Overexpression Of GDNF Mediated By Lentivirus In Bone Marrow Stromal Cells And Neuroprotection In The Lactacystin-induced Parkinson's Disease Model

Parkinson's disease (PD) is a common neurodegenerative disease and its exact pathogenesis is not clear. The pathological hallmarks of PD are reduction or loss of the dopaminergic neurons in the SNc and the presence of Lewy body in the degenerated neurons. The inability of ubiquitin-proteasome system (UPS) to degrade alpha-synuclein and other unwanted proteins in SNc in idiopathic PD may contribute to the formation of Lewy body. In sporatic parkinson's disease, proteasomal function is impaired in SNc. Mutations in alpha-synuclein, Parkin and ubiquitin C-terminal hydrolase L1, cause the familial PD. These results suggests that dysfunction of the ubiquitin-proteasome system may underlie nigral degeneration and Lewy body formation in PD. Our research team had set up the Lactacystin-induced Parkinsonian model in rattus. The next step is to search and confirm an effective therapy to alleviate the damage of dopaminergic neurons induced by proteasomal inhibitor. GDNF has been shown that it can protect dopaminergic neurons from 6-OHDA-or MPTP-induced damage and be serves as a promising candidate of neuroprotective therapy for PD. Taking consideration of the chronic and progressive nature of PD, the therapy should be administered continuously to sustain dopaminergic neuron's survival and maintain normal function longer. Therefore, bone marrow stromal cells (BMSCs) overexpressing lentivirus-mediated GDNF was studied in this research to evaluate their neuroprotection to dopaminergic neurons in vivo and in vitro.PartⅠConstruction of PLenti6/V5-GDNF Expressing Plasmid and Production of Pseudo-Lentivirus in 293FT CellsAim: To construct the pLenti6/V5-GDNF expressing plasmid and produce the pseudolentivirus in 293FT cells. Methods: The pLenti6/v5-GDNF plasmid was set up by the digestion of BamH I and Xho I double restriction enzyme and ligation of T4 ligase, and then the plasmid was transformed into Top10 cells. Several clones were picked for enzymatic digestion and only the clones with correct result of enzymatic digestion were chosen for sequencing. Cotransfecting the 293FT with the pLenti6/V5-GDNF and the packaging mixture by Lipofectamine2000, the purified plasmid from the positive clone was used to produce the lentivirus. Viral supernatant was harvested and then its titer was determined. Results: The pLenti6/V5-GDNF plasmid was constructed and confirmed by sequencing. The lentivirus expressing GDNF in-frame with the V5 epitope tag was produced and the titer of GDNF-Lentivirus was 5．6×10~5 TU/ml. Conclusion: Lentivirus-engineered GDNF was constructed successfully and GDNF virus stock was produced.PartⅡOverexpression of Lentivirus-mediated GDNF in BMSCs and Its Neuroprotection to the Lactacystin-Induced Damage of PC12 cellsAim: To infect bone marrow stromal cells by lentivirus-engineered GDNF and investigate its neuroprotection in lactacystin-induced PC12 cells. Methods: The overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection in lactacystin-induced PC12 cells was evaluated by MTT assay. Results: Lentivirus-engineered GDNF can effectively infect BMSCs (MOI=1, percent of positive cells >95%). GDNF-BMSCs can secret GDNF in supernatant with higher concentration (800pg/ml) than that of BMSC (<100pg/ml). The supernatant of GDNF-BMSC can significantly alleviate the damage induced by proteasome inhibitor Lactacystin (10μm) in PC12 cells. Conclusion: Overexpression of lentivirus-mediated GDNF in BMSCs can effectively protect the PC12 cells from the injury of proteasome inhibitor.PartⅢIntrastriatal Transplantation of GDNF-engineered BMSCs and its neuroprotection in Lactacystin-induced Parkinsonian Rat Model.Aim: To evaluate the neuroprotective effect of GDNF-engineered BMSCs by intrastriatal transplantation in Lactacystin-induced Parkinsonian Rat Model.Methods: Female Sprague-Dawley 64 rats were used and randomly devided into four groups (control: n=16; lactacystin: n=16; LacZ: n=16; GDNF: n=16) . GDNF-BMSCs and LacZ-BMSCs was transplanteded into right SNc, which followed by the injection of Lactacystin (2μg) in median forebrain bundles (MFB) at the same side one week later, with exception of control group. The neuroprotective effect was evaluated by behavior test, tyrosine hydroxylase (TH) immunohistochemistry in SNc and striatum, TH western blot in SNc and HPLC in striatum. Results: The intrastriatal transplantation of GDNF-BMSCs can significantly rescue the dopaminergic neurons from lactacystin-induced neurotoxicity in terms of behavior recovery, TH immunohistochemistry and western blot, and striatal dopamine level by HPLC.Conclusion: Transplantation of GDNF-engineered BMSCs may be beneficial as an alternative therapeutic strategy in the treatment of PDSummary1．We set up the GDNF-Lentivirus expressing system and produce the pseudo-lentivirus supernatant for gene therapy.2．BMSCs transduced by the GDNF-lentivirus can secret sufficient and effective GDNF in supernatant, and the concentration from it is much higher than that produced by BMSCs. The supernatant of GDNF-BMSCs can statistically protect the survival of PC12 from proteasome inhibitor.3．A single injection of lactacystin into MFB at a lower dose than direct injection into SNc can induce the damage of dopaminergic neurons. The novel rat model can mimic the pathogenesis of Parkinson's disease.4．Transplantation of GDNF-BMSCs can be helpful to protect the dopaminergic neurons in SNc damaged by a single injection of lactacystin into MFB, its efficacy is better than that of LacZ-BMSCs.