Decreased Transketolase And Dysfunction Of Pentose Phosphate Cycle Are Involved In Pathogenesis Of Brain Regionally Selective Damage Induced By Thiamine Deficiency

Wernicke's encephalopathy induced by thiamine deficiency(TD) is a neurodegenerative disease,which is characterized by regionally selective damage of brain peri-midline.The pathogenesis of brain regionally selevtive lesion induced by TD has not been fully elucidated.Most researchers focused on diminished energy metabolism and oxidative stress because of the reduced activities of the thiamine-dependent mitochondrial enzymes,a-ketoglutarate dehydrogenase(KGDH) and pyruvate dehydrogenase(PDH).However,the mechanism cannot exactly interpret why brain peri-midline is typically damaged,whereas neorcortical areas rich in energy metabolism are not susceptible.Thiamine(vitamin B1),which its biologically active form is thiamine pyrophosphate(TPP),is an essential coenzyme in several biochemical pathways in the brain.TD also decreased the activity of transketolase(TK),another thiamine-dependent enzyme.TK is a key enzyme of the nonoxidative branch of the pentose phosphate cycle,which is adominant pathway responsible for synthesizing ribose-5-phosphate and reducing equivalents(NADPH). Both ribose-5-phosphate and NADPH are essential for various synthetic pathways such as nucleic acids.The damage induced by decreased activity of TK should be apt to take place at the region that cell proliferation is abundant.The researches about tumor cell had confirmed this phenomenon.The region of brain peri-midline presents actively cell proliferation.Our previous study has demonstrated that hippocampal neurogenesis is vulnerable to TD.Are decreased activity of TK and the dysfunction of pentose-phosphate cycle involved in the pathogenesis of brain regionally selective damage induced by TD? We established TD mouse model and investigated the changes of the thiamine-dependent enzymes activities and pentose-phosphate cycle function.The effects of siRNA against TK and TK inhibitor on hippocampal stem cell were observed.Part 1.Effects of thiamine deficiency on activities of brain thiamine-dependent enzymes and function of pentose-phosphate cycle.Objection:To explore the mechanism of brain regionally selective damage induced by TD through studying the effects of thiamine deficiency on the activities of brain PDH,KGDH,TK,and glucose-6-phosphate dehydrogenase(G6PD) as well as the contents of NADP~+ and NADPH.Method:TD mouse model was produced according to previous described methods.The mice were divided into TD9,TD14,TD21 subgroups and normal control group.There were five mice in each subgroup and normal control group.After being anesthesized and decapitated,mouse brain was taken out rapidly.The brain mitochrondfia were extracted according to the method reported by Stephan.The activities of PDH,KGDH,TK,and G6PD were determined according to the methods reported by Humphries,Hinman,Hecqquet and Ninfali respectively.The contents of NADP~+ and NADPH were determined according to the method reported by the literatures。Result:The alterations of the activities of brain PDH and KGDH at TD9 and TD14 groups were not significant compared with control group(p＞0.05). Hippocampal TK activity and expression were higher than that of neocortical areas (p＜0.05).TD decreased significantly the activities of hippocampal TK and the contents of brain NADPH both TD9 and TD14,but did not significantly affect brain G6PD and neocortical TK activities.Conclusion:Energy failure and oxidative stress induced by decreased activities of PDH and KGDH hardly explain exactly why TD leads to specific regionally selective damage of brain peri-midline.Decreased activity of TK and the dysfunction of pentose phosphate cycle induced by TD may be involved in the mechanism of regionally selective damage. Part2.Construction and selection of siRNA against TK through lentiviral vectorObjection:To establish the method of siRNA against TK of hippocampal stem cell through recombinant lentivirus tagged with green fluorescent protein(GFP).Method:According to the design principle of RNAi sequence,we designed several RNAi sequence of transketolase gene.Double strands DNA oligo containing TK-RNAi sequence was ligated with lentiviral vector that had been enzymatic digested.The synthetic product was transformed into competent cells of Bacillus coli and,then,the clones were identified by RT-PCR.Lentiviral vectors containing TK-RNAi sequence and GFP gene were constructed.The neural stem cells from hippocampus were plated in uncoated 12-well plate,and divided into several groups according to the lentiviral vectors containing different TK-RNAi sequences.Three days after being transfected by lentiviral vectors, the percentage of GFP positive stem cells was evaluated.Five days after being transfected,the cells were collected and the expression of TK gene were measured by real-time PCR.The interference efficiency of different RNAi sequences was determined by the expression of TK. RNAi sequence with highest inhibiting efficiency was selected for further study.Result:Lentiviral vectors containing TK-RNAi sequence were constructed successfully;the rate of transfected cells was more than 50%according to the expression of GFP.The reduced rates of TK gene expression of neural stem cells were 65%estimated by real-time PCR.Conclusion:The lentivirus containing TK-RNAi sequence was constructed successfully and inhibited efficiently the expression of TK gene in hippocampal stem cell. Part 3.The effects of siRNA against TK and TK inhibitor on hippocampal stem cellsObjection:To explore the effects of siRNA against TK and TK inhibitor on hippocampal stem cell.Method:Fetal rat on 15th day of embryo were isolated from their mothers. Hippocampus tissues were carefully micro-dissected and triturated with Pasteur pipet to obtain a single cell suspension cultured in the medium for neural stem cells.The cells were exposed to oxythiamine,TK inhibitor,for 1,4,and 7 days,the growth of neurospheres was observed and the proliferation of neural stem cells was evaluated by CCK-8 assay and Brdu incorporation assay.Recombinant lentivirus containing transketolase-RNAi tagged with green fluorescent protein(GFP) and control virus containing negative-RNAi expressing GFP were used to transfect neural stem cells isolated from neurospheres.Three days after being transfected,the transfected rate of the cells was estimated according to the expression of GFP.If the rate reached 50%, the knockdown of TK gene of neural stem cells was evaluated by Real-Time PCR at the fifth day.The number,size and migratory level of neurospheres transfected by lentivirus were observed,and the counting of viable cells was carried out by CCK-8 assay.Result:The growth of the neurospheres explored to oxythiamine was inhibited. The speed of neural stem cell proliferation was also inhibited according to the results of CCK-8 and Brud assay.The reduced rates of TK gene expression in neural stem cells transfected with lentivirus containing transketolase-RNAi reached 65%.The number,size,migratory level and shape of the neurospheres transfected with lentivirus containing transketolase-RNAi were distinctly affected compared with control group.The result of CCK-8 assay showed that RNAi against TK inhibited significantly the proliferation of neural stem cells in vitro.Conclusion:Both siRNA against TK and TK inhibitor inhibit significantly the growth and proliferation of hippocampal stem cells.