The Influence Of Targeted Suppression Of Glutaryl-CoA Dehydrogenase And Excessive Intake Of Lysine On Neurotoxicity In Primary Cultured Striatal Neurons

Part I The in vitro study of neurotoxicity induced by lysine in primary rat striatal neuronsObjective To culture and identify primary rat striatal neurons. To investigate the neurotoxic effect of lysine on rat striatal neurons in vitro.Methods Striatum tissues were separated from SD rats born within24h. Trypsin digestion and serum free cultivation method were used to culture primary striatal neurons. The purity of neurons was identified by using immunohistochemical method of neuron specific enolase (NSE), using immunofluorescence and flow cytometry methods of microtubule associated protein (MAP2).24h after the intervention of different concentrations of lysine (0,5,10,15,20mmol/L) with neurons, the cell viability was determined by MTT method. Discussed the neurotoxicity induced by different concentrations of lysine in primary rat striatal neurons.Results This method cuccessfully cultured rat striatal neurons. MAP2immunofluorescence showed that the purity of neurons was92.4±1.6%. MAP2staining flow cytometry showed that the purity of neurons was94.3±2.5%. NSE immunohistochemistry showed the purity of neurons was96.2±3.3%. The neurotoxicity induced by lysine in primary rat striatal neurons was concentration dependent. After incubated with lysine for24h, the results of MTT showed the concentration less than10mmol/L had no effect on neuron viability,15mmol/L and20mmol/L lysine reduced the viability of neurons to84.63%and45.60%respectively.Conclusion The rat striatal neurons with high purity were successfully cultured by using trypsin digestion and serum free cultivation method. Lysine intervention for24h, the concentration of not more than10mmol/L had no neurotoxic effect on neurons. When the concentration reached more than15mmol/L, the neurotoxicity was concentration dependent. In view of that high lysine diet does not promote neurotoxicity in normal human, the concentrations of5mmol/L and10mmol/L were used in the following experiments. Part II The neurotoxicity of targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine on rat striatal neuronsObjective To investigate the neurotoxicity induced by excessive lysine in GCDH silencing neurons by using lentivirus-mediated shRNA and excessive intake of lysine.Methods Lentiviral vector was constructed by using a known siRNA sequence targeted suppression of GCDH gene expression. The appropriate multiplicity of infection was determined by using GFP carried by virus vector. The interference efficiency of lentivirus was determined by using Real-time PCR and Western blotting. The viability of GCDH silencing neurons incubated with lysine or not was determined by using MTT. The nuclear morphological changes in rat neurons were detected by using Hoechst33342staining. The quantity of neural apoptosis was analysed by using Annexin V-PE/7-AAD and flow cytometry.Results shRNA was transfected into primary rat striatal neurons successfully by using lentiviral vector, and the transfection efficiency was as high as96.5%. The vector can effectively suppressed the GCDH gene expression. The expression of protein was reduced as much as80.78%. The targeted suppression of GCDH by lentivirus-mediated shRNA reduced the neuron viability to68.62%,5mmol/L and10mmol/L lysine enhanced this effect markedly, and the neurotoxicity was concentration dependent. Hoechst33342 staining showed nuclear apoptosis. Annexin V-PE/7-AAD flow quantitative analysis further verified the results of MTT analysis.Conclusion Lentivirus-mediated shRNA vector suppressed the expression of GCDH gene effectively and stably.5mmol/L lysine had no effect on neuron viability, but induced GCDH silencing neurons apoptosis. GCDH silencing neurons incubated with addition of5mmol/L lysine can simulate the hypermetabolic condition in GA1patients. A novel model of GA1was established. PartⅢ the mechanism of the neurotoxicity induced by lysine in GCDH silencing neurons was investigatedObjective To investigate the the mechanism of the neurotoxicity induced by lysine in GCDH silencing neurons.Methods Mitochondrial membrane potential was monitored using TMRM staining, confocal microscope and flow cytometry. The levels of caspase3,8,9, AIF and Hsp70expression were determined by Western bloting.100μmol/L Z-VAD-FMK was added to the medium1h prior to lentiviral infection, and then the quantity of neural apoptosis was analysed by using Annexin V-PE/7-AAD and flow cytometry.Results TMRM staining, confocal microscope and flow cytometry all showed suppression the expression of GCDH gene significantly decreased the neuron mitochondrial membrane potential, and5mmol/L lysine enhanced this decrease. The protein levels of caspases3and9were significantly upregulated by lentivirus. The combination of lysine and lentivirus intensified the upregulation of caspases3and9. Neither lentivirus nor5mmol/L lysine alone changed the level of caspase8expression, but exposure to both increased the protein level of caspase8. With Z-VAD-FMK pretreatment, the apoptotic cell fraction in cells infected with lentivirus decreased to21.87%in cells not exposed to lysine and41.66%in cells exposed to5mmol/L lysine.Conclusion Mitochondrial membrane potential changes may be involved in the the mechanism of the neurotoxicity induced by lysine in GCDH silencing neurons. And the neurotoxicity is caspase pathway dependent.