Abstract: Parkinson’s disease (PD) is a prevalent neurodegenerative disorder with limited therapeutic options and no cure, underscoring the urgent need for novel treatment strategies. Our previous work demonstrated that an engineered strain ofClostridium butyricum-pMTL007-glucagon-like peptide-1 (C. butyricum-pMTL007-GLP-1) alleviated PD symptoms by enhancing mitophagy, though the exact molecular mechanisms remained incompletely understood. In this study, we further investigated the neuroprotective effects and underlying mechanisms of this engineered strain using an A53T α-synuclein (α-syn) transgenic mouse model of PD. Specifically, we evaluated its impact on motor function, gut α-syn expression, intestinal barrier function, gut microbial composition, and neuropathological changes, with a focus on the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. Our findings revealed thatC. butyricum-pMTL007-GLP-1 ameliorated motor deficits in PD mice by reducing intestinal α-syn accumulation, restoring gut barrier function, and modulating microbial diversity—notably increasing the relative abundance ofPrevotella at the genus level. Furthermore, the engineered strain attenuated neuropathological alterations by decreasing phosphorylated α-syn (p-α-syn) in the substantia nigra while upregulating tyrosine hydroxylase (TH), dopamine-transporter (DAT), and glucagon-like peptide-1-receptor (GLP-1R) expression. These neuroprotective effects were associated with suppressed proinflammatory responses and enhanced anti-inflammatory and anti-apoptotic signaling, likely mediated through PI3K/AKT/GSK-3β pathway activation. In conclusions,C. butyricum-pMTL007-GLP-1 exerts significant neuroprotective effects in PD mice by reshaping gut microbiota composition and activating the PI3K/AKT/GSK-3β pathway. These findings provide further theoretical support for the potential application of probiotic-based therapies in PD treatment.

工程菌Clostridium butyricum-pMTL007-GLP-1通过PI3K/AKT/GSK-3β信号通路对A53T α-突触核蛋白小鼠模型的神经保护作用

[1]BohnenNI,RoytmanS,van der ZeeS,et al.,2025.A multicenter longitudinal study of cholinergic subgroups in Parkinson disease.Nat Commun,16:5655.

[2]ChangEES,HoPWL,LiuHF,et al.,2022.LRRK2 mutant knockin mouse models: therapeutic relevance in Parkinson’s disease.Transl Neurodegener,11:10.

[3]ChenTT,TianPY,HuangZX,et al.,2018.Engineered commensal bacteria prevent systemic inflammation-induced memory impairment and amyloidogenesis via producing GLP-1.Appl Microbiol Biotechnol,102(17):7565-7575.

[4]ChidambaramSB,EssaMM,RathipriyaAG,et al.,2022.Gut dysbiosis, defective autophagy and altered immune responses in neurodegenerative diseases: tales of a vicious cycle.Pharmacol Ther,231:107988.

[5]CredleJJ,GeorgeJL,WillsJ,et al.,2015.GSK-3β dysregulation contributes to Parkinson’s-like pathophysiology with associated region-specific phosphorylation and accumulation of Tau andα-synuclein.Cell Death Differ,22(5):838-851.

[6]CuiC,CuiNS,WangP,et al.,2016.Neuroprotective effect of sulfated polysaccharide isolated from sea cucumberStichopus japonicus on 6-OHDA-induced death in SH-SY5Y through inhibition of MAPK and NF-κB and activation of PI3K/Akt signaling pathways.Biochem Biophys Res Commun,470(2):375-383.

[7]FangX,ZhouXT,MiaoYQ,et al.,2020.Therapeutic effect of GLP-1 engineered strain on mice model of Alzheimer’s disease and Parkinson’s disease.AMB Express,10:80.

[8]GolpichM,AminiE,HemmatiF,et al.,2015.Glycogen synthase kinase-3 beta (GSK-3β) signaling: implications for Parkinson’s disease.Pharmacol Res,97:16-26.

[9]GowayedMA,El-SayedNS,MatarNA,et al.,2022.The α7 nAChR allosteric modulator PNU-120596 amends neuroinflammatory and motor consequences of parkinsonism in rats: role of JAK2/NF-κB/GSk3β/TNF-α pathway.Biomed Pharmacother,148:112776.

[10]GrahamDR,SidhuA,2010.Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior.J Neurosci Res,88(8):1777-1783.

[11]HigashiS,BiskupS,WestAB,et al.,2007.Localization of Parkinson’s disease-associated LRRK2 in normal and pathological human brain.Brain Res,1155:208-219.

[12]HölscherC,2018.Novel dual GLP-1/GIP receptor agonists show neuroprotective effects in Alzheimer’s and Parkinson’s disease models.Neuropharmacology,136:251-259.

[13]HolstJJ,2007.The physiology of glucagon-like peptide 1.Physiol Rev,87(4):1409-1439.

[14]KozikowskiAP,GaisinaIN,PetukhovPA,et al.,2006.Highly potent and specific GSK-3β inhibitors that block Tau phosphorylation and decrease α-synuclein protein expression in a cellular model of Parkinson’s disease.ChemMedChem,1(2):256-266.

[15]LaiF,JiangR,XieWJ,et al.,2018.Intestinal pathology and gut microbiota alterations in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease.Neurochem Res,43(10):1986-1999.

[16]LaiZL,TsengCH,HoHJ,et al.,2018.Fecal microbiota transplantation confers beneficial metabolic effects of diet and exercise on diet-induced obese mice.Sci Rep,8:15625.

[17]LeiP,AytonS,BushAI,et al.,2011.GSK-3 in neurodegenerative diseases.Int J Alzheimers Dis,2011:189246.

[18]LiangF,ChenCY,LiYP,et al.,2022.Early dysbiosis and dampened gut microbe oscillation precede motor dysfunction and neuropathology in animal models of Parkinson’s disease.J Parkinsons Dis,12(8):2423-2440.

[19]LinCH,ChenCC,ChiangHL,et al.,2019.Altered gut microbiota and inflammatory cytokine responses in patients with Parkinson’s disease.J Neuroinflammation,16:129.

[20]LiptakR,GromovaB,GardlikR,2021.Fecal microbiota transplantation as a tool for therapeutic modulation of non-gastrointestinal disorders.Front Med,8:665520.

[21]LiuML,FanGH,MengLK,et al.,2025.New perspectives on microbiome-dependent gut-brain pathways for the treatment of depression with gastrointestinal symptoms: from bench to bedside.J Zhejiang Univ-Sci B (Biomed & Biotechnol),26(1):1-25.

[22]LiuXR,LiuSY,TangY,et al.,2021.Intragastric administration of casein leads to nigrostriatal disease progressed accompanied with persistent nigrostriatal-intestinal inflammation activited and intestinal microbiota-metabolic disorders induced in MPTP mouse model of Parkinson’s disease.Neurochem Res,46(6):1514-1539.

[23]LorenzM,EversA,WagnerM,2013.Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity.Bioorg Med Chem Lett,23(14):4011-4018.

[24]LvLL,TanXL,PengXK,et al.,2020.The relationships of vitamin D, vitamin D receptor gene polymorphisms, and vitamin D supplementation with Parkinson’s disease.Transl Neurodegener,9:34.

[25]MalageladaC,JinZH,GreeneLA,2008.RTP801 is induced in Parkinson’s disease and mediates neuron death by inhibiting Akt phosphorylation/activation.J Neurosci,28(53):14363-14371.

[26]MengJR,MaX,WangN,et al.,2016.Activation of GLP-1 receptor promotes bone marrow stromal cell osteogenic differentiation through β-catenin.Stem Cell Rep,6(4):579-591.

[27]MosharovEV,LarsenKE,KanterE,et al.,2009.Interplay between cytosolic dopamine, calcium, and α-synuclein causes selective death of substantia nigra neurons.Neuron,62(2):218-229.

[28]MulvaneyCA,DuarteGS,HandleyJ,et al.,2020.GLP-1 receptor agonists for Parkinson’s disease.Cochrane Database Syst Rev,7(7):CD012990.

[29]ReichN,HölscherC,2022.The neuroprotective effects of glucagon-like peptide 1 in Alzheimer’s and Parkinson’s disease: an in-depth review.Front Neurosci,16:970925.

[30]RojoAI,InnamoratoNG,Martín-MorenoAM,et al.,2010.Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson’s disease.Glia,58(5):588-598.

[31]SharmaN,SoniR,SharmaM,et al.,2022.Chlorogenic acid: a polyphenol from coffee rendered neuroprotection against rotenone-induced Parkinson’s disease by GLP-1 secretion.Mol Neurobiol,59(11):6834-6856.

[32]SmidtMP,AsbreukCHJ,CoxJJ,et al.,2000.A second independent pathway for development of mesencephalic dopaminergic neurons requiresLmx1b.Nat Neurosci,3(4):337-341.

[33]SnigdhaS,HaK,TsaiP,et al.,2022.Probiotics: potential novel therapeutics for microbiota-gut-brain axis dysfunction across gender and lifespan.Pharmacol Ther,231:107978.

[34]StoevaMK,Garcia-SoJ,JusticeN,et al.,2021.Butyrate-producing human gut symbiont,Clostridium butyricum, and its role in health and disease.Gut Microbes,13(1):1907272.

[35]SuYF,LiuNN,ZhangZJ,et al.,2022.Cholecystokinin and glucagon-like peptide-1 analogues regulate intestinal tight junction, inflammation, dopaminergic neurons and α-synuclein accumulation in the colon of two Parkinson’s disease mouse models.Eur J Pharmacol,926:175029.

[36]SubbarayanMS,HudsonC,MossLD,et al.,2020.T cell infiltration and upregulation of MHC II in microglia leads to accelerated neuronal loss in an α-synuclein rat model of Parkinson’s disease.J Neuroinflammation,17:242.

[37]SunJ,LiHJ,JinYJ,et al.,2021.ProbioticClostridium butyricum ameliorated motor deficits in a mouse model of Parkinson’s disease via gut microbiota-GLP-1 pathway.Brain Behav Immun,91:703-715.

[38]TanseyMG,GoldbergMS,2010.Neuroinflammation in Parkinson’s disease: its role in neuronal death and implications for therapeutic intervention.Neurobiol Dis,37(3):510-518.

[39]TarsyD,2012.Treatment of Parkinson disease: a 64-year-old man with motor complications of advanced Parkinson disease.JAMA,307(21):2305-2314.

[40]TripodiF,LambiaseA,MoukhamH,et al.,2024.Targeting protein aggregation using a cocoa-bean shell extract to reduce α-synuclein toxicity in models of Parkinson’s disease.Curr Res Food Sci,9:100888.

[41]VaughanCP,MorleyJF,LehositJ,et al.,2025.Behavioral compared with drug therapy for overactive bladder symptoms in Parkinson disease: a randomized noninferiority trial.JAMA Neurol,14:e251904.

[42]WangQ,LuoYQ,Ray ChaudhuriK,et al.,2021.The role of gut dysbiosis in Parkinson’s disease: mechanistic insights and therapeutic options.Brain,144(9):2571-2593.

[43]WangQQ,LiuYJ,ZhouJW,2015.Neuroinflammation in Parkinson’s disease and its potential as therapeutic target.Transl Neurodegener,4:19.

[44]WangY,ChenS,XuZ,et al.,2018.GLP-1 receptor agonists downregulate aberrant GnT-III expression in Alzheimer’s disease models through the Akt/GSK-3β/β-catenin signaling.Neuropharmacology,131:190-199.

[45]WangY,ChenWJ,HanYY,et al.,2023.Neuroprotective effect of engineeredClostridiumbutyricum-pMTL007-GLP-1 on Parkinson’s disease mice models via promoting mitophagy.Bioeng Transl Med,8(3):e10505.

[46]WangZQ,ZhangY,ZhangS,et al.,2011.DJ-1 can inhibit microtubule associated protein 1 B formed aggregates.Mol Neurodegener,6:38.

[47]WuH,WeiJ,ZhaoXM,et al.,2023.Neuroprotective effects of an engineeredEscherichia coli Nissle 1917 on Parkinson’s disease in mice by delivering GLP-1 and modulating gut microbiota.Bioeng Transl Med,8(5):e10351.

[48]WuX,LiS,XueP,et al.,2018.Liraglutide inhibits the apoptosis of MC3T3-E1 cells induced by serum deprivation through cAMP/PKA/β-catenin and PI3K/AKT/GSK3β signaling pathways.Mol Cells,41(3):234-243.

[49]XuWX,QiYZ,GaoYJ,et al.,2021.Benzo(a)pyrene exposure in utero exacerbates Parkinson’s Disease (PD)-like α-synucleinopathy in A53T human alpha-synuclein transgenic mice.Toxicol Appl Pharmacol,427:115658.

[50]XuX,XuTC,WeiJ,et al.,2024.Gut microbiota: an ideal biomarker and intervention strategy for aging.Microbiome Res Rep,3(2):13.

[51]YanJQ,FuQZ,ChengLN,et al.,2014.Inflammatory response in Parkinson’s disease (Review).Mol Med Rep,10(5):2223-2233.

[52]YangJCS,WuSC,RauCS,et al.,2014.Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice.J Biomed Sci,21:20.

[53]YangJL,ChenWY,ChenYP,et al.,2016.Activation of GLP-1 receptor enhances neuronal base excision repair via PI3K-AKT-induced expression of apurinic/apyrimidinic endonuclease 1.Theranostics,6(12):2015-2027.

[54]YaoMY,ZhangJ,LiZH,et al.,2021.Liraglutide protects nucleus pulposus cells against high-glucose induced apoptosis by activating PI3K/Akt/mTOR/caspase-3 and PI3K/Akt/GSK3β/caspase-3 signaling pathways.Front Med,8:630962.

[55]ZengCY,ChenTT,ZhangY,et al.,2017.Hedgehog signaling pathway regulates ovarian cancer invasion and migration via adhesion molecule CD24.J Cancer,8(5):786-792.

[56]ZhangYC,BaileyTS,HittmeyerP,et al.,2024.Multiplex genetic manipulations inClostridium butyricum andClostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.Microb Cell Fact,23:119.

[57]ZhaoBT,XiaB,LiXH,et al.,2020.Sesamol supplementation attenuates DSS-induced colitis via mediating gut barrier integrity, inflammatory responses, and reshaping gut microbiome.J Agric Food Chem,68(39):10697-10708.

[58]ZommitiM,FeuilloleyMGJ,ConnilN,2020.Update of probiotics in human world: a nonstop source of benefactions till the end of time.Microorganisms,8(12):1907.