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A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis.

ABSTRACT: A major impediment to CNS repair is the glial scar, which forms following damage and is composed mainly of ramified, 'reactive' astrocytes that inhibit neuronal regrowth. The transition of astrocytes into this reactive phenotype (reactive gliosis) is a potential therapeutic target, but glial scar formation has proved difficult to study in monolayer cultures because they induce constitutive astrocyte activation. Here we demonstrate a 3D collagen gel system in which primary rat astrocytes were maintained in a persistently less reactive state than comparable cells in monolayer, resembling their status in the undamaged CNS. Reactivity, proliferation and viability were monitored and quantified using confocal, fluorescence and time-lapse microscopy, 3D image analysis, RT-PCR and ELISA. To assess the potential of this system as a model of reactive gliosis, astrocytes in 3D were activated with TGFbeta1 to a ramified, reactive phenotype (elevated GFAP, Aquaporin 4, CSPG, Vimentin and IL-6 secretion). This provides a versatile system in which astrocytes can be maintained in a resting state, then be triggered to undergo reactive gliosis, enabling real-time monitoring and quantitative analysis throughout and providing a powerful new tool for research into CNS damage and repair.

SUBMITTER: East E

PROVIDER: S-EPMC2842570 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.

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A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis.

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