Neuroprotective Effect Of Recombinant Lenti-virus Encoding Rab9 CDNA On Niemann-Pick Disease Type C Mice

Partâ… Localization of Rab9 and NPC1 in cultured microgliaObjectiveTo observe the localization of Rab9 and NPClin cultured microglia,and to investigatethe positional relationship between Rab9 and different organelles,including lysosome,Golgi apparatus and late endosome.MethodsMouse microglia cell line BV-2 were employed in this study.Triple-fluoresencelabeled immunohistochemical staining and laser scanning confocal microscopy wereadopted.Resultsâ‘ The Rab9 and NPC1 colocalized in cytoplasm of microglia.â‘¡Rab9 expressed in lysosome,Golgi apparatus and late endosome.ConclusionRab9 may have important influence on function of NPC1 protein. Partâ…¡Packaging and functional identification of Lentivirus encodingRab9 cDNA by Lentivector Expression System(SBI)ObjectiveRab9 plays an important role in transport of vesicle between different organelles.Asthe first step toward exploring the possibility of Rab9 gene therapy for Niemann-pickdisease type C,recombinant lentivirus encoding Rab9 cDNA was packed.MethodsThe expressing plasmid pCDH1-MCF1-RAB9-EF1-copGFP was constructed bymolecular biological techniques.The LV encoding Rab9 cDNA(Lentivirus-Rab9 cDNA)was packed by Lipofectamine 2000^TM mediated cotransfection of pCDH1-MCF1-Rab9-EF1-copGFP,pPACKH1-GAG,pPACKH1-REV and pVSV-G into 293T cells.ResultsNCBI blast showed that the sequence of Rab9 in lentiviral expression vector matchedto that of murine Rab9 gene(Genebank accessing number:NM₀04251)100%.Afterdigestion with restrication endonuclease,the 678 bp fragment of Rab9 gene and the7.544kb vector fragment of pCDH1-MCF1-EF1-copGFP were observed with gelelectrophoresis.The recombinant lentivirus infected BV-2 cell effectively.The transfectionrate 72 hours after infection was approximately 80%.Compared to the control group,expression of GFP and remarkbly increased Rab9 level(P=0.021) could be observed inbilateral cerebella of virus group mice four week after injection.ConclusionOur data indicate that the packaging of lentivirus encoding Rab9 cDNA is successful.The recombinant lentivirus carrying Rab9 cDNA could infect BV-2 cell line and deliver theRab9 into the Balb/c mouse brain efficaciously,which might offer a novel means fortreating Niemann-Pick disease type C. Partâ…¢Effect of recombinant lentivirus encoding Rab9 cDNA on thebehavior and body weight of Niemann-Pick disease type C miceObjectiveTo observe the effect of bilateral lateral ventricle injection of recombinant lentivirusencoding Rab9 cDNA on the behavior of Niemann-Pick disease type C(NPC) mice.MethodsThe lentivirus encoding Rab9 cDNA(lentivirus-Rab9 cDNA) was injected into thebilateral lateral ventricle of postnatal day 3 npc^-/- mice.The mice were weighed,and coathanger test and footprint test were performed to assess their motor ability from 4 to 8 weeksage.Resultsâ‘ Weight loss of female npc^-/- mice was significantly delayed in the lentivirus-Rab9group.â‘¡Coat hanger test showed that the motor defect was remarkably ameliorated in thelentivirus-Rab9 group.â‘¢Footprint test showed that the relative stride in lentivirus-Rab9 group(68.1%±3.8%)was markedly longer than that in the controlled viral group(35.7%±8.1%) and thenon-surgical group(40.2%±4.8%,P＜0.05).ConclusionThe bilateral lateral ventricle injection of lentivirus encoding Rab9 cDNA improvesweight and motor ability of npc^-/- mice,which may offer a novel means for treating NPC. Partâ…£Effect of recombinant lentivirus encoding Rab9 cDNA on theneuropathology of Niemann-Pick disease type C miceObjectiveTo assess the effect of bilateral lateral ventricle injection of recombinant lentivirusencoding Rab9 cDNA on the neuropathology of Niemann-Pick disease type C(NPC) mice.MethodsLentivirus encoding Rab9 cDNA was injected into the bilateral lateral ventricle ofpostnatal day 3 npc^-/- mice.The neuropathology in the treated mice was evaluated by HEstaining,immunohistology and immunoblotting.Resultsâ‘ Lentivirus mediated Rab9 cDNA was widely expressed in npc^-/- mice brain.â‘¡Lentivirus encoding Rab9 cDNA ameliorated the deletion of purkinje cells,andremarkably reduced the number of axonal spheroids in npc^-/- mice.â‘¢Lentivirus encoding Rab9 cDNA markedly increased the expressions of Rab9 andinhibited the phosphorylation of neurofilament(recognized by SMI31),tau(recognizedby CP-13) and mitotic associated proteins(recognized by MPM-2).ConclusionThe Lentivirus encoding Rab9 cDNA injected into the bilateral lateral ventricle ofpostnatal day 3 npc^-/- mice can ameliorate the neuronal cytoskeletal pathology andneurodegeneration,which implys that Rab9 might be a novel therapeutic target in NPC.