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

Singhrao, Sim K., Consoli, Claudia, Dennison, Sarah R., Kanagasingam, Shalini and Welbury, Richard 2024. 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. Journal of Alzheimer's Disease Reports 8 (1) , pp. 1055-1067. 10.3233/adr-240066

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Abstract

Background: Glycogen synthase-3 kinase (GSK3) is one of the major contributors of tau hyperphosphorylation linked to neurofibrillary tangles in Alzheimer’s disease (AD). Objective: 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 conditioned media were collected. IMR-32 cells were challenged for 48 h 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 overexpressed in IMR-32 cells treated with Pg.LPS with a 2.09-fold change (p = 0.0005), while A. naeslundii treated cells demonstrated 1.41-fold change (p = 0.004). Western blotting of the cells challenged with Pg.LPS (p = 0.01) and A. naeslundii 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 A. naeslundii alone demonstrated cytoplasmic and nuclear localization. Conclusions: Exposure to various bacterial factors upregulated the gene expression of GSK-3β. Western blotting for GSK-3β confirmed the presence of the cleaved fragment by Pg.LPS (37 kDa band p = 0.01) and A. naeslundii conditioned medium (37 kDa band p = 0.001). Immunostaining demonstrated both cytoplasmic and nuclear localization of GSK-3β. Therefore, Pg.LPS and an unknown factor from the A. naeslundii 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.

Item Type:	Article
Date Type:	Published Online
Status:	Published
Schools:	Medicine
Publisher:	IOS Press
ISSN:	2542-4823
Date of First Compliant Deposit:	19 November 2024
Date of Acceptance:	19 June 2024
Last Modified:	19 Nov 2024 11:00
URI:	https://orca.cardiff.ac.uk/id/eprint/174138

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