Investigating the Neuroprotective Properties of Keap1-Nrf2 Protein-Protein Interaction Disruptors in Alzheimer’s Disease
Authors:   , Elsharkasi Mohamed Moftah Omer 
Published: July 2024

Alzheimer’s disease (AD) is a complex and multifactorial neurodegenerative condition. Beside accumulation of β-amyloid (Aβ) and the formation of neurofibrillary tangles, oxidative stress plays a crucial role in its development and progression. A major challenge in AD research and drug development is the development of physiologically relevant models that accurately reflect human AD. To address this, this thesis aimed to develop a screenable, human-derived, neuronal model to investigate the potential neuroprotective effects of activating the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in vitro. The toxic effects of secretomes from human-induced pluripotent stem cell (hiPSC)-derived neurons from a patient with APPV717I mutation (APP-CM) and synthetic Aβ42 oligomers (AβO) on Lund human mesencephalic (LUHMES)-derived neurons was explored. Results revealed that APP-CM did not promote morphological damage to the development of LUHMES neurons, whereas 5 μM AβO significantly impaired axonal integrity, synaptic networks and reduced cell viability by approximately 50% within 24 hours. Based on the conditions optimised to establish the neuronal AD model, a co-culture model of LUHMES-derived neurons and human primary cortical astrocytes was developed. In this co-culture model, AβO (5 μM) led to a less pronounced (~20%) reduction in cell viability compared to the mono-culture neuronal model. Additionally, AβO significantly reduced ATP levels in both neurons and astrocytes. This study further assessed the efficacy of a newer class of Nrf2 activators known as Keap1-Nrf2 protein-protein interaction (PPI) disruptors, compared to classical electrophilic Nrf2 activators, in astrocytes alone and the co-culture system. The study found them effective in astrocytes, as measured by the NAD(P)H Quinone Dehydrogenase 1 (NQO1) assay, but not in the co-culture. A notable discovery was that the neuronal media used in the co-culture system activated astrocytic Nrf2, as confirmed by NQO1 assay and Nrf2 knockdown experiments. This has limited the pharmacological activation of Nrf2 in this co-culture system and therefore the potential therapeutic effects against AβO in this study. Concurrently, a systematic review and meta-analysis study was conducted to investigate the relationship between Nrf2 target genes and cognitive impairment in mouse models of amyloid toxicity. A total of 50 studies were included, with 12 studies qualifying for inclusion in quantitative analyses. Meta-analysis findings confirmed a correlation between Nrf2 activation (SMD= 2.54, 95% CI: 1.85 to 3.23) and improved cognitive function (Morris Water Maze (MWM) acquisition phase: SMD = 1.39, 95% CI: 0.82 to 1.95; probe phase: SMD = 1.2175, 95% CI: 0.7480 to 1.6869) in these models, highlighting the haem oxygenase-1 (HO-1) gene (SMD = 2.67; 95% CI: 1.4 to 3.86) as a mediator of Nrf2-dependent protection, and potential therapeutic target for improved specificity and reduction of off-target effects in neurodegenerative disease. Overall, this research offers significant insights into Nrf2's potential in mitigating Aβ-induced neurotoxicity and cognitive decline in AD. The unexpected findings from the co-culture experiments indicate a critical issue of media incompatibility with a commonly used system in neurodegenerative research. This study not only advances our knowledge of AD but also highlights the need for refined approaches in AD modelling, paving the way for future investigations into the broader impacts of Nrf2, and identifying the HO-1 gene as a promising therapeutic target.