UC Santa Cruz

Protein misfolding and amyloid formation is associated with several disorders, including type II diabetes (T2D), Alzheimer’s disease (AD) and Parkinson’s disease (PD). While these diseases are some of the most common and costly pathologies in the modern world, effective treatments to prevent and reverse them are still lacking. There is an urgent unmet need for novel approaches to understand the molecular mechanisms driving these diseases and to develop therapeutic strategies to prevent and stop these pathological processes. A common mechanistic feature of protein misfolding disorders is a complex aggregation pathway that leads to the fibrillar state, where multiple, rapid-interconverting aggregation intermediates, i.e. oligomeric species, are generated upon the self-association of unfolded or partially folded conformations. These transient oligomeric entities are thought to be the most toxic species during the aggregation cascade. Nonetheless, oligomers are exceedingly difficult to target therapeutically due to their heterogeneous and dynamic nature, and thus classical structure-activity relationships are exceedingly hard to determine and to implement in drug design. One of the most prominent characteristics of AD pathology is the deposition of amyloid fibrils of the amyloid β (Aβ) peptide in brain tissue. Aβ is an intrinsically disordered peptide, majorly consisting of 40-42 amino acids, and its assembly into oligomers and amyloid fibrils is thought to lead to the development of AD. The molecular mechanisms by which Aβ oligomerizes and interacts with the cellular environment are still not fully understood. The work performed in this thesis aims to provide new insights into these mechanisms using molecular chirality as the main mechanistic tool, and combined techniques ranging from chemical synthesis to biophysics and cellular assays. In Chapter 2, we designed a focused chiral mutant library (FCML) of Aβ, and identified several point D-substitutions that allowed us to modulate Aβ aggregation propensity and biological activity. Surprisingly, the reduced propensity towards aggregation and the stabilization of oligomeric intermediates did not always correlate with an increase in toxicity. This directly challenges the current working hypothesis of AD research, where these soluble aggregation intermediates are thought to represent the most neurotoxic species of Aβ. Additionally, we found that the subtle L-Ser26 to D-Ser26 mutation (S26s) led to reduced fibril formation propensity and inhibited toxicity, which appears to be related to the resultant peptide’s lack of ability to adopt a fibril-seeding conformations based on NMR and DFT results. In Chapter 3, we employed mirror-image Aβ as a strategy to enhance fibril formation and prevent oligomer formation of the Aβ peptide. This was accompanied by an almost complete abolishment of toxicity, setting one of the few examples of enhancing aggregation as an alternative approach to inhibit Aβ toxicity. Furthermore, we determined that the non-aggregating segment comprising amino acids 1-30 of Aβ, i.e., Aβ(1-30), is taken up by cells in a stereoselective fashion (about 3-fold difference), and found Aβ(1-30) cellular internalization to depend on cellular prion protein PrPC in the cellular membrane. To the best of our knowledge, this is the first time that Aβ aggregation and its cellular, receptor-mediated neuronal uptake have been disentangled. The work performed in this thesis highlight how chirality can be a powerful tool for studying Aβ structure-activity relationships. Additionally, the concepts presented here should be broadly applicable to study many other amyloidogenic proteins and peptides.