(KIF1A). Consistent with the RNA levels from our RNAseq data, overexpression of RBM8 a in CAD cells also produces corresponding level of proteins. In the other hand, knockdown of RBN8 a generates reversal level of proteins of these genes in CAD cells. 3. We utilized the DAVID Bioinformatics tool to analyze our dataset of RBM8 a regulated genes. DAVID determined that RBM8 a regulates genes involved in several processes vital for embryonic brain development, including the MAPK pathway, growth factor signaling, the Rho signaling, extracellular matrix receptors including integrins and collagens and Ca2+ signaling. 4. Our dataset identified that 371 alternative splicing events in 101 protein coding genes were significantly altered. And most of 101 protein coding genes alternative splicing events are associate with ASD.5. Finally we found that 814 genes out of 1788 differentially expressed genes were determined to possess features that make the RNA susceptible to NMD。Conclusion Unbiased RNA-seq analysis reveals that RBM8 a regulates many risk genes involved in neurodegenerative/neuropsychiatric diseases and multiple important functional processes that are important for early neurodevelopment.