LPS and Conditioned Medium Enhance the Release of a Low Molecular Weight, Transcriptionally Active, Fragment of Glycogen Synthase-3 Kinase in IMR-32 Cell Line.

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

FDC381 and ATCC10301 conditioned media were collected. IMR-32 cells were challenged for 48 h with the conditioned media alongside (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 ( = 0.0005), while treated cells demonstrated 1.41-fold change ( = 0.004). Western blotting of the cells challenged with Pg.LPS ( = 0.01) and conditioned medium ( = 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 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  = 0.01) and conditioned medium (37 kDa band  = 0.001). Immunostaining demonstrated both cytoplasmic and nuclear localization of GSK-3β. Therefore, Pg.LPS and an unknown factor from the 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.