The role of glial fibrillary acidic protein (GFAP) in astrocyte and central nervous system (CNS) dysfunction
Publication Date
July 27, 2026
Creator
Lewis, Renee
Abstract
Astrocytes are a subtype of glial cells in the central nervous system (CNS), acting as key regulators of homeostasis, neuronal support, and signalling regulation, and maintaining blood–brain barrier (BBB) integrity. Astrocytes undergo astrogliosis in response to inflammatory stimuli, which involves undertaking a ‘reactive’ phenotype responsible for neurotoxic and neuroprotective functions that influence neuronal microenvironments.
Reactive astrocytes can be categorised into two forms; A1 (neurotoxic) or A2 (neuroprotective), where A1 astrocytes promote synaptic loss as well as neuronal and oligodendrocyte death, while A2 astrocytes provide neurotrophic support, synaptogenesis, and tissue repair. However, this binary classification is increasingly recognised as an oversimplification, as more recent research has indicated that astrocyte reactivity represents a spectrum of states, rather than two fixed phenotypes.
Notably, glial fibrillary acidic protein (GFAP), the hallmark cytoskeletal protein in astrocytes, is upregulated in reactive astrocytes, with increased expression observed in a variety of neurodegenerative diseases. One of these is Alexander Disease (AxD), a rare leukodystrophy caused by single-point mutations in the GFAP gene in astrocytes, leading to the accumulation of GFAP aggregates, known as Rosenthal fibres. These aggregates result in the dysregulation of astrocytic activity, causing neurodegeneration in patients; currently, no effective treatments exist. Notably, AxD is the only known genetic disorder affecting only astrocyte cells, providing a unique opportunity to investigate astrocyte-driven mechanisms of CNS dysfunction.
This project aimed to explore the effects of altered GFAP expression on astrocyte function and the broader impact this has on the CNS. In vitro models of human primary astrocytes were produced using lentiviral transduction to overexpress either wild-type (WT) GFAP (OVER-GFAP) or the AxD-associated R79H mutant variant (MUT-GFAP), alongside a GFP-control, where qualitative analysis demonstrated high levels of GFAP expression in both mutant and WT conditions; however, protein expression was not quantitatively measured and therefore could not be directly compared with in vivo levels. Using these models, astrocyte-conditioned media (ACM) were profiled via cytokine arrays and applied to endothelial cells, cortical neurons, and naïve astrocytes to assess how GFAP alterations influence astrocytic interactions and communication with the surrounding CNS, with cortical neurons used to evaluate astrocyte-derived trophic support, endothelial cells to assess astrocyte signalling at the Blood-brain barrier, and naïve astrocytes to examine astrocytic network signalling. This involved the optimisation of a calcium imaging assay to evaluate astrocytic responsivity to various neurotransmitters after ACM treatment, providing a functional readout of astrocyte signalling behaviour.
Significant differences were observed between ACM conditions effect on cortical neuronal viability and neurite outgrowth. MUT-ACM produced the greatest reduction in neuronal survival and axonal length, whereas OVER-ACM increased neuronal cell counts and partially restored neurite length relative to GFP-ACM, indicating that AxD-associated GFAP mutations disrupt astrocyte-derived neurotrophic support, while GFAP overexpression displayed restorative effects, indicating that GFAP overexpression can enhance the release of neurotrophic factors relative to GFP-control and MUT-GFAP astrocytes. While ACM did not significantly alter E-selectin expression in CRISPR-edited Human Umbilical Vein Endothelial Cells (HUVECs), indicating no significant endothelial inflammatory activation, cytokine profiling identified distinct differences in secretome profiles between control, OVER, and MUT conditions. Most notably, eight cytokines (ANGPT2, CD26, IFNG, IL-5, IL-17, KLK3, CSF1 and CCL19) were significantly altered, with all eight downregulated in MUT-ACM relative to OVER-ACM, and IL-5 and CSF1 additionally reduced compared to GFP-ACM. This pattern suggests a loss of neurotrophic and immune-modulatory support in MUT-GFAP astrocytes, whereas OVER-ACM showed a trend toward increased expression of the same mediators.
Calcium imaging further demonstrated reduced astrocyte responsivity following exposure to MUT-ACM, specifically showing a reduction in histamine-evoked Ca²⁺ response intensity, while ATP-evoked responses remained unaffected. The efficacy of this optimised calcium imaging assay was reinforced by the detection of clear differences in calcium signalling between reactive-like and quiescent-like astrocytes, including increased proportions of responding cells and enhanced glutamate-evoked Ca²⁺ responses in reactive-like astrocytes, highlighting the heterogeneity of calcium signalling dynamics depending on astrocyte phenotype changes.
Overall, these findings indicate that AxD pathology extends beyond a toxic gain-of-function and instead involves a loss of essential astrocyte support throughout the CNS, underscored by a breakdown of astrocytic communication. Notably, GFAP mutations and WT-GFAP upregulation produced markedly different functional outcomes, highlighting the need to distinguish between these mechanisms when investigating AxD pathology. More broadly, by integrating molecular, cellular, and functional analyses this project reinforces the central role of astrocytes in maintaining neuronal microenvironments and identifies how astrocyte dysfunction and impaired astrocyte signalling are key contributors to CNS dysfunction, highlighting restoration of astrocyte homeostatic support as a promising therapeutic strategy for neurodegenerative disease.
Item Type
ethesis
Thesis Type
MRes
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Subjects (LC)
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