Mechanism Of F-actin Remodeling In Synapse Caused By Street Rabies Virus Infection

Objective: Rabies is an acute viral infection of the central nervous system (CNS) and istypically fatal in humans and animals. Despite its lethality, only relatively mildhistopathological lesions are typically found under natural conditions. Dendritic spinesconstitute major postsyanptic sites for excitatory synaptic transmission. Dendritic spines arehighly motile and can undergo remolding; In addition, their structural plasticity is tightlycoordinated with synaptic function, leading to the concept that neuronal synaptic dysfunctonis responsible for the clinical features and fetal outcome. F-actin is concentrated in thedendritic spines. The F-actin changes have effects on the morphology of dendritic spines.F-actin is relative stable and central to numberous celluar processes involving membranedynamics by its depolymerization and polymerization, also plays an important roles inremoldeling of dendritic spines. So our research was focused on the F-actin remoldelingcaused by MRV infection to elucidate the pathogenesis of rabies.Methods:(1) In vivo: The C57/BL mice were randomly divided into experimentalgroup(n=10) and control group(n=10). The mice in experimental group were inoculated byintracranial inoculation at10TCID50/30μl MRV suspension, the mice in control group wereinoculated with indentity volume of cell maintenance medium.1) Immunohistochemicalanalysis of antigen distribution in brain with an MRV strain of the street rabies virus. Thechanges of neuron were demonstrated by HE staining and nissl staining.2) Morphology ofcell bodies and dendrites were studied by fluorescent staining against MAP2antibodies.3)Alexa488-conjugated-Phalloidin were used to explore the changes of F-actin.4)12genesinvoved in depolymerization and polymerization of F-actin were analyzed by real time PCR.5) The level of cofilin, p-cofilin and the ratio of F-actin to G-actin were analyzed by Westernblot.(1) In vitro: The primary neuronal cultures were prepared and innoculated by MRV.1)The infected primary hippocampal neurons were detected by immunofluorescent staining ofMAP2and RV antigen.2) Phalloidin were used to explore the changes of dendritic spines at96h after MRV infection.3) Some genes which might imply to the depolymerization andpolymerization of F-actin were choosed and confrmed by real-time PCR.4) The level of cofilin, p-cofilin and the ratio of F-actin to G-actin were analyzed by Western blot.(1) The signal pathway of F-actin depolymerization1) Preparation of synaptoneurosome: The synapstoneurosome was detected underelectrical microscope and Western blot. Rho-GTPase activity in hippocampalsynaptoneurosome was analyzed by pull-down assays and Western blot.2) Western blotanalysis p-PAK, p-cofilin and the total levels of PAK and cofilin.Result:(1) In vivo1) The distribution of MRV antigen in CNS: MRV antigen could be detected in thecortices, the thalamuses, the cerebulla and hippiocampi by Immunohistochemical analysis.Hippocampus was suggested as the optimal site for enrichment of dendritic spines in CA1region of hippocampus.The MRV infected cells were detected in CA1、CA2and CA3ofhippocampus.2)Neuronal changes caused by MRV infection: The changes of neuron weredemonstrated by HE staining and nissl staining and morphology of cell bodies and dendriteswere studied by fluorescent staining against MAP2antibodies. The results showed that noobvious morphological changes are seen in the cell bodies or dendritic processes in the CA1region of the hippocampus, suggested that both the cell bodies and dendrites remained intactin the infected area.3)The changes of F-actin caused by MRV infection: Phalloidin were used to exploredendritic spine of CA1regions in hippocampus. The punctate labeling of phalloidin, which istypical for the location of F-actin in dendritic spines, was observed in the PBS-injected mice.By contrast, a significant decrease in fluoresent intensity and punctate labeling was observedin the MRV-infected mice. In addition, many rope-like structures were observed to appearalong the dendrites in the brains of the infected mice, indicative of a reorganization of F-actinoccuring post-synaptically.4) F-actin remolding caused by MRV infection: In order to discuss the mechanism ofF-actin remolding.12genes invoved in depolymerization and polymerization of F-actin wereanalyzed by real time PCR.4genes, such as cfll3，ssH1，gsn，ppp3a were up-regulated and2genes, such as limk and Pfn1were down-regulated. The level of cfll3is up-related highersignificently than the control.5) F-actin dynamic changes: The intensity and ratio were significantly decreased in the CA1region of the infected mice when compared with that in the PBS controls. Western blotanalysis revealed that an MRV infection has no effect on the total levels of actin and cofilin;However, it can increase the levels of p-cofilin in the hippocampi, maybe due to theorgernization of F-actin in postsynaptic membrane.(2)In vitro:1) Primary hippocampal neurons were inoculated by MRV: Immunofluorescent stainingof MAP2and RV antigen indicated the higher virus load was reached at96h after MRVinfection.2) The changes of dendritic spines: The coculture of primary hippocamal and corticalneurons were developed. Phalloidin staining revealed that most pyramidal hippocamalneurons showed mushroom-like structures at28days after plating, suggesting the presence ofwell-defined dendritic spines. Infection with MRV resulted in dramatic decrease of spines inboth size and number, as revealed by phalloidin staining. A significant decrease of spine innumber was found in the infected cell（s4±2.0/um n=12）when compared whose those in thecontro（l12±3.0/um，n=10，p<0.01,t-test）. A significent decrease in fluoresence intensity wasalso observed in the MRV-infected primary hippocampal neurons;3) F-actin remolding caused by MRV infection:. The gene related to F-actindepolymerization, as cfll3，ssH1，gsn and ppp3a, were up-regulated in vivo and in vitro,however, limk and Pfn1genes related to F-actin polymerization were down-regulated.4) F-actin dynamic changes: The ratio of F-actin to G-actin were significantly decreasedcompared with control. Western blot analysis revealed that an MRV infection has no effect onthe total levels of actin and cofilin; However, it can decrease the levels of p-cofilin in thehippocampi.(3) The signal pathway of F-actin depolymerization1) Preparation of synaptoneurosome: The synapstoneurosome was detected underelectrical microscope and Western blot. We investigated the effects of MRV infection onRho-GTPase activity in hippocampal synaptoneurosome with pull-down assays. MRVinfection markedly reduced the level of GTP-bound Rac1, produced a modest inhibition ofCdc42activity, and have no detectable effect on RhoA. MRV infection did not affect totallevels of the three GTPases。2) Signal pathway of F-actin remolding: PAK and cofilin phosphorylation are regulatedby Rho family small GTPases in many cell system. Western blot analysis revealed that an MRV infection has no effect on the total levels of PAK and cofilin; However, it can decreasethe levels of p-PAK and p-cofilin in the hippocampi.Conclusion: In our study, In spite of no abvious injury of neuronal bodies, we havedetermined that an MRV infection can decrease in the number of dendritic spines in vivo andin vitro, MRV infection also markedly reduced levels of GTP-bound Rac1, p-PAK andp-cofilin, which indicated the F-actin depolymerization. The decresed in the number of thedendritic spine caused by F-actin depolymerization that it may be lead to neuronaldysfunction.The relationship between F-actin and MRV infection was discovered by our research, asit is an important to illuminate the pathogenisis of rabies.