Neuroprotective Effect And Molecular Mechanism Of The GLP-1 Engineered Commensal Bacterium On Neurodegeneration In Mice

Background and Aims:Neurodegenerative diseases(NDD)is one of the important factors affecting the health of the elderly,which causes nervous system dysfunction by irreversible damage to neurons,such as the Alzheimer's disease(AD),Parkinson's disease(PD)and so on.At present,the pathogenesis of these diseases is not completely clear,and there is no satisfactory radical therapy.Therefore,the development of new drugs for such diseases has far-reaching practical significance.It has been found that type 2 diabetes mellitus(T2DM)is associated with neurodegenerative diseases in neuropathological manifestations and clinical treatment.Drugs for treating T2DM have become a new strategy for prevention and treatment of NDD.At present,Glucagon-like peptide-1(GLP-1)and its analogues are mainly used in the treatment of T2DM,and its neuroprotective effects are also receiving increasing attention.Endogenous GLP-1 has a very short half-life,making it unable to perform physiological functions well.GLP-1 analogues are derivatives that modify natural forms,which retain biological activity but are burdened by intravenous injectionon to patients.In order to overcome the above defects,this paper attempt to construct long-lasting,oral GLP-1 supplements by bacteria as drug carriers.Two engineering strains of Lactococcus lactis MG1363-pMG36e-GLP-1(prokaryotic expression system)and attenuated Salmonella typhimurium VNP20009-pLIVE--GLP-1(eukaryotic expression system)were constructed.Our aim is to effectively prevent the development of neurodegenerative diseases from multiple aspects and multiple angles.In this study we first established the AD mice model,trying to evaluate the efficacy of the two engineered bacteria and the efficacy of the combined treatment,the results showed that the MG1363-pMG36e-GLP-1 group had the best curative effect.Then in the PD mice model,the differences among various doses,the effects of prophylaxis before therapy and simple therapy were evaluated again.The results showed that the low-dose administration group was more effective,and there was no significant difference between preventive treatment group and treatment group.Method one:AD mice model1.The construction of two engineered strains and the verification of secreted GLP-1 by two engineered strains in vitro.MG1363-pMG36e-GLP-1 and VNP20009-pLIVE-GLP-1 were constructed by genetic engineering methods.GLP-1secretion and expression in MG1363-pMG36e-GLP-1 bacterial fermentation supernatant were detected by Western-blot and ELISA,the shuttling ability of VNP20009-pLIVE-GLP-1 in eukaryotic cell was verified by 293-T cells,and then the expression level of GLP-1 of VNP20009-pLIVE-GLP-1 in eukaryotic cell culture supernatant was detected by Western-blot and ELISA;2.Establishment of AD mice model.Eight-week-old male C57BL/6 mice were divided into five groups(n=10 per group):control group(C),LPS group(L),LPS+MG1363-pMG36e-GLP-1 group(LL),LPS+VNP20009-pLIVE-GLP-1 group(LV),and LPS+MG1363-pMG36e-GLP-1 group+VNP20009-pLIVE-GLP-1 group(LLV).The mice were intraperitoneally injected with LPS at 0.25 mg/kg body weight per day for 9 days;3.Treatment of AD mice model.The LL group,LV group and LLV group were given the corresponding bacterial as prophylaxis treatment for 2 weeks before establishment of the model.The LL group,LV group and LLV group were treated with the corresponding bacteria during the modeling process and behavioral training.That is,the total administration time of the above three treatment groups lasted for 4weeks.The administration methods were MG1363-pMG36e-GLP-1 10~9 CFU/100?L/day,VNP20009-pLIVE-GLP-1 10~7 CFU/100?L,once every other day;4.Therapeutic effects and mechanism of engineered strains on AD mice.The behavioral tests on learning and memory capabilities were evaluated by Barnes maze.The brain slides were used for evaluation of tissue damage,glial cell activation and aggregation of A?.The protein expression of inflammatory signaling pathway(COX-2,TLR-4,NF-?B,MAPKs,PI3K/AKT)in brain tissue were detected by Western-blot.Then,inflammatory cytokines(TNF-?,IL-1?)in brain tissue and serum were measured using q-PCR and ELISA kit at gene level and protein level.q-PCR and high-through-put sequencing were used to determine the microbial diversity and difference in mice intestine.Method two:PD mice model1.Establishment of PD mice model.Eight-week-old male C57BL/6 mice were divided into six groups(n=12 per group):control group treated with saline alone(C),a group treated with MPTP alone(M),a group pretreated with 10~7 CFU MG1363-pMG36e-GLP-1(till the end of experiment)and then treated with MPTP(PTL),a group treated with MPTP in combination with 10~7 CFU MG1363-pMG36e-GLP-1(TL),a group pretreated with 10~9 CFU MG1363-pMG36e-GLP-1(till the end of experiment)and then treated with MPTP(PTH),a group treated with MPTP in combination with 10~9 CFU MG1363-pMG36e-GLP-1(TH).Mice received continuously intraperitoneal injections of MPTP(20 mg/kg body weight)for 7 days;2.Treatment of PD mice model.For prevention group(PTL and PTH),MG1363-pMG36e-GLP-1 strains(containing in drinking water)were administered 7days before the injection of MPTP and were daily given till the end of experiment.For treatment group(TL and TH),MG1363-pMG36e-GLP-1 strains(containing in drinking water)were administered daily since the injection of MPTP till the end of experiment.That is,the total administration time of the PTL,PTH group and TL,TH group lasted for 2 weeks and 1 week,respectively;3.Therapeutic effect and mechanism of engineering strains on PD mice.The behavioral tests on exploratory and locomotor activity were evaluated using open-field test.The brain slides were used for evaluation of the TH-positive neurons reduction and glial cell activation.The protein expression of inflammatory signaling pathway(GFAP,Iba1,TLR-4,p-NF-?B/NF-?B)and PD related protein(?-syn)aggregation in brain tissue were detected by Western-blot.Then,inflammatory cytokines(TNF-?,IL-1?)in brain tissue and serum were measured using q-PCR and ELISA kit at gene level and protein level.High-through-put sequencing was used to determine the microbial diversity and difference in mice intestinal.Metabolite profiling the intestinal contents difference among groups.Result one:AD mice model1.The Barnes maze test showed that mice in LL group treated with MG1363-pMG36e-GLP-1 had significantly recovered the damage caused by LPS,showing the closest level to the C group.Regardless of whether VNP20009-pLIVE-GLP-1administered alone or combination of these two strains,LL group possessed a better effect;2.The same results were obtained in the pathological section of brain slides.Supplementation of MG1363-pMG36e-GLP-1 in LL group could obviously reverse tissue damage(HE staining),glial cell activation(GFAP immunohistochemistry)and aggregation of A?(Congo red staining);3.Western-blot results showed that the administration of MG1363-pMG36e-GLP-1alone in LL group down-regulated key proteins expression in inflammatory signaling pathways(COX-2,TLR4,NF-?B,MAPKs,PI3K/AKT)in brain tissue.Simultaneously inhibited the expression of inflammatory factors(TNF-?,L-1?)at the gene level(q-PCR)and protein level(ELISA),respectively;4.High-through-put sequencing results showed that the administration of MG1363-pMG36e-GLP-1 alone in LL group could restore the reduction of bacterial diversity caused by LPS to the greatest extent,decrease the degree of AD pathogenic bacteria(Bacteroides)and increase the abundance of probiotics(Lactobacillus).q-PCR results also confirmed that supplementation of MG1363-pMG36e-GLP-1 had significantly enhanced the beneficial genus of Lactobacillus,and reduced the pathogenic genus of Enterobacteriaceae,Fusobacterium spp.,and C.perfringers.Result two:PD mice model1.The open field assessment showed that taking low dose of MG1363-pMG36e-GLP-1 enhanced the exploratory activity and locomotor activity better,and no significant change was observed between treated group and pre-treated group;2.The same results were obtained in the pathological section of brain slides and western-blot.Low dose of MG1363-pMG36e-GLP-1 had significantly increased TH-positive neurons,and inhibited the activation of glial cells(GFAP,Iba1);3.Western-blot results showed that low dose of MG1363-pMG36e-GLP-1down-regulated key proteins expression in inflammatory signaling pathways(TLR-4,NF-?B)and?-syn accumulation in substantia nigra in brain tissue.Meanwhile,inhibited the expression of inflammatory factors(TNF-?,L-1?,IL-6)at the level of gene transcription(q-PCR)and protein expression(ELISA),respectively;4.High through-put sequencing results indicated that low dose of MG1363-pMG36e-GLP-1 could reverse the reduction of microbial diversity caused by MPTP,while oral taking this strain formed a new dynamic balance of microbiota compared with C group.In addition,low-dose of MG1363-pMG36e-GLP-1 also reduced the increase in the abundance of Prevotella,Enterobacter and Escherichia coli caused by MPTP,and had a positive effect on the increase of Lactobacillus and Akkermansia,two most important probiotics in human intestinal tract;5.Finally,although the composition of intestinal contents in each treatment group showed significant differences,no significant change of Acetate,Propionate,Butyrate,GABA and Glycine were observed.Conclusion one:AD mice model1.Both MG1363-pMG36e-GLP-1 and VNP20009-pLIVE-GLP-1 can successfully express GLP-1;2.MG1363-pMG36e-GLP-1 administered alone has the most positive therapeutic effect on LPS-induced AD mice models;3.MG1363-pMG36e-GLP-1 can alleviate cognitive deficits,preventing neuron damage,suppressing glia activation,down regulating A?aggregation and excessive activation of glial cells;4.MG1363-pMG36e-GLP-1 plays an anti-inflammatory role by down-regulating the expression of COX-2,TLR-4,NF-?B,MAPKs,and PI3K/AKT in the inflammatory signaling pathway,decreasing the transcription and protein expression of pro-inflammatory factors TNF-?and IL-1?in brain tissue and serum;5.MG1363-pMG36e-GLP-1 can restore the reduction of bacterial diversity caused by LPS to normal composition,as well as decrease AD pathogenic bacteria and increase probiotics abundance;Conclusion two:PD mice model1.Low dose of MG1363-pMG36e-GLP-1 administered has the most positive therapeutic effect on MPTP-induced PD mice models,and no significant change is observed between treated group and pre-treated group;2.Low dose of MG1363-pMG36e-GLP-1 can alleviate memory impairment,suppressing glia activation,down regulating?-syn aggregation and excessive activation of glial cells;3.Low dose of MG1363-pMG36e-GLP-1 plays an anti-inflammatory role by down-regulating the expression of TLR-4,NF-?B in inflammatory signaling pathways,decreasing the transcription and protein expression of pro-inflammatory factors TNF-?,IL-1?and IL-6 in brain tissue and serum;4.Low dose of MG1363-pMG36e-GLP-1 can reverses the decrease in the diversity of intestinal flora caused by MPTP and forms new microbial homeostasis different from that of the normal control group,moreover,it enhances the abundance of Akkermansia and Lactobacillus;5.Metabolomics results showed that MG1363-pMG36e-GLP-1 has no significant effect on the expression of PD related metabolites between the groups;6.The engineered bacteria can directly(effector protein action)and indirect(probiotics restore intestinal flora homeostasis)enhance the treatment effect on PD and AD.