| Background:Alzheimer’s disease（AD）,the most prevalent type of dementia among the elderly,has become one of the major social and economic burdens worldwide.Over the past three decades,there has been constant postulation regarding the infectious etiology of Alzheimer disease.The“microbial hypothesis”suggests that chronic infection with viral,bacterial,and/or fungal pathogens may promote AD development.Recently,Helicobacter pylori（H.pylori）infection has been found associated with AD.However,the molecular mechanism by which H.pylori induced AD-like pathology is still unknown.Previous studies have reported that the filtrates of cultured H.pylori could induce AD-like pathologies and cognitive deficit in rats.However,the pathogenic factors in the filtrates and the underlying pathogenesis remain unclarified.Outer membrane vesicles（OMVs）are spherical,bilayered,membranous structures with an average diameter of 20–250 nm,which are secreted by a wide variety of gram-negative bacteria during all stages of bacterial growth.With bacterial-originated lipids,proteins,lipopolysaccharides（LPS）,DNA,RNA,metabolites and lots of signaling molecules equipped,OMVs exert several biological functions such as cell-to-cell signal transduction,toxins transferring and immune response elicitation in host cells.Whether the OMVs are the pathogenic factors of H.pylori in promoting AD-like pathologies,and the underlying mechanisms,are worthy of further exploration.Objective:To explore the effects of intraperitoneally injected H.pylori OMVs on AD-related pathologies in APP/PS1 mice and wild type mice,and to disclose the underlying mechanisms.Methods:H.pylori were cultured at 37℃in a microaerophilic environment for 72 h,E.coli were cultured at 37°C in an aerobic environment for 24 h.OMVs were prepared by ultracentrifugation.Transmission electron microscopy（TEM）was used to determine the quality of the OMVs preparation.OMVs were fluorescently-labeled with lipophilic fluorescent dye Di D and administrated intraperitoneally into APP/PS1 mice and wild type mice for monitoring their entry into the brain.To identify the entry of exogenous OMVs into the mouse brain,we visualized brain vascular endothelial cells using fluorescein isothiocyanate（FITC）following injection of Di D-labeled OMVs.Learning and memory function of mice was determined by Morris water maze and contextual discrimination test.The amyloid burden in mice brains was measured by staining with Thioflavin-S（THS）and ELISA.Levels of Tau phosphorylation were determined by Western blotting.Neuroinflammation was measured by immunostaining for Iba-1 and ELISA.Synaptic impairment was evaluated by Western blotting and Golgi staining,the number of neurons was detected by Nissl staining.To explore the effects of H.pylori OMVs on Aβaggregation and neurocytotoxity in vitro,Aβmonomers were incubated alone or with OMVs.Aβaggregation was measured by Thioflavin-T（THT）fluorescence assay,and end-point incubation products were visualized by TEM.Different Aβaggregates were applied to primary neurons,the neurotoxicity was determined by LDH cytotoxicity assay.Synaptic impairment was measured by Western blotting and immunostaining.Ultra-high performance liquid chromatography-tandem mass spectrometry（UPLCMS/MS）was performed to characterize the lipid profile in OMVs.The intracellular calcium level of neurons was measured by intracellular calcium imaging.Results:Di D-labeled H.pylori OMVs and E.coli OMVs can be detected in the hippocampus after intraperitoneal injection to mice,indicating that the OMVs entered the brain.The fluorescence results showed co-localization of cerebral vascular with some OMVs,indicating that the OMVs can be taken by brain vascular endothelial cells to enter the brain.Using the dendritic marker MAP-2 to identify neurons,we found that Di D-labeled H.pylori OMVs or E.coli OMVs co-localized with neuronal cells in the hippocampus.We further used Aβantibody 4G8 to probe amyloid plaques and found that OMVs were mostly co-localized with amyloid plaques in the hippocampus of APP/PS1 mice.H.pylori OMVs significantly increased amyloid burden without influencing APP phosphorylation and cleavage,induced Tau hyperphosphorylation,enhanced neuroinflammation and system inflammation,increased synaptic impairment and exacerbated cognitive deficits in APP/PS1 mice,indicating H.pylori OMVs promoted AD pathologies.Besides,H.pylori OMVs also induced Tau hyperphosphorylation and neuroinflammation,but did not induce cognitive dysfunction and synaptic impairments in wild type mice.Therefore,we proposed that the severer cognitive deficits and synaptic impairments in H.pylori OMVs-injected APP/PS1 mice were mostly related to enhanced Aβaccumulation in brain.To identify this hypothesis,Aβmonomers were incubated alone or with H.pylori OMVs/E.coli OMVs to explore the effects of OMVs on Aβaggregation and neurotoxicity in vitro.THT fluorescence assay showed that H.pylori OMVs increased Aβ-induced THT fluorescence intensity compared with Aβalone or E.coli OMVs in a dose-dependent manner.TEM showed that Aβmonomers incubated alone or with E.coli OMVs mostly assembled into aggregates and little fibrils at the end of incubation,however a large amount of amyloid fibrils were observed when Aβmonomers were incubated with H.pylori OMVs.We next examined the effects of H.pylori OMVs on Aβ-induced neurotoxicity.Aβmonomers were pre-incubated alone or with H.pylori OMVs/E.coli OMVs to form different Aβaggregates,then primary neurons were treated with the incubation mixture.LDH cytotoxicity assay showed H.pylori OMVs-induced Aβaggregates showed significantly increased neurotoxicity.Using the neuronal marker MAP-2 to show the neuronal morphology,we found that H.pylori OMVs-induced Aβaggregates dramatically damaged neuronal dendrites.Western blotting showed the level of post-synaptic protein PSD95 was dramatically decreased in neurons treated with H.pylori OMVs-induced Aβaggregates.These results indicated that H.pylori OMVs promoted Aβaggregation and enhance Aβ-induced neurotoxicity in vitro.Since H.pylori OMVs consist of protein,RNA,DNA and lipid,to identify which components in H.pylori OMVs are involved in enhancing Aβneurotoxicity,H.pylori OMVs were given different treatments to remove the above components respectively before being incubated with Aβ.Western blotting showed that only pretreatment with silica to remove lipid rescued Aβ-OMVs induced synaptic toxicity,suggesting that H.pylori OMVs-enhanced Aβneurotoxicity was most likely attributed to lipid components.Furthermore,ultra-high performance liquid chromatography-tandem mass spectrometry（UPLCMS/MS）was performed to characterize the lipid profile in OMVs based on the absolute quantitative lipid technology,which showed that the content of lysophosphatidylcholine（LPC）in H.pylori OMVs was significantly higher than that in E.coli OMVs.Last,calcium imaging showed that H.pylori OMVs-induced Aβaggregates significantly increased intracellular calcium level in neurons.In addition,when extracellular Ca2+was removed,or cells were preincubated with a blocker of Aβaggregates-formed calcium channels,decrease of synaptic protein PSD95 in neurons treated with H.pylori OMVs-induced Aβaggregates was also reversed.These data collectively indicated that H.pylori OMVs enhanced Aβneurotoxicity through promoting Aβaggregation,thus increasing the Ca2+influx through the Aβaggregates-formed calcium channels.Conclusions:We conclude that bacterial H.pylori OMVs have the ability to enter the brain,promote Aβaggregation and amyloidosis.Mechanically,H.pylori OMVs enhance Aβneurotoxicity through the lipid interaction with Aβand in a Ca2+-dependent manner,thus exacerbating neuronal damage and cognitive deficits in AD mice. |
