Abstract

BACKGROUND: Alzheimer’s disease (AD) is a neurodegenerative disease clinically presenting with deterioration in memory and, neuropathologically by the presence of insoluble amyloid-beta (Aβ) plaques and phosphorylated tau (p-Tau) binding neurofibrillary tangles (NFTs).

AIM: This study was aimed at investigating the complex interaction between periodontitis, its pathogens, especially Porphyromonas gingivalis and mechanisms of AD hallmark lesion pathogenesis.

METHODOLOGY: The experimental protocols used include light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), immunohistochemical staining, in vitro cell culture using SH-SY5Y cell line challenged with P. gingivalis (Pg381) conditioned medium in the presence and absence of cytokines and immunoblotting. An obese, diabetic (db/db) mouse model inoculated with P. gingivalis and Fusobacterium nucleatum were assessed via light microscopy and immunostaining to assess for neuroinflammation. Antibacterial assays for P. gingivalis were performed to assess if gingipains-fragmented tau peptides displayed antimicrobial properties. Circular dichroism (CD) was undertaken to assess tau peptide secondary structure. Paired helical/straight filament (PHF/SF) formation with implications for the NFT lesion was evaluated using TEM and polarising light microscopy.

RESULTS: SEM demonstrated a polymicrobial biofilm with extracellular polymeric substance (EPS) and water channels within the calculus. Immunohistochemistry on rehydrated paraffin wax embedded calculus and diseased tooth sections confirmed the presence of Aβ TEM revealed electron dense fibrils of variable sizes, similar to human Aβ fibrils. Western blotting of the cell lysates with selected APP antibodies demonstrated variable molecular weight bands corresponding to full length APP, C99 and C83 dimerized fragments. Anti-Aβ antibody detected statistically significant increase in Aβ40/42 levels in experimental groups with P. gingivalis and cytokines. TEM examination of supernatants demonstrated insoluble Aβ40 and Aβ42 fibrils. No Aβ plaques were detected in the db/db mice, however neuroinflammation was confirmed by immunohistochemistry in the infected groups. Phosphorylated tau peptide A showed significant antimicrobial activity against planktonic P. gingivalis. The CD results demonstrated that phosphorylated and non-phophorylated tau peptide A adopted mainly-β pleated sheet structures. The peptides investigated appeared fibrillar, with helical twists under TEM. Tau peptides were birefringent.

CONCLUSIONS: This study detected both soluble and insoluble Aβ fibrils within the EPS of periodontal and endodontic natural biofilm, strongly suggesting its role as a host antimicrobial peptide. However, its potential risk for cross-seeding into the brain may have implications to AD development. P. gingivalis infection in the presence of pro-inflammatory cytokines elevated the production of Aβ. Periodontitis and comorbidities of obesity and diabetes can instigate neuroinflammation. Phosphorylation of tau peptides physically changed their tertiary structure into PHFs with potential for self-aggregation and binding to the NFT lesion. These findings support the bidirectional relationship between periodontal disease and AD.