Dendritic Cell-induced Neural Stem Cell Survival Is Dependent On NT-3/TrkC Signaling Pathway

The spinal cord injury (SCI) often causes severe nerve dysfunction and traditionaltreatments make very little improvements. However, the discovery of neural stem cells(NSPCs) which are self-renewable and potent in nerve differentiation, makes the repairmentof SCI possible and NSPCs mainly locate in the olfactory bulb, hippocampus andsubependymal zone of central nervous system (CNS). But experiments using NSPCs aloneto repair SCI obtain poor effects as most of the transplanted NSPCs disappear or end inapoptosis within2weeks after implantation, which is believed due to the strong inhibitoryfactors and the lack of beneficial factors to secure the survival of CNS in the damagedspinal sites.Dendritic cells (DCs) are the key in regulating immune responses, but also secrete avariety of cytokines. DCs are the strongest antigen-presenting cells (APCs). Our previousstudies found that transplanting DCs to lesion site could improve the local plausibleenvironment and enhance the proliferation of endogenous neural stem cells to promote thefunctional recovery of SCI. However, the underlying mechanism in this events is stillunknown.Neurotrophin-3(NT-3) belongs to the neurotrophic factors (NTFs) family, and it isresponsible for the differentiation, development and maintenance of mature neurons. NT-3can protect the damaged neurons by promoting their survival and regeneration. ElevatedNT-3can promote the proliferation of NSPCs and their differentiation into neurons. Thebiological effects of NT-3is dependent on the ligation with its specific tyrosine kinasereceptor C (TrkC) expressed on the surface of NSPCs. Combination of NT-3with TrkCinitiates signal transduction cascades, including Ras pathway, and induces the proliferation,survival, and differentiation of peripheral nervous system (PNS) and CNS neurons.According to the above phenomena, we propose a hypothesis: DCs can promote thesurvival of NSPCs and this biological effect is dependent on the interaction of DC-secreted NT-3with its specific receptor, TrkC.In this study, we observed the concentration of NT-3in the lesion site and the survival of the transplanted NSPCs,both in vivo and in vitro. Theeffect and interaction of NT-3and TrkC were also detected in the presence of a NT-3specific antiboy in a co-culture system.Materials and methods:1. DC cultureBone marrow-derived immature DCs were generated from SD rats, by a previouslydescribed method (Hauben et al.2003) with minor modifications. Bone marrow tissues infemur and tibia from4-6week-old female SD rats were cultured in the presence ofGM-CSF and IL-4, and culture medium was refreshed each other day and cells wereharvested on day7.2. NSPC culturePrimary NSPCs were generated from full brain of enhanced green fluorescence protein(EGFP) transgenic rats or1-3-day newborn SD rats. The neural spheres were harvested onday7and kept for further analysis. The third generation cells were detected fordifferentiation markers (Oilg2, tublin, GFAP) after10%serum stimulation, and then NSPCstemness was observed.3. Rat model of SCIRat spinal cords were exposed by laminectomy at the level of thoracic (T)9to T10.The model was established successfully and then DCs and NSPCs were transplanted intothe leision site on day9.4. Immunofluorescence stainingAfter fixation, suspended cells, adherent cells and tissue sections were incubated inprimary antibody over night at4℃，washed with PBS and incubated in secondary antibodyfor2h at37℃and then observed by laser scanning confocal microscope.5. Co-culture of DCs and NSPCs.In co-culture, to verify the effect of DCs on NSPC survival and proliferation, NSPCsderived from primary neurospheres were added into transwells at a concentration of1×106cells/ml. Four groups were set. The first group was NSPCs (1×106/ml,2.5ml/well) in thelower chamber with DCs (1×106cells/ml,1.5ml) in the upper chamber; the second group was2.5ml DMEM/F12medium in the lower chamber with DCs (1×106cells/ml,1.5ml) inthe upper chamber, the third group was NSPCs (1×106/ml,2.5ml) in the lower chamberwith2.5ml DMEM/F12cultural medium in the lower chamber and, the fourth group waswith NSPCs/DCs of the same concentration and amount as the first group but in thepresence of20ug/ml NT-3antibody in the upper chamber. Cells were collected and testedafter48h culture.6. AnnexinⅤ analysisAnnexinV was used to as a survival marker of NSPCs.1×106NSPCs were suspendedin medium and then adjusted to5×105/ml after washing with PBS. Cell suspension wasstained with AnnexinⅤ-FITC for5min, and PI (propidine iodide)(20μg/ml,10μl) for10min at room temperature. The proportion of live NSPCs were analyzed with flowcytometry.7. Western blottingNSPCs of each group were collected and total protein was extracted for Westernblotting assay to detect the expression of TrkC protein which is known to destribute on theNSPCs menbrane.8. qRT-PCR (quantitative real-time reverse transcriptional polymerase chain reaction)TrkC and NT-3mRNA were detected by qRT-PCR, all the primers were designed bySangon LTD.9. Statistical analysisData were statistically analyzed with SPSS13.0software and data are shown as mean±SD. Difference between two groups was analyzed by student test. p<0.05is consideredas significant difference.Results:1. DCs increase the survival of NSPCs in vivo. More transplanted NSPCs from EGFPrats in DCs/NSPCs group were observed with immunofluorescence than NSPCs alonegroup (NSPCs group:33.33±6.11/high power field (HPF);NSPCs/DCs group:57.33±5.13/HPF).2. Coculture of DCs enhances the survival of NSPCs. Two weeks afterco-transplantation of DCs and NSPCs, the survived NSPCs in DCS+NSPCs group (57.33±5.13, n=3) were much more than NSPCs group (33.33±6.11, n=3)(p<0.05) 3. DCs promote NSPC survival through NT-3in vitro. The proportion of doublenegative cells in DC/NSPC group was much higher than DCs/NSPCs+NT-3antibody group(37.77%±2.73%). Moreover, early apoptotic AnnexinV+PI-cells (19.45%±0.94%) inNSPC/DC group was significantly reduced, compared with the NSPC group (25.30%±1.63%), and DCs/NSPCs+NT-3antibody group (34.14%±3.62%).4. NPSCs promote the expression of NT-3in DCs in vitro and in vivo. Mature DCswere observed in co-culture system but not in DC alone system. The level of NT-3mRNA(2.36±0.14) of DCs in co-cultured group was much higher than DC alone group (1.17±0.04)(p<0.05).Two weeks after combined transplantation, immunofluorescence showedthat the distributing area of NT-3at lesion sites of DCs/NSPCs group was significantlylarger than HBSS group (119042.21±10130.23μm2)(p<0.05), DC group (128985.04±8342.58μm2) and NSPC group (169729.32±12091.18μm2)..5. Co-culture with DCs increases the proliferation and TrkC expression of NSPCs.Immunofluorescence revealed that NSPC spheres in DCs/NSPCs group enlarged and TrkCexpression significantly higher than NSPC alone group and NSPC/DC+NT-3antibodygroup, which was consistent with RT-PCR results. Protein expression of TrkC confirmed byWestern blotting also upregulated in DC/NSPC co-culture system.6. Co-cuture of DCs and NSPCs upregulateds the phosphorylation of TrkC. Westerrnblotting showed higher level of phopsphorylated-TrkC in NSPCs/DCs group, comparedwith other two groups (p<0.05).Conclusions:Based on the above study, the following conclusions can be reached:1. Co-culture of DCs and NSPCs upregulate the expression of NT-3in DCs andpromotes the survival of NSPCs.2. DCs promote the survival of NSPCs in a NT-3/TrkC-dependent manner.In conclusion, DCs can promote the survival of NSPCs through the NT-3/TrkC signalpathway..