The amyloidoses are a group of debilitating disorders which include neurodegenerative diseases such as Alzheimer’s disease and systemic diseases such as dialysis-related amyloidosis (DRA). Amyloidoses are associated with the aggregation of proteins into amyloid fibrils with a highly organised cross-β structure. Amyloid fibrils are formed by a variety of proteins and peptides despite differences in sequence and native structure. Amyloid formation occurs by a nucleated growth mechanism and proceeds via oligomeric intermediates into mature fibrils. Despite intense research, the molecular mechanisms involved in disease pathogenesis remain unclear. This thesis discusses the mechanism by which amyloid fibrils cause cellular disruption.
Chapter 3 describes work performed to validate the use of β2-microglobulin (β2m), the protein that self-associates into amyloid fibrils found in DRA deposits, as a model to study amyloidosis. Fragmentation of mature β2m fibrils, increased their internalisation and access to intracellular compartments, and were therefore used to investigate mechanisms of cellular disruption. Building on previous work in the laboratory showing trafficking of β2m fibrils to the lysosome, chapter 4 examined the effect of fragmented fibrils on lysosomal function and demonstrates that fragmented fibrils impair lysosome-mediated degradation of endocytosed proteins. Following on from this, chapter 5 discusses the effect of fragmented fibrils on membrane trafficking. Fragmented fibrils perturbed the trafficking of lysosomal membrane proteins and also reduced the trafficking of endocytosed cargo to lysosomes. This may rationalise the impairment in degradation of endocytosed proteins. The molecular chaperone, heat shock protein 70 (Hsp70) has been shown to be protective in amyloid disease. The role of Hsp70 in fibril-mediated cell disruption was investigated in chapter 6. Hsp70 protected fibril-treated cells from impairment in degradation of endocytosed protein but not from membrane trafficking defects. This work demonstrates that fragmented fibrils are nanoparticles which target lysosomes and implicates the lysosome in the pathogenesis of amyloidosis
