S. Singhrao, Claudia Consoli, Sarah R. Dennison, S. Kanagasingam, R. Welbury
{"title":"牙龈卟啉菌 LPS 和奈氏放线菌条件培养基可增强 IMR-32 细胞系中糖原合成酶-3 激酶片段的低分子量、转录活性的释放","authors":"S. Singhrao, Claudia Consoli, Sarah R. Dennison, S. Kanagasingam, R. Welbury","doi":"10.3233/adr-240066","DOIUrl":null,"url":null,"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.","PeriodicalId":73594,"journal":{"name":"Journal of Alzheimer's disease reports","volume":null,"pages":null},"PeriodicalIF":2.8000,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"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\",\"authors\":\"S. Singhrao, Claudia Consoli, Sarah R. Dennison, S. Kanagasingam, R. Welbury\",\"doi\":\"10.3233/adr-240066\",\"DOIUrl\":null,\"url\":null,\"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.\",\"PeriodicalId\":73594,\"journal\":{\"name\":\"Journal of Alzheimer's disease reports\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.8000,\"publicationDate\":\"2024-07-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Alzheimer's disease reports\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3233/adr-240066\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"NEUROSCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Alzheimer's disease reports","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3233/adr-240066","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"NEUROSCIENCES","Score":null,"Total":0}
引用次数: 0
摘要
背景:糖原合成酶-3 激酶(GSK3)是导致与阿尔茨海默病(AD)神经纤维缠结相关的 tau 过度磷酸化的主要因素之一。研究目的确定两种牙周细菌激活 GSK-3β 的机制,这两种细菌在阿兹海默症(AD)尸检大脑中得到了一致证实。方法:收集牙龈卟啉菌 FDC381 和奈氏放线菌 ATCC10301 条件培养基。在既定的细胞培养条件下,将条件培养基与牙龈卟啉菌(ATCC33277)超纯化脂多糖(LPS)(命名为 Pg.LPS)单独或混合使用,对 IMR-32 细胞进行 48 小时的挑战。对 GSK-3β 进行了基因表达和蛋白质分析。结果:qPCR 显示,经 Pg.LPS 处理的 IMR-32 细胞中 GSK-3β 基因过表达 2.09 倍(p = 0.0005),而经 A. naeslundii 处理的细胞中 GSK-3β 基因过表达 1.41 倍(p = 0.004)。对接受 Pg.LPS (p = 0.01)和 A. naeslundii 条件培养基(p = 0.001)处理的细胞进行 Western 印迹,结果显示每种处理都有 37 kDa 的条带,不同培养基对照的条带强度不同。对单独使用 Pg.LPS 和 A. naeslundii 的 IMR-32 细胞进行的 GSK-3β 免疫组织化学显示了细胞质和细胞核定位。结论暴露于各种细菌因子会上调 GSK-3β 的基因表达。针对 GSK-3β 的 Western 印迹证实,Pg.LPS(37 kDa 带 p = 0.01)和 A. naeslundii 条件培养基(37 kDa 带 p = 0.001)裂解片段的存在。免疫染色显示了 GSK-3β 在细胞质和细胞核中的定位。因此,Pg.LPS 和来自 A. naeslundii 条件培养基的未知因子通过其具有转录活性的裂解片段介导 GSK-3β 激活。体内的这些毒力因子似乎不利于大脑健康。
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
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.