Role Of Helicobacter Pylori In Alzheimer’s Disease And The Underlying Mechanisms

Alzheimer’s disease (AD) is the most common type of dementia in clinical surveys, which affects more than35million people in the world. AD is characterized by the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles (NFTs) and neuronal degeneration within the afflicted brains. However, AD etiology has not been fully elucidated. Only5%of patients were found to have certain related disease genes,95%are sporadic, implying that environmental factors play a key role in AD pathogenesis.Helicobacter pylori(H.pylori) is a gram-negative bacterium infecting more than one half of the world’s population. Chronic H.pylori infection is accepted as the major cause of chronic gastritis, peptic ulcer, and gastric cancer. H.pylori has also been associated with extradigestive disorders, such as polyradiculoneuropathy, cardiovascular and/or cerebrovascular ischemia, hypertension, and stroke. Recently, a possible association between H.pylori infection and AD was disclosed by several clinical case-control or interventional studies, but the underlying mechanism is still unknown.In the present study, we aimed to investigate the role of H.pylori infection in tau phosphorylation, cognitive function and β-amyloid production, and to explore the underlying mechanisms. The main findings are as the following:PartⅠ Helicobacter pylori filtrate induces Alzheimer-like tau hyperphosphorylation by activating glycogen synthase kinase-3βAbnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative disorders including AD. H.pylori infection has been reported to be related with a high risk of AD, but the direct laboratory evidence is lacking.[Aim]:To explore the role H.pylori infection in tau phosphorylation, and disclose the underlying mechanisms. [Materials and Methods]:The experiments were performed on the animal and cell levels. For the concentration-dependent effect research, rats were intraperitoneally injected with middle or high concentrations of H.pylori or E.coli filtrate, respectively (280ul/rat/day, n=7for each group) for7days. For the long time effect investigation, rats were intraperitoneally injected with middle concentration of H.pylori filtrate for7,14or21days (280ul/rat/day, n=7for each group). Four of the animals in each group were sacrificed24hours after the final injection and the brains were isolated, half of the hippocampus was homogenized for Western blotting. The other hemisphere was homogenized for ELISA. Three of the rats in each group were used for immunohistochemistry. For the cell experiments, N2a cells were incubated with the prepared H.pylori or E.coli filtrate in low, middle or high concentrations for different time. In order to explore the role of GSK-3P in H.pylori-induced tau phosphorylation, cells were pre-incubated with GSK-3inhibitors SB216763(7μM) or LiCl (10mM) for30minutes, then treated with middle concentration of H.pylori filtrate for24hours. At the end of incubation, cells were harvested for Western blotting.[Results]:H.pylori filtrate induced tau hyperphosphorylation at several AD-related sites both in vitro and in vivo with a simultaneous activation of glycogen synthase kinase3β (GSK-3β), while inhibition of GSK-3could rescue H.pylori-induced tau hyperphosphorylation. Treatment of the cells or injection of the rats with H.pylori filtrate did not affect protein phosphatase-2A (PP2A). Escherichia coli (E.coli), another common intestinal bacterium, had no effect on tau phosphorylation.[Conclusion]:We demonstrate that H.pylori filtrate can induce Alzheimer-like tau hyperphosphorylation both in vitro and in vivo by activating GSK-3β in neuronal cells. Our data provide the first laboratory evidence to link H.pylori infection with AD. PartⅡ Helicobacter pylori filtrate impairs spatial learning and memory in rats and increases β-amyloid by enhancing expression of presenilin-2H.pylori infection is related with a high risk of Alzheimer’s Disease, previous study indicated that H.pylori filtrate induces Alzheimer-like tau hyperphosphorylation by activating glycogen synthase kinase-3β, but whether or not H.pylori has an effect on cognitive function and β-amyloid production is still unknown.[Aim]:To explore the effect of H.pylori infection on cognitive function and β-amyloid production, and to disclose the underlying mechanisms.[Materials and Methods]:For the animal experiments, three months old male Sprague Dawley rats (220±20g, Grade:SPF) were pre-trained in Morris water maze (MWM) to search a hidden platform under the water for7days. At the end of pre-training, all the rats could find the platform in15s. Then the rats were randomly divided into three experimental groups (n=9) and received intraperitoneal injection of H.pylori, E.coli filtrate or the same volume of DMEM/Opti-MEM medium (1:1) as control (280μ1/rat/day) for7days. On day4of injection the spatial memory of the rats in the MWM was measured. Then the rats were trained again in MWM for3days, with the platform placed in a new quadrant (re-learning). Spatial memory retention for the second learning was measured24hours later (day8, one day after the last injection). On day9, motor ability of the rats was tested in the MWM with a visible platform. At the end of behavior tests, the rats were sacrificed and the density and morphology of dendritic spines in the dentate gyrus of hippocampus were detected by using Golgi staining. The cell number and density in both the hippocampus and cortex in the rat brains were detected by using Nissl staining. The hippocampus and cortex was homogenized and membrane proteins of hippocampus were prepared, then the expression level of functional synaptic receptors,scaffolding proteins,p-secretase (BACE-1),γ-secretase (presenilin (PS)-1and PS-2) were detected by Western blotting. Aβ level of hippocampus and cortex were test by Sandwich enzyme linked immunosorbent assay (ELISA). For the cell experiments, N2a-APP cell were treated with H.pylori or E.coli filtrate for24hours, and then the levels of Aβ, β and y-secretase were detected.[Results]:Intraperitoneal injection of H.pylori filtrate induced spatial learning and memory deficit in rats with a simultaneous retarded dendritic spine maturation in hippocampus. Injection of H.pylori filtrate significantly increased A042both in the hippocampus and cortex, together with an increased level of PS-2. Incubation of H.pylori filtrate with N2a cells which over-express APP also resulted in increased PS-2expression and Aβ42overproduction. Injection of E.coli filtrate had no effect on cognitive function in rats and Aβ production in rats and cells.[Conclusion]:Soluble surface fractions of H.pylori may promote Aβ42formation by enhancing the activity of y-secretase, thus induce cognitive impairment through interrupting the synaptic function.