The Role Of Anaphase-promoting Complex And Its Subunit Cdh1 In The Proliferation And Differentiation Of Neural Stem Cell

Background and objectiveNeural stem cells are defined as immature cells with the capacity for self-renewal,proliferation and differentiation into multiple specialized cell types. During the past decade,cell transplantation therapies for neurological diseases have been widely researched andhave progressed toward clinical trials. For both endogenous and transplanted progenitorcells, survival, differentiation and the establishment of effective synaptic connectionswithin injured regions of the nervous system are key issues in the recovery of neurologicalfunctions. Previous studies indicated that mutated IκBαgene can increase the cell survivalrate of immortal neural progenitor cells and lessen the cell damage caused byoxygen-glucose deprivation by down-regulating the activity of NF-κB and the expressionof some NF-κB-driven cytokines, but the differentiation of immortal neural progenitor cellsand neurological functions were not improved.Anaphase-promoting complex (APC)/cyclosome is an E3 ubiquitin ligase that controlsthe cell cycle by directing the ubiquitin-dependent proteolysis of cyclins and other cellcycle regulatory proteins in proliferating cells, Cdh1 is one of its co-activators, which isrequired for APC activity during late mitosis and G1. Recent studies show that Cdh1 and core components of APC are highly expressed in mature neurons in many parts of thecentral nervous system. Cdh1-APC plays roles in the regulation of axonal growth andpatterning, synaptic development and function and neuronal survival. But only limited datasuggest possible functions of Cdh1-APC in the control of neural stem cell proliferation anddifferentiation in the developing and mature brain and in the realm of central nervoussystem injury and disease.In the mammalian central nervous system, the proliferation and differentiation ofneural stem cells are associated with cell cycle regulators. On the one hand, neuroepithelialstem cells withdraw from the cell cycle and differentiate into post-mitotic neurons. On theother hand, Cdhl-APC function is important in maintaining the G1 phase of the cell cyclein dividing cells. Therefore, we hypothesized that Cdh1-APC activity may be involved inthe differentiation of neural stem cells. Firstly, we investigated the expression and cellulardistribution of Cdh1 in the central nervous system both in vitro and in vivo. Secondly, wecultured primary neural stem cells from postnatal Sprague-Dawley (SD) rats, and analyzedchanges in Cdh1 expression during the induction of their differentiation with all-transretinoic acid (ATRA). Thirdly, the target sequences for Cdh1 RNA interference werescreened. Fourthly, lentiviral vectors of RNA interference for Cdh1 were constructed.Fifthly, the effects of lentivirus-mediated Cdh1 RNAi on the proliferation anddifferentiation of neural stem cells were investigated. This study elucidates additional rolesof Cdh1 in the central nervous system, and it provides evidence that Cdh1 may be a newtarget for gene therapies of neurological diseases.Methods and Results1. The characteristics of APC-Cdh1 expression in central nervous systemMethods Fresh frozen sections were prepared from brain tissue of healthy adult maleSD rat aged 2～3 months. The expression of Cdh1 was examined by immunohistochemistry.The cell types expressing Cdh1 were determined by double immunofluorescence labeling: neurons were identified by NeuN immunostaining; neural stem cells were examined bynestin immunostaining; MAP2 and GFAP immunostaining were used to respectivelyindentify differentiated neurons and astrocytes. Primary neurons and neural stem cells wereisolated from hippocampus of postnatal rat pups 24 hours after birth.The mRNA of neuronsand neural stem cells was extracted by the Trizol method. The expression of Cdh1 andCDC20 (another cofactor of APC during mitosis) was examined by quantitative real-timePCR.Results The immunostaining showed that Cdh1 is highly expressed in cerebral cortexand hippocampus. The double-immunofluorescent staining showed that Cdh1 is mainlylocated in neurons, but that it is almost absent in glial cells. About 90% of the cells inprimary cultures of hippocampal neurons were NeuN-positive and extended long neurites.Neural stem cells isolated from hippocampus formed floating neurospheres on the 4^th or 5^thdays of culture, They were nestin-positive and differentiated either into neurons orastrocytes when cultured with fetal bovine serum. Compared with neural stern cells, theexpression of Cdh1 mRNA was significantly higher in neurons (neural stem cells: 1.00±0.05; neurons: 2.10±0.07, arbitrary units, P＜0.05 by Student's test), while the expressionof CDC20 mRNA in mature neurons was very low (neural stem cells: 1.00±0.04; neurons:0.02±0.01, P＜0.05).2. Changes in Cdh1 expression in neural stem cells during the induction of neuronaldifferentiation with all-trans retinoic acid (ATRA)Methods Neural stem ceils isolated from hippocampus of 24-hour-old SD rats wereisolated and cultured as neurospheres in serum-free neurobasal medium containing B27,epidermal growth factor and basal fibroblast growth factor. After three passages, they wereharvested and cultured on poly-L-ornithine-coated dishes. They were randomly divided intothree groups: control group, 1μmol/L ATRA and 10μmol/L ATRA groups. As ATRA wasdissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM, 0.1% of DMSO was added to the culture medium of the control group. At the beginning and on the 4^th and 8^thdays of ATRA treatment, total RNA was extracted, and the expression of Cdh1 mRNA wasexamined by quantitative real-time PCR. On the 8^th day of induction, the treated cells werefixed and immunostained with antibodies against MAP2 or GFAP to evaluate theproportions of neural stem cells differentiated into neurons or astrocytes. The mRNA of Id2(inhibitor of differentiation 2, a downstream substrate of Cdh1) was also examined byquantitive real-time PCR.Results Analysis by immunocytochemistry showed that the percentage of MAP2positive neurons increased after ATRA-induced neuronal differentiation (control group:19.1±2.8%; 1μmol/L ATRA: 41.4±0.8 %*; 10μmol/L ATRA: 34.3±1.0 %* (*P＜0.05when compared to the control group by chi-square test). The percentage of neural stem cellswhich differentiated into neurons was significantly higher in the 1μmol/L ATRA inductiongroup when compared to the other two groups. Thus, ATRA treatment significantlyincreased the number of MAP2 positive cells. In the control group, the expression of Cdh1was similar at the beginning of the treatment and after 4 or 8 days. In contrast, theexpressions of Cdh1 was significantly increased in the groups treated with 1μmol/L or 10μmol/L ATRA at the 4^th and 8^th days of induction (P＜0.05 when compared to the beginningof the treatment by ANOVA/LSD post-hoc tests). The expression of Cdh1 on the 8^th day ofinduction with 1μmol/L ATRA was significantly higher than at any other point (P＜0.05).On the contrary, the expression of Id2 mRNA was decreased in neural stem cells during theinduction with 1μmol/L ATRA.3. Screening the effective target site for Cdh1 small interfering RNA in ratimmortalized neural progenitor cells (INPC)Methods The three bands of interfering sequences and the control sequence of Cdh1small hairpin RNA (shRNA) were designed based on the Cdh1 coding sequence. Followingthe instruction for pENTR/H1/TO vector, the oligonucleotides were synthesized, annealed and ligated into linearized pENTR/H1/TO vector. After confirmation by DNA sequencing,positive recombinant plasmids including three RNAi vectors and one control vector weretransfected into immortalized neural progenitor cells respectively by the liposome method.The pEGFP (Enhanced Green Fluorescent Protein) plasmid was transfected to evaluate theefficiency of transfection according to the expression of green fluorescent protein.Forty-eight hours after transfection, total cellular RNA was extracted and the expression ofCdh1 was analyzed by quantitative real-time PCR.Results The recombinant plasmids were primarily identified by 1% agarose gelelectrophoresis. Their sizes were about 3kb. The three eukaryotic vectors for Cdh1 siRNAand the control vector were successfully constructed as verified by DNA sequencing. Theefficiency of cell transfection was 54±5 % at 24h and 36±4 % at 48h after transfection.When compared to INPC transfected with control vector (value set at 1), the expression ofCdh1 in two of the three INPC transfected with Cdh1 siRNA vectors was significantlyreduced (vector shRNA 1: 1.15±0.31; vector shRNA2:0.37±0.06; vector shRNA3:0.51±0.08).4. Construction and identification of lentiviral vector for RNA interference of ratCdh1 geneMethods The effective sequence of siRNA targeting Cdh1 gene was confirmed in ourprevious study. The complementary DNA containing both sense and antisense Oligo DNAof the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFPvector. The recombinant plasmid was confirmed by PCR and DNA sequencing. Then, itwas cotransfected into 293T cells with pHelper 1.0 and pHelper 2.0 by the liposomemethod. The titer of virus was tested by infecting 10⁵ human embryonic kidney cells, 293Tcells, with a serial dilution of the LV vector according to the expression level of GFP.Proliferative PC12 cells were infected either with control lentivirus or with Cdh1 RNAilentivirus. Seventy-two hours later, total cellular RNA and total protein were extracted and the expression of Cdh1 was analyzed by RT-PCR and Western blot.Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector ofCdh1 (LV-Cdh1 RNAi) was constructed successfully. The titer of concentrated virus was7×10⁸TU/ml. RT-PCR and Western blot showed that the expression of Cdh1 in PC12 cellsinfected with Cdh1 RNAi lentivirus was significantly decreased when compared with cellsinfected with control lentivirus or with uninfected cells (P＜0.05).5. Effect of lentivirus-mediated Cdh1 RNAi on neural stem cellsMethods Neural stem cells from hippocampus of postnatal 24 hours-old SD rats wereisolated and cultured with serum-free neurobasal medium containing B27, epidermalgrowth factor and basal fibroblast growth factor. After three passages, they were infectedwith control lentivirus containing GFP in different MOI (Multiplicity of Infection=ratio ofinfectious virus particles to cells). Seventy-two hours later, the appropriate MOI for neuralstem cells was determined by examining GFP expression by fluorescent microscopy. In thisstudy, three groups were involved, uninfected group, LV-control group (neural stern cellswere infected with control lentivirus) and LV-Cdh1 RNAi group (neural stem cells wereinfected with lentivirus for Cdh1 RNAi). Viable cells were analyzed by using the MTTassay at 24h, 48h and 72h after viral infection. Three days after viral infection, the cellswere induced to differentiate with 2% fetal bovine serum. Four days later, total cell proteinswere extracted for Western blot analysis. The expressions of MAP2 and GFAP wereexamined to evaluate the differentiation of neural stem cells respectively into neurons andastrocytes.Results The appropriate lentivirus MOI for neural stem cells ranged from 10 to 50.The MTT assay showed that there was no significant difference in the absorption betweenthe LV-Cdh1 RNAi group and the LV-control group at 24h and 48h after viral infection(P＞0.05), while the absorption in LV-Cdh1 RNAi group was significantly increasedcompared with the LV-control group and uninfected group at 72h after viral infection (P＜0.05). The expression of MAP2 protein in LV-Cdh1 RNAi group was significantlydecreased when compared with the LV-control group and uninfected group (P＜0.05). Therewas no significant difference in GFAP expression between the three groups (P＞0.05).Conclusions1. APC-Cdh1 is highly expressed in brain tissue and is mainly located in neuron. Theexpression of Cdh1 in neurons is higher than in neural stem cells in vitro.2. The primary neural stem cells are successfully induced to differentiate into neurons byATRA, and 1μmol/L ATRA can produce the highest ratio of differentiated neurons.When neural stem cells are differentiated into neurons, the expression of Cdh1 mRNAis up-regulated, whereas Id2 mRNA is down-regulated.3. By using rat immortal neural progenitor cells and pENTR/H1/TO vector system, theeffective target sequence for Cdh1 RNA interfering was successfully screened.4. The lentivirus RNAi vector of the rat Cdh1 gene was successfully constructed and thetiter of concentrated virus was 7×10⁸TU/ml. It can produce effective down-regulationof Cdh1 at both mRNA and protein levels.5. Lentivirus-mediated Cdh1 RNAi can promote an increase in neural stem cells andinhibit their differentiation into neurons.SummaryThis study shows that the differential expression of Cdh1 in different types of neuralcells analyzed by in vivo and in vitro experiments. Cdh1 is mainly located in neurons, andthere is only little expression in neural stem cells and in astrocytes. In addition, we foundthat the expression of Cdh1 mRNA is increased when the neural stem cells are induced todifferentiate into neurons by all-trans retinoic acid. This observation suggests that Cdh1may be involved in the differentiation of neural stem cells into neurons. Then, screening theeffective target sequence for Cdh1 RNA interference and constructing lentivirus RNAi vectors of rat Cdh1, allowed us to observe the effects of lentivirus-mediated Cdh1 RNAi onthe proliferation and differentiation of neural stem cells. The results suggest that thedown-regulation of Cdh1 can promote the proliferation of neural stem cells and inhibit theirdifferentiation into neurons. They further demonstrate a role for Cdh1 in the differentiationof neural stem cells. This study thus extends the understanding of Cdh1 functions in thecentral nervous system, and suggests that Cdh1 may also play an important role in neuraldevelopment and differentiation. To pave the way for new gene therapies for neurologicaldiseases, it will be very interesting to also explore additional functions of Cdh1-APC in theinjured or diseased nervous system and during neuronal regeneration.