Helicobacter Pylori Outer Membrane Vesicles Induce AD-like Tau Pathology

Alzheimer’s disease(AD)is the most common neurodegenerative disease,with progressive memory and cognitive dysfunction as the main clinical symptoms.One of the characteristic pathological changes of AD is neurofibrillary tangles(NFTs).The hyperphosphorylated microtubule-associated protein tau is the major component of NFTs.The etiology of AD is not yet clear.99% of clinical AD is sporadic,suggesting that the occurrence of AD is closely related to environmental factors.Helicobacter pylori(H.pylori)is a Gram-negative bacterium,which has a high infection rate in the population and is the main pathogenic bacteria of gastritis and gastric ulcer.Clinical epidemiological investigations in recent years have shown that H.pylori infection is associated with AD.Our previous study also showed that H.pylori filtrate can induce hyperphosphorylation of tau in vivo and promote the AD pathogenesis.However,taking into account the fact that H.pylori infection in the population is mainly oral infection and through bacteria colonization in the stomach,it is suspected whether chronic colonization of H.pylori can cause AD-like lesions through the intestinal brain axis or other pathways.And if it is so,which components of H.pylori filtrate are mainly involved in the induction of AD-like lesions remains unknown.On one hand,we considered the main pathogenic factors of H.pylori,such as cytotoxin associated protein(CagA)and urease(Ure)et al to be the possible upstream etiologic factors.In the experiment we detected Ure in both the H.pylori lysate and filtrate by immunoblot,and observed a higher level of Ure in the H.pylori lysate than that in filtrate.Thus,it was asked whether H.pylori lysate can also induce AD-like lesions as in the case of filtrate.At last,H.pylori can secrete outer membrane vesicles(OMVs).OMVs contain a large number of bacterial components and participate in the transmission of pathogenic factors to distant organs or tissues.In the experiment we found that OMVs can be isolated from the H.pylori filtrate,suggesting that OMVs might be involved in the induction of AD-like lesions.【Aims】 1.To study the effects of long-term colonization of H.pylori on learning and memory and tau phosphorylation in rats.2.To study the effect of H.pylori lysate on the phosphorylation of tau.3.To study the effect of H.pylori OMVs on the phosphorylation of tau.【Methods】 1.Healthy female SD rats with no significant difference in body weight were subjected to intragastric administration of H.pylori bacteria to establish a long-term gastric colonization model of H.pylori.Colonization of the bacteria in the stomach was detected by PCR and Gram staining.Open-field and water maze tests were used to evaluate memory and learning ability.Western blotting was used to detect the phosphorylation level of tau at different sites in brain homogenates.2.Healthy male SD rats with no significant difference in body weight were injected intraperitoneally with H.pylori lysate or filtrate,then brain tissues were collected,homogenized and used for Western blotting to detect the phosphorylation level of tau at different sites.3.OMVs were separated and purified from H.pylori filtrate.Healthy female SD rats with no significant difference in body weight was injected with the purified OMVs through the tail vein.Brain tissue protein was extracted for Western blotting to detect the tau phosphorylation at different sites.【Results】 1.16S-rRNA and caga were detected in stomach tissue from the H.pylori group but not in the untreated control group.Gram-stained red short rod-shaped bacteria were obseved in the gastric mucosa of the H.pylori group.These results suggested that the establishment of gastric H.pylori colonization model in SD rats was successful.2.In open-field trials,no difference was observed in motor ability between the two groups of rats.The water maze experiment showed no significant difference in latency between the two groups during the training.In the memory test,there was also no significant difference in the number of crossing times or the latency between the two groups.3.Tau phosphorylation levels in hippocampal and cortical tissue were detected in the Con and the H.pylori groups by Western blotting.No significant differences in tau phosphorylation at the detected sites such as Thr205,Thr231,Ser396,and Ser404 were observed between the two groups.4.Tau phosphorylation levels in hippocampal tissue were detected in the H.pylori lysate or filtrate-injected rats.Intraperitoneal injection of H.pylori lysate did not induce significant changes in tau phosphorylation in the rat brain;Intraperitoneal injection of H.pylori filtrate induced significantly(P<0.05)increased tau phosphorylation level compared to the Con group.5.OMVs were purified and observed in the filtrate of H.pylori and E.coli.Intraperitoneal injection of H.pylori but not E.coli OMVs induced tau hyperphosphorylation at Ser396 and Ser404(P<0.05).【Conclusion】 1.H.pylori long-term stomach colonization rat model does not show pathogeneis in the brain,thus is not suitable for studying the role of H.pylori infection in brain disorders.2.Ure B is not the principle etiologic factor inducing tauopathology in H.pylori.filtrate-injected rats.3.H.pylori OMVs induce tau hyperphosphorylation in the brain.