Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo

Abstract

Alzheimer's disease (AD) is considered the most common neurodegenerative disorder in people over the age of 60, characterized by dementia combined with memory deficit. Its characteristic histopathological changes in the patients’ brain are the formation of senile plaques - deposits of beta-amyloid peptide (Aβ) - and neurofibrillary tangles constituted by the deposition of hyperphosphorylated tau protein. The function of the tau protein, normally associated with microtubules in neuronal axons, is regulated by phosphorylation and dephosphorylation mechanisms, with more than 80 reported phosphorylation sites. The literature reported that the Helicobacter pylori culture’s filtrate, obtained without causing lysis of the bacteria, induces hyperphosphorylation of the tau protein at different sites, in vivo and in vitro. As there was no significant cerebral inflammatory response, a hypothesis was raised that exotoxins produced by the bacteria, capable of penetrating the blood-brain barrier (BBB), would directly induce tau phosphorylation. The gram-negative bacterium Helicobacter pylori is a gastric pathogen responsible for chronic gastritis, peptic and duodenal ulcers, and gastric cancer, infecting approximately 60% of the world population. This bacterium produces large amounts of urease (HPU), an enzyme considered as an important virulence factor of the bacterium. Our group has previously demonstrated the ability of HPU to induce the activation of human neutrophils, protecting them against apoptosis, and contributing to the promotion of tissue damage. The present work sought to investigate possible changes promoted by HPU in phosphorylation sites of tau protein in vivo and possible pro-inflammatory mechanisms in vitro on cells of the nervous system. In in vivo tests, male 30-days Wistar rats received an intraperitoneal dose of 5 g of this protein daily, for 7 days, then euthanized on the eighth day, and had their brains stored at -80 ºC for further analysis. In the control group, only the vehicle (sterile saline) was administered. The treatment regimen was designed to mimic a chronic H. pylori infection in young individuals. Western blotting assays were performed, using anti-Tau5, anti-pTau396, anti-pTau199 and anti-pTau205 antibodies, specific for the identification of tissue total tau protein and phosphorylated tau at Ser396, Ser199 and Thr205 sites, respectively. The levels of GSK-3β and NFκβ-p65 and the presence of HPU in brain tissue were also evaluated by Western blotting. The results indicated that HPU significantly increased tau phosphorylation at Thr205, Ser396 and Ser199 sites, however there was no detection of HPU in brain tissue, suggesting that HPU may interfere in tau phosphorylation indirectly. After in vivo treatment, HPU did not alter or decrease the GSK-3β total protein content in brain tissue. However, it increased the levels of NFκβp65 in brain tissue, indicating neuroinflammation. Such findings reinforced an association between H. pylori infection and Alzheimer's disease tauopathy, as indicated in epidemiological studies, and suggest a contribution of HPU to the pathogenesis of this neurodegenerative disease.