The Expression And Function Study Of Sema4C In NSC Development

Sema4C is a transmembrane protein and belongs to class IV group of Semaphorins family. Now little was known on expression and function of Sema4C, especially the role of Sema4C in the nervous system. Studies on the roles of semaphorins were concentrated on axon guidance and synaptic plasticity in the nervous system. Detailed bioinformatics analysis on Sema4C showed that Sema4C was expressed in embryonic tissues and neuronal tissues and involved in development process. Our in situ hybridization results also showed that Sema4C was expressed in E11.5 and E13.5 embryonic brain tissues. These suggest Sema4C may function in the nervous system development. Moreover, Sema4C has been reported to interact with GIPC, PSD95, SAP97, SAP 102 and Norbin. GIPC and SAP97 have been involved in Wnt or Notch signal pathway, respectively. As we all kown, Wnt signal pathway play an important role in central nervous system and control cell growth and differentiation, and Notch signal play decisive roles in NSC maintance and differentiation. The nervous system development is that NSC self-renewing and differentiationed into neurons, astrocytes and oligodentrocytes. The NSC is the important model to study nervous system development. So we investigated the expression and function of Sema4C in NSC development.By RT-PCR, western blot and immunoflurescence analysis, we showed that Sema4C was highly expressed in NSC. The level of Sema4C expression was down-regulated during induction of NSC differentiation, and Sema4C was expressed in neurons, but not in astrocytes. These results suggest that the possible function of Sema4C in NSC.Next, we constructed adenoviral expression system to infect NSC. RT-PCR and western blot results showed that Ad-S4C infected NSC successfully and Sema4C was overexpressed in NSC. Then we test the effects of overexpressed Sema4C on NSC: 1. Brdu incorporation assay was performed to reflect the proliferation of NSC, It was shown that the incorporation of Brdu was significantly decreased after 48 h of infection with Ad-S4C compared to control Ad-EGFP, The results suggest that Sema4C possibly inhibit NSC proliferation.2. NSC was infected with Ad-EGFP or Ad-S4C with different multiplicity of infection (MOIs). After 4 days, NSC infected with Ad-S4C at MOI of 50 and 100 were adherent and dendrites grew out as comparison with control cells infected with Ad-EGFP. The total number of adherent neurospheres had 3-5 fold increases as comparison with control; Moreover, the percentage of adherent neurospheres infected with Ad-S4C at MOI of 50 and 100 were 67%±4.8153% and 63%±3.3693% respectively as compared with control infected with Ad-EGFP (24%±5.099%). However, there were no significant difference among those infected with Ad-EGFP with different multiplicity of infection of 50,100 and 150. This suggests Sema4C significantly induced NSC adhesion.3. In order to determine possible effects of Sema4C on the neuron population differentiated from NSC, co-immunohistochemical staining for TuJ1 was used to reveal the number of TuJ1 -positive cells. The results showed that the TuJ1-positive cells were significantly decreased in Ad-S4C-infected cultures of NSC compared to that for Ad-EGFP-infected cultures. The finding suggests that Sema4C possible inhibit NSC differentiation toward neurons.GIPC and DLG2 were identified as the interacting proteins of Sema4C by the yeast two-hybrid system. Our results showed that GIPC was expressed in NSC and terminal differentiation cells. Also, plexinB1, the Sema4C possible receptor, was also expressed in NSC and terminal differentiation cells. These suggest that sustaining expression of GIPC and plexinB1 in NSC may mediate the role of Sema4C in NSC development.DLG2 and other Sema4C interacting proteins PSD95, SAP97, SAP102 and Norbin are synaptic proteins and play important roles in synaptic plasticity. Hypoxia has been an important model to investigate synaptic plasticity. Our results confirmed the up-regulation of SAP97 and GluR2 in mRNA level after treatment of hypoxia (IH, 4h/d) at 3km or 5km for 2 weeks. But no change of Sema4C could be observed. This suggests Sema4C could not be involved in synaptic plasticity change made by hypoxia. So, we think that Sema4C mainly function in NSC development.