Franklin, Hannah Daisy; (2023) Investigating the role of hypoxic stress in determining astrocyte function in health and ALS. Doctoral thesis (Ph.D), UCL (University College London).

Text Hannah Franklin Post-Viva Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 January 2025. Download (10MB)

Abstract

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease, with emerging evidence highlighting the pivotal role of astrocytes in its pathogenesis. While hypoxia is considered a hallmark of ALS, the underlying mechanisms are poorly understood. Using hiPSC-derived astrocytes and motor neurons from control (CTRL) and VCP-mutant (VCPMUT) ALS patients, this thesis investigates the impact of hypoxic stress on astrocyte function in ALS. Central to my findings is the alteration of hypoxia-inducible factor 1-alpha (HIF-1ɑ) function in VCPMUT astrocytes, both under basal conditions and upon exposure to hypoxia. This phenotype, indicative of hypoxic stress, is paired with multiple dysfunctional traits shared with CTRL astrocytes exposed to hypoxia, including heightened susceptibility to HIF-1ɑ-dependent glutamate uptake reduction and ROS accumulation, mitochondrial dysfunction, lipid droplet accumulation, and metabolic alterations. This hypoxic stress state marks the first functional characterisation of basal hypoxia activation in a human ALS astrocyte model and suggests potential early involvement of hypoxia pathways in ALS given the developmental nature of hiPSC-derived cells. I also identify hypoxia-mediated alterations in RNA-binding proteins (RBPs) known to be implicated in ALS pathogenesis. Hypoxia significantly modulated the subcellular distribution of these proteins in astrocytes, with their differential responses between CTRL and VCPMUT astrocytes highlighting potential interactions between hypoxia and RNA dyshomeostasis in ALS. Non-cell autonomous experiments highlight the complex interactions between astrocytes and motor neurons, and the influence of hypoxia. VCPMUT astrocyte-conditioned media (ACM) induces notable mislocalisation of RBPs in CTRL motor neurons, mirroring changes seen in untreated VCPMUT motor neurons. Surprisingly, ACM from hypoxic astrocytes shows a reduced capacity to rectify RBP mislocalisation in VCPMUT motor neurons, suggesting hypoxia-induced modifications in the astrocytic secretome, diminishing their neuroprotective capacity. This is further emphasised by VCPMUT motor neurons exhibiting reduced survival when exposed to hypoxic ACM. This research underscores the intricate and dynamic astrocyte-motor neuron interactions in ALS, with hypoxia emerging as a potentially critical modulator. Therefore, this work paces the way for further exploration of hypoxia’s role in ALS pathogenesis and potentially informs therapeutic strategies targeting astrocyte-mediated neurodegeneration.

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