Investigating the in vivo aggregation mechanisms of Amyloid-β and Islet Amyloid Polypeptide in Caenorhabditis elegans

Fibrillar protein aggregation is a pathological hallmark of several neurodegenerative and metabolic disorders, including Alzheimer’s disease (AD) and Type 2 Diabetes Mellitus (T2DM). The prevalence of these complex diseases continues to rise globally, driven by an ageing population, environmental factors and lifestyle choices. Chapter 1 provides a detailed overview of the molecular and genetic background of these diseases, along with their potential pathological mechanisms. In AD, one of the earliest pathological markers is the accumulation of amyloid-β (Aβ42) plaques in the brain, associated with neurodegeneration and progressive cognitive decline. T2DM is characterised by insulin resistance and hyperglycaemia, resulting in overproduction of pancreatic hormones, including insulin and islet amyloid polypeptide (IAPP). Elevated IAPP levels can cause fibrillar deposition of the IAPP hormone, causing loss of pancreatic β-cells that regulate glucose metabolism. The conversion of soluble monomeric peptides into insoluble fibrillar aggregates has become a great interest for developing therapeutic strategies. Although in vitro approaches have provided insight into aggregation kinetics and toxicity, they fail to replicate the complexity of multicellular organisms with intact protein homeostasis machinery. Given the financial, technical and ethical limitations of mammalian models, Caenorhabditis elegans was employed to investigate the aggregation mechanisms of Aβ42 and IAPP. Several nematode models exist for studying Aβ42 aggregation, but a tractable IAPP-expressing model was lacking. In Chapter 2, we presented our novel IAPP C. elegans models expressing fluorescently labelled IAPP in muscle or intestinal tissue. We characterised our model using biochemical and microscopic assays. We demonstrated intracellular IAPP-GFP aggregation and partial co-localisation to mitochondria. Our data suggest that IAPP-GFP expression causes modest motility toxicity and the formation of filamentous structures. In Chapter 3, we investigated the in vivo effects of Urolithin B, a metabolite derived from ellagitannin-rich foods such as berries and pomegranate, previously identified in silico as a potential IAPP aggregation inhibitor. We used our IAPP-GFP models described in Chapter 2 and performed lifespan assays, along with biochemical and microscopic techniques. We observed a decrease in IAPP-GFP aggregation load upon treatment. Our data suggests an interplay between Urolithin B and IAPP-GFP. We believe Urolithin B shows potential as a therapeutic agent against IAPP aggregation and toxicity. In Chapter 4, we explored the aggregation kinetics of Aβ42 in vivo using a muscle-specific C. elegans model and investigated how protein homeostasis machineries modulate Aβ42 toxicity. We demonstrated that Aβ42 toxicity follows the accumulation of fibrils, underlying the pathological role of fibrillar aggregates in nematodes. We showed that Aβ42 aggregation and toxicity are strongly dependent on protein concentration, indicating a critical threshold for Aβ42 fibril formation. Our data revealed the role of the proteasomal degradation pathway in Aβ42 toxicity. We also demonstrated the protective role of DNAJB6 against Aβ42 toxicity in vivo. Altogether, our work provides molecular insights into the behaviour of Aβ42 and its interplay with protein homeostasis components in C. elegans, contributing to our current understanding of AD pathogenesis. Finally, in Chapter 5, we discussed the results and insights gained from the previous chapters in the context of existing knowledge.