Modelling age-associated protein aggregation and neurodegeneration in C. elegans

Abstract

Protein aggregation is a hallmark of many neurodegenerative diseases. Structurally and functionally distinct proteins aggregate in different disorders, ultimately leading to neuronal death. Diseases such as Alzheimer’s and Parkinson’s are largely sporadic, whereas Huntington’s disease has a genetic basis. The disease-associated proteins tau, α-synuclein, and huntingtin perform diverse physiological roles in their native soluble states. However, the mechanisms that drive their transition into highly ordered, β-sheet-rich aggregates, and how this relates to neuronal toxicity, remain poorly understood. In this thesis, we developed Caenorhabditis elegans models to study protein aggregation and neurodegeneration associated with tau, mutant huntingtin, and α-synuclein. Using these models, we investigated aggregation behavior, neuronal toxicity, and the influence of factors such as ageing, post-translational modifications, and molecular chaperones on disease-associated proteins. Chapter 1 provides an overview of age-associated protein aggregation. Proteostasis pathways normally prevent protein misfolding and aggregation, but their decline with age is thought to promote aggregation and cellular death. We discuss Alzheimer’s, Parkinson’s, and Huntington’s diseases, focusing on the aggregation-prone properties of tau, α-synuclein, and huntingtin exon1 fragments. We highlight gaps in understanding the factors that trigger and modulate aggregation and review age-related proteostasis changes that contribute to pathology. Finally, we discuss the advantages of C. elegans as a model organism for mechanistic studies of protein aggregation and neurodegeneration. In Chapter 2, we evaluated fluorescent proteins as aggregation tags in C. elegans neurons. We generated strains expressing mNeonGreen or mEYFP, both reported as monomeric. We found that mNeonGreen aggregates upon neuronal overexpression and cannot be detected by available antibodies in worm lysates. In contrast, mEYFP remains diffuse and is readily detectable by western blotting. These findings demonstrate that mEYFP is a more suitable tag and underscore the importance of validating fluorescent proteins in vivo. Chapter 3 examines the effects of human tau expressed at near-physiological levels using single-copy insertion models. Tau localizes to neuronal processes, consistent with its microtubule-binding function. Age-dependent structural defects, including buckling of the ventral nerve cord, were observed and exacerbated by ageing. Exposure to stressors such as UV, heavy metals, and ER stress further increased this phenotype. Phosphomimic tau and expression of Alzheimer’s-associated kinases also enhanced neuronal buckling, with tau inclusions appearing in some animals. These results suggest that phosphorylation initially disrupts microtubule-associated tau, with inclusions forming later. In Chapter 4, we studied huntingtin exon1 with polyQ lengths of 20 and 80. Expression of Htt1Q80 caused aggregation and motility defects. While CCT1 did not reduce aggregation, the chaperone DNAJB6 effectively inhibited Htt1Q80 aggregation. Proteome analysis revealed no major chaperome differences but showed upregulation of energy metabolism pathways in Htt1Q80 animals, linking metabolic changes to aggregation and toxicity. Chapter 5 describes single-copy α-synuclein models showing synaptic localization and age-dependent dopaminergic dysfunction, as assessed by basal slowing assays, while motor neuron function remained largely intact. This model recapitulates key Parkinson’s disease features. Chapter 6 discusses these findings in the context of neurodegeneration, emphasizing the importance of low-expression models to study early pathological changes preceding overt aggregation and their relevance for therapeutic intervention.