Singhrao Sim K, Consoli Claudia, Dennison Sarah R, Kanagasingam Shalini, Welbury Richard
School of Medicine and Dentistry, University of Central Lancashire, Preston, UK.
Central Biotechnology Services, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK.
J Alzheimers Dis Rep. 2024 Jul 18;8(1):1055-1067. doi: 10.3233/ADR-240066. eCollection 2024.
Glycogen synthase-3 kinase (GSK3) is one of the major contributors of tau hyperphosphorylation linked to neurofibrillary tangles in Alzheimer's disease (AD).
To determine a mechanism of GSK-3β activation by two periodontal bacteria consistently confirmed in AD autopsied brains.
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.
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.
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.
糖原合酶-3激酶（GSK3）是与阿尔茨海默病（AD）神经原纤维缠结相关的tau蛋白过度磷酸化的主要促成因素之一。
确定两种在AD尸检大脑中一直被证实的牙周细菌激活GSK-3β的机制。
收集FDC381和ATCC10301的条件培养基。在既定的细胞培养条件下，将IMR-32细胞分别用条件培养基单独处理或与超纯化脂多糖（LPS）（指定为Pg.LPS）联合处理48小时。对GSK-3β进行基因表达和蛋白质分析。
qPCR显示，用Pg.LPS处理的IMR-32细胞中GSK-3β基因过度表达，变化倍数为2.09倍（P = 0.0005），而用处理的细胞变化倍数为1.41倍（P = 0.004）。用Pg.LPS（P = 0.01）和条件培养基（P = 0.001）处理的细胞进行蛋白质免疫印迹显示，每种处理在培养基对照中均出现37 kDa条带，强度各异。用Pg.LPS和单独处理的IMR-32细胞的GSK-3β进行免疫组织化学显示，其定位于细胞质和细胞核。
暴露于各种细菌因子会上调GSK-3β的基因表达。GSK-3β的蛋白质免疫印迹证实了Pg.LPS（37 kDa条带，P = 0.01）和条件培养基（37 kDa条带，P = 0.001）存在切割片段。免疫染色显示GSK-3β定位于细胞质和细胞核。因此，Pg.LPS和来自条件培养基的未知因子通过其转录活性切割片段介导GSK-3β的激活。体内的这些毒力因子似乎对大脑健康有害。
