MicroRNA and Nrf2 pathway manipulation in human astrocytes: towards a therapeutic approach for Amyotrophic Lateral Sclerosis (ALS)
Publication Date
July 31, 2026
Creator
Hannah, Bailey
Abstract
Dysfunction of the Nrf2 antioxidant pathway is a hallmark of ALS, with Nrf2 activation suggested as potential therapeutic strategy. The Nrf2 pathway plays a critical role in glia, with astrocyte-specific activation shown to be neuroprotective in ALS mice. Dysregulation of miRNAs is also reported in ALS, yet their roles in regulating astrocyte reactivity or Nrf2 signalling remain poorly understood. Here, potential links between miRNAs and these pathways were explored in human astrocyte models, to investigate if inhibiting Nrf2-targeting miRNAs could be a viable astrocyte-targeted Nrf2-activating ALS therapy.
Proteomic analysis of cultured human primary astrocytes detected ~4000 proteins, including 55/154 ALS-associated proteins in the ALSoD database. Twelve Nrf2 target gene products were downregulated in ‘reactive’ (FBS-exposed) astrocytes, compared to ‘quiescent’ serum-free astrocytes, while no Nrf2 target gene products were upregulated. Reduced expression of NFE2L2 (the Nrf2 gene) was also detected by RT-qPCR. This reactive astrocyte model therefore offered a disease-relevant platform to explore Nrf2 rescue strategies.
Parallel small non-coding RNA sequencing (sncRNAseq) was conducted, with analysis extended to astrocyte-derived extracellular vesicles (ADEVs) due to their potential as circulating ALS biomarkers. This enabled rational selection of highly expressed Nrf2-targeting miRNAs (miR-340-5p and miR-27b-3p) for inhibition in reactive astrocytes. Transfection of a GFP plasmid proved ineffective, prompting use of cell-permeable antisense miRNA ‘power inhibitors’ to upregulate Nrf2. However, presumed low inhibitor uptake limited their utility. Lentiviral delivery modestly improved transduction (5-20%), with GFP expression allowing single-cell analysis, though no Nrf2 pathway upregulation was observed. Nucleofection achieved ~50% delivery of a GFP plasmid, however proteomic analysis of reactive astrocytes nucleofected with a miR-340-5p inhibitor revealed no significant protein changes.
Regardless, the reactive astrocyte model proved effective for screening Nrf2 activators, as demonstrated with the commercial activator tBHQ, which upregulated Nrf2 pathway components and increased cell viability, ATP-induced Ca²⁺ signalling, and reduced ROS (non-significantly). These assays were extended to the ALS therapeutics riluzole and edaravone, which modestly upregulated the Nrf2 pathway. Riluzole’s effect may reflect oxidative stress, as high doses reduced viability. Edaravone had similar effects to tBHQ, with the enhanced Ca2+ response to ATP predicted to promote astrocyte reactivity and glutamate release, potentially contributing to its limited efficacy in ALS.
This thesis evaluated a practical human astrocyte model for screening Nrf2 activators. Multi-omic analysis identified ALS-associated proteins and miRNAs altered in astrocyte reactivity, highlighting possible therapeutic targets to combat neuroinflammation. Although miRNA-based Nrf2 activation was unsuccessful, the astrocytic effects of riluzole and edaravone were evaluated, to work towards identification of astrocyte-targeted treatments for ALS.
Item Type
ethesis
Thesis Type
PhD
Supervisors
University of Nottingham
University of Nottingham
University of Nottingham
University of Nottingham
Subjects (LC)
Associated Schools / Departments
UoN Repository URI
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BAILEY Hannah PhD thesis.pdf
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