Porphyromonas gingivalis Conditioned Medium Induces Amyloidogenic Processing of the Amyloid-β Protein Precursor upon in vitro Infection of SH-SY5Y Cells

Shalini Kanagasingam; Christopher von Ruhland; Richard Welbury; Sasanka S Chukkapalli; Sim K Singhrao

doi:10.3233/ADR-220029

Porphyromonas gingivalis Conditioned Medium Induces Amyloidogenic Processing of the Amyloid-β Protein Precursor upon in vitro Infection of SH-SY5Y Cells

J Alzheimers Dis Rep. 2022 Sep 20;6(1):577-587. doi: 10.3233/ADR-220029. eCollection 2022.

Authors

Shalini Kanagasingam¹, Christopher von Ruhland², Richard Welbury¹, Sasanka S Chukkapalli³, Sim K Singhrao¹

Affiliations

¹ Brain and Behavior Centre, Faculty of Clinical and Biomedical Sciences, School of Dentistry, University of Central Lancashire, Preston, UK.
² Electron and Light Microscopy Facility, College of Biomedical and Life Sciences, Cardiff University, Wales, UK.
³ Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA.

Abstract

Background: Cleavage of the amyloid-β protein precursor (AβPP) mediated by host secretase enzymes, releases several fragments including amyloid-β (Aβ₄₀ and Aβ₄₂).

Objective: To determine if Porphyromonas gingivalis conditioned medium cleaved AβPP to release Aβ₄₀ and Aβ₄₂.

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 AβPP cleavage fragments by immunoblotting and transmission electron microscopy.

Results: Western blotting of the cell lysates with the anti-AβPP C-terminal antibody demonstrated variable molecular weight bands corresponding to full length and fragmented AβPP 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 Aβ₄₀ and Aβ₄₂, levels when these two Aβ species were pooled across test samples and compared to the untreated group. Electron microscopic examination of the supernatants demonstrated insoluble Aβ₄₀ and Aβ₄₂.

Conclusion: These observations strongly imply that AβPP is an infection responsive protein cleaved via the amyloidogenic pathway on exposure to conditioned medium and in the presence of pro-inflammatory mediators.

Keywords: Amyloid-β; Porphyromonas gingivalis conditioned medium; amyloid-βprotein precursor.