Porphyromonas gingivalis LPS and Actinomyces naeslundii conditioned medium enhance the release of a low molecular weight, transcriptionally active, fragment of glycogen synthase-3 kinase in IMR-32 cell line

Abstract

Background: Glycogen synthase-3 kinase (GSK3) is one of the major contributors of tau hyperphosphorylation linked to neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD).
Objectives: To determine a mechanism of GSK-3β activation by two periodontal bacteria consistently confirmed in AD autopsied brains.
Methods: Porphyromonas. gingivalis FDC381 and Actinomyces naeslundii ATCC10301 (designated An) conditioned media were collected. IMR-32 cells were challenged for 48h with the conditioned media alongside P. gingivalis (ATCC33277) ultrapurified lipopolysaccharide (LPS) designated Pg.LPS under established cell culture conditions either, alone or combined. Gene expression, and protein analyses for GSK-3β were carried out.
Results: qPCR demonstrated that GSK-3β gene was over-expressed in IMR-32 cells treated with Pg.LPS with a 2.09 fold change (p=0.0005) whilst An treated cells demonstrated 1.41 fold change (p=0.004). Western blotting of the cells challenged with Pg.LPS (p=0.01) and An conditioned medium (p=0.001) demonstrated the 37 kDa band for each treatment with variable intensity across the medium control. Immunohistochemistry with the GSK-3β of the IMR-32 cells challenged with Pg.LPS and An alone demonstrated cytoplasmic and nuclear localisation.
Conclusions: Exposure to various bacterial factors up-regulated the gene expression of GSK-3β. Western blotting for GSK-3β confimed the presence of the cleaved fragment by Pg.LPS (37 kDa band p=0.01) and An conditioned medium (37 kDa band p=0.001). Immunostaining demonstrated both cytoplasmic and nuclear localisation of GSK-3β. Therefore, Pg.LPS and an unknown factor from the An conditioned medium mediated GSK-3β activation via its transcriptionally active, cleaved, fragment. These virulence factors in the body appear to be detrimental to brain health.