Mechanism Of MiR-30c/sema3A In Adult Neurogenesis And Their Impact On Olfaction And Memory

BackgroundMost neurons in the adult brain of rodents and primates cannot be renewable. However, the astrocyte like cells confined to just two regions:the subgranular zone of the hippocampal dentate gyrus and the subventricular zone, located at the walls of lateral ventricle still reserve the capacity of neurogenesis, besides the adult neurogenesis is declined with age. Recent researches find that the neurogenic diseases, especially the neurodegenerative diseases have close relationship with adult neurogenesis. So, disclosing the mechanism of adult neurogenesis would be meaningful in neuroscience per se and provide a novel avenue in treating neurogenic diseases.Axon guidance cues are a kind of secreted and transmembrane proteins, navigating the neuronal axons to the accurate synapses. They are specifically expressed by neuronal precursor or neural stem cells in the niche of adult stem cells and loss of specific cue could give rise to changes of cell cycle and abnormal migration of neuronal precursors, which directly impacts the adult neurogenesis capacity.microRNAs is a kind of endogenous non-coding small RNA, they are involved in processes of cell proliferation, differentiation and apoptosis by transcriptional inhibition or degradation of their targets. Comparing with the embryos stage, some microRNAs are specifically expressed in adult tissues and evidences suggest that microRNAs play critical roles in adult neurogenesis, which is hopeful in achieving the gene therapy in the adult neurogenesis by microRNAs.To detailed explain the functions of microRNAs in adult neurogenesis and find their interactive targets involved in adult neurogenesis,74 differentially expressed microRNAs during different developmental stages were screened with the high throughput sequencing results. Among these microRNAs, miR-16 and miR-30c are the two microRNAs with the most-significantly differentially expressed and they are declined with age. The followed target predictions and the functional annotation results demonstrated that the targets of these microRNAs are mainly located in the brain and mainly play roles in cell differentiation and axon guidance pathways. The microRNA-mRNA interaction network further indicated that sema3 A participates in the cell differentiation pathway by the regulating of two potential microRNAs:miR-16 and miR-30c. Up to now, there is no report about functions of microRNA/sema3 A in adult neurogenesis. Whether miR-16 and miR-30c take roles in the proliferation and differentiation of adult stem cells by negatively regulation of sema3 A? What the other molecules and mechanism that renders the downstream signal of sema3A to the cell proliferation and differentiation? The thesis discusses these.Objectives1. Screening of the microRNAs related with cell differentiation and axon guidance based on the high throughput sequencing database;2. Validation of the interactions between sema3A and the two potential regulatory microRNAs-miR-16 and miR-30c by bioinformatics and experimental methods;3. Study the role of miR-30c/sema3A in the adult-born neurons of subventricular zone;4. Disclosure of the mechanism of miR-30c/sema3A in adult neurogenesis;5. Examination the impact of miR-30c in adult newborn neurons in olfactory bulb;6. Revealing the downstream mechanism of miR-30c/sema3A in adult neurogenesis;7. Examination the changes of olfaction and memory that influenced by changes of adult neurogenesis induced by miR-30c/sema3 A.Methods1. Using of the Gene Expression Omnibus (GEO) for high throughput sequencing database, hierarchical clustering and Self-organization Mapping (SOM) algorithms to screen and analyze the differential expressed microRNAs during different stages;2. Construction of microRNA over-expression, knockdown vectors and vectors of dual luciferase reporting assay. Designing and synthesizing the microRNA mimics. The interaction of sema3A and microRNAs was validated by dual luciferease reporting assay and qRT-PCR;3. Construction of microRNA over-expression and knockdown lentiviral vectors and package of lentiviruses. Detection changes of adult born neurons in the subventricular zone by brain steretaxic microinjection and examination the expression of microRNAs and sema3 A by qRT-PCR after flow cytometry sorting (FACS);4. Analysis the morphology of neurons in subventricular zone by brain sections of subventricular zone and the primary neurons of subventricular zone treated with microRNA overexpression and knockdown vectors. Detection the proliferation and cell cycle with Real-time cellular analyzer (RTCA) and flow cytometry respectively;5. Using BrdU to trace the newborn neurons and detection the stem cells and differentiated cells in the subventricular zone and olfactory bulb by their specific protein markers. Using the TUNEL kit to examine the apoptosis in the subventricular zone;6. Design of GSK3β and c-myc siRNAs and examination of the morphology and proliferation of Neuro2A that treated with above siRNAs with Laser scanning confocal microscope and Real-time cellular analyzer respectively;7. Examination of the effects of miR-30c to the whole structure of olfactory bulb and olfaction by the overall anatomy of olfactory bulb and food burying experiments respectively. Using of the brain stereotaxic microinjection and BrdU incorporation for detection of the newborn neurons in the dentate gyrus. Detect the effect of miR-30c level on the memory of hippocampus with conditional fear memory and water maze experiments.Results1. Screening of microRNAs that simultaneously participate in cell proliferation and axon guidanceWe retrieved the high throughput sequencing in different developmental stages from the database of GEO and analyzed these data by One-way ANO VA. The results showed that 74 microRNAs were differentially expressed. The further HCL and expression patterns analysis with SOM algorithm, two classes of expression patterns were obtained, one pattern with gradual decline (11 microRNAs) while the other one with continuously increasing (63 microRNAs). Among these microRNAs, miR-16 and miR-30c were the two most differentially expressed microRNAs that belong to the type of class one. The bioinformatics results showed that the targets of these microRNAs were mainly located in the nucleus, endosome and axons, playing critical roles in cell differentiation and axon guidance. The further microRNA-mRNA network analysis with Cytoscape revealed that all the microRNAs were simultaneously involved in the above most enriched pathways except miR-17. It is noted that miR-16 and miR-30c that the two most significant differentially expressed microRNAs, connected the axon guidance and cell differentiation pathways together by targeting with sema3A and the other two proteins Raflkinase and MAP3K5 respectively.2. miR-30c is a specific regulator of sema3ASema3A, a notable axon guidance cue, has been verified to play roles in neuronal polarity (cell differentiation). As cell proliferation and differentiation are just the two faces of one coin, in order to explore the mechanism that controlling the adult neurogenesis in the subventricular zone, we selected sema3A for further study. We detected that miR-30c can significantly inhibit the expression of sema3 A ((55±1.2%, p <0.05), while when the binding sites between miR-30c and sema3A were mutated, the effect of miR-30c on sema3A was disinhibted (78±4%, p>0.05). However, no matter co-transfected with the wild type of sema3A or the mutated sema3A, miR-16 has no effect on sema3A. The effects of different concentration of microRNAs mimics on sema3A verified the above results. When the neuro2A were treated with above microRNA mimics and vectors, we found the expression of miR-30c was negatively correlated with that of sema3A detected by qRT-PCR. However, the expression of miR-16 has no correlation with that of sema3A. Taken together, miR-30c is the regulator of sema3A.3. Effect of miR-30c/sema3A on the newborn neurons in the subventricular zoneUsing BrdU to trace the newborn neurons in the mice that microinjected with miR-30c overexpression and knockdown lentiviruses with brain stereotaxic operation, we found the elevated level of miR-30c in the subventricular zone can significantly increase the number of newborn neurons in the subventricular zone and the rostral migration stream (SVZ:4.59±1.74 fold, p<0.01; RMS:8.69±1.32 fold, p<0.001), when miR-30c level was knocked down by miR-30c-KD(miR-30c knockdown), the results were opposite with that of miR-30c over-expression. The quantification results of miR-30c and sema3A by qRT-PCR for the cells that separated by flow cytometry sorting showed that the expression of sema3A was significantly decreased in miR-30c overexpression group (0.41±0.012 fold, p< 0.001). But in miR-30c knockdown group, the expression of sema3A was remarkably increased (2.48±0.023 fold, p< 0.001). These results indicated that miR-30c modulated the newborn neurons in the subventricular zone though negatively regulation of sema3A.4. miR-30c/sema3A influence the number of newborn neurons by regulating the cell proliferation and differentiationIn order to explore the way that miR-30c/sema3A influence newborn neurons in the subventricular zone, we using the miR-30c over-expression and knockdown lentiviral vectors to treated the primary neurons of subventricular zone. The morphological results revealed that neuronal differentiation was reduced when miR-30c level was elevated. In contrast, the neuronal differentiation was enhanced when miR-30c level was knocked down. The neuronal morphology of subventricular zone of the in vivo brain section that treated with miR-30c overexpression and knockdown lentiviruses was consistent with above results. Neuro2A were transfected with miR-30c overexpression and knockdown vectors respectively, the proliferation results detected with Real-time cellular analysis (RTCA) showed that the proliferation was increased when miR-30c level was elevated. In contrast, the proliferation was reduced when miR-30c level was knocked down. The further cell cycle analysis by flow cytometry revealed that cell cycle was prompted by miR-30c overexpression, while the miR-30c was knocked down, cell cycle was significantly postponed.5. Effect of miR-30c/sema3A on cell proliferation and differentiation pathway was mediated by GSK3β and c-mycBased on the analysis of the related papers and signal pathways, we focus on GSK3β and c-myc as candidate mediators to further explore the mechanism of miR-30c on cell proliferation and differentiation. Cell morphology and cell proliferation that treated respected with siRNA of GSK3β and c-myc revealed that GSK3β can prompt cell differentiation and inhibited proliferation. In opposite, c-myc has the function of prompts cell proliferation and inhibits differentiation. Combined with the changes of morphology and cell proliferation induced by miR-30c overexpression and knockdown, as well as the protein immunobloting resuts, we found that expression of sema3A was inhibited by over-expression of miR-30c, and then the activity of GSK3β was inhibited. Thus pThr58 of c-myc was reduced and the total c-myc was increased which further prompts cell proliferation. When miR-30c was knocked known, the results was in opposite with that of miR-30c over-expression.6. Impact of miR-30c on the structure and function of olfactory bulb and hippocampus by the role in cell proliferation of differentiationChanges of miR-30c level give rise to difference speed in cell proliferation and differentiation, which final results to changes in number of newborn neurons. To investigate the effect of miR-30c on the migration of newborn neurons to the target regions, we microinjected the miR-30c overexpression and knockdown lentiviruses to the subventricular zone and dentate gyrus of hippocampus with brain stereotaxic operation. The results of newborn neurons and neuronal precursor migration tracing by BrdU demonstrated that there is no difference between treated group and the control group. Later the immunofluorescence results showed that the proportion of stem cells was remarkably increased, while the differentiated cells that NeuN-and GFAP-positively reactive neurons were reduced. However, miR-30c overexpression gives rise to a proportional increase of stem cells and the differentiated cells in the olfactory bulb. Also, when miR-30c level was elevated, astrocytes in the hippocampus have increased. In contrast, miR-30c level was reduced, the results were opposite to above. The further ethology results revealed that the level of miR-30c was reduced in the subventricular zone, olfactory sensitivity was reduced (p< 0.05). When the level of miR-30c was reduced in the hippocampus, all the conditional fear memory and the spatial memory were reduced (p< 0.05 and P<0.01 respectively). However, there is no significantly difference between the miR-30c overexpression group and the control group in the ethological performances. These results indicated that a proportional number of newborn neurons is essential for the maintenance of normal structure and function. But the surplus newborn neurons cannot give rise to much more improvement further.ConclusionsBased on above results, our results revealed that 1) miR-30c play roles in adult neurogenesis by negative regulation of sema3A; 2) miR-30c/sema3A are critical in controlling the number of adult newborn neurons through regulating the proliferation and differentiation of adult stem cells; 3) GSK30 and c-myc probably as downstream molecules mediated the signal pathway of adult neurogenesis induced by miR-30c/sema3A; 4) Aberrant expression of miR-30c/sema3A in the subventricular zone and the dentate gyrus of hippocampus leads to the changes of olfaction sensitivity and learning and memory abilities.