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Gulgun Tezel, Xiangjun Yang; In Vitro Analysis of the Effects of IκKβ Deletion on Glaucoma-Related Alterations in the Bioenergetic Profile of Astrocytes. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1209.
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© ARVO (1962-2015); The Authors (2016-present)
Our recent studies of experimental glaucoma showed that astroglia-targeted immunomodulation impedes the glaucoma-related metabolic shift of astroglia in addition to anti-inflammatory/neuroprotective outcomes. This study aimed to further explore bioenergetic responses of astrocytes in culture.
Retina and optic nerve astrocytes were derived from GFAP/IκKβ (crossbreds of IκKβ-f/f and GFAP-Cre/ERT2) and WT mice, and cultured with tamoxifen for conditional recombination. Transgenic effects on survival (by viability assay) and inflammatory phenotype (by glial morphology, GFAP labeling, and cytokine/chemokine profiles) were analyzed after 48-hour incubation with/without glaucoma-related stressors, including hypoxia or oxidative stress. Immunoblotting and immunocytochemistry analyzed a set of metabolic enzymes, and cellular glycogen content and glycolytic activity (extracellular acidification) were analyzed by specific assays.
WT astrocytes exposed to glaucoma-related stress exhibited a metabolic shift towards glycolysis, parallel to increased secretion of pro-inflammatory cytokines (>four-fold increase in IL-1, IL-2, IFN-γ, TNF-α; P<0.001). Increased glycolytic activity was characterized by >three-fold increase in expression of GLUT1 (involved in glucose uptake), hexokinase, pyruvate kinase (involved in catalysis of glycolysis), pyruvate dehydrogenase complex (involved in conversion of pyruvate to acetyl-CoA), and glycogen phosphorylase (involved in glycogenolysis). However, IκKβ-deleted astrocytes exhibited recovery of both the stress-induced pro-inflammatory phenotype and metabolic shift (~three-fold down-regulation of inflammatory and glycolytic activities; P<0.01). In addition, compared to WT, IκKβ-deleted astrocytes exhibited >two-fold increase in expression of glycogen synthase (involved in glycogenesis), LDHA (involved in conversion of glycolytically-derived pyruvate to lactate), and MCT1 (involved in monocarboxylate export). Also detectable was increased cellular glycogen content of transgenic astrocytes versus WT (P<0.05).
These in vitro findings, in support of recent in vivo observations, encourage astrocyte-targeted modulation of neuroinflammation (such as by transgenic inhibition of NF-κB as herein) to reduce neurodegeneration by recovering the glial metabolic support as well as decreasing the inflammatory neurotoxicity in glaucoma.
This is a 2020 ARVO Annual Meeting abstract.
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