Porphyromonas gingivalis conditioned medium induces amyloidogenic processing of the amyloid -β precursor protein upon in vitro infection of SH-SY5Y cells

Abstract

Background: Cleavage of the amyloid precursor protein (APP) mediated by host secretase enzymes, releases several fragments including amyloid-beta (Aβ1-40/42).
Objective: To determine if Porphyromonas gingivalis conditioned medium cleaved APP to release Aβ1-40/42.
Methods: The SH-SY5Y cell line was challenged, in vitro, with P. gingivalis (Pg381) conditioned medium in the presence/absence of cytokines. The cells and their supernatants were assessed for APP cleavage fragments by immunoblotting and transmission electron microscopy.
Results: Western blotting of the cell lysates with the anti-APP C-terminal antibody demonstrated variable molecular weight bands corresponding to full length and fragmented APP in lanes treated with the following factors: Tryptic soy broth (TSB), Pg381, IL-6, Pg381+IL-1β and Pg381+TNF-α. The low molecular weight bands corresponding to the C99 dimerized fragment were observed in the Pg381 and interlukin-6 (IL-6) treated groups and were significantly more intense in the presence of Pg381 with either IL-6 or TNF-α. Bands corresponding to the dimerized C83 fragment were observed with cells treated with TNF-α alone and with Pg381 combined with IL-1β or IL-6 or TNF-α. The anti-Aβ antibody detected statistically significant Aβ1-40/42, levels when Aβ1-40/42 species were pooled across test samples and compared to the untreated group. Electron microscopic examination of the supernatants demonstrated insoluble Aβ40/42.
Conclusion: These observations strongly imply that APP is an infection responsive protein cleaved via the amyloidogenic pathway on exposure to conditioned medium and in the presence of pro-inflammatory mediators.