UC Irvine

Amyloid-β (Aβ) oligomers are a major cause of neurodegeneration in Alzheimer’s disease (AD). These soluble aggregates of the Aβ peptide have proven difficult to study due to their inherent metastability and heterogeneity. Strategies to isolate and stabilize homogenous Aβ oligomer populations have emerged such as mutations, covalent cross-linking, and protein fusions. These strategies along with molecular dynamics simulations have provided a variety of proposed structures of Aβ oligomers, many of which consist of molecules of Aβ in β-hairpin conformations. β-Hairpins are intramolecular antiparallel β-sheets composed of two β-strands connected by a loop or turn. Chapter 1 details three decades of research that suggests Aβ peptides form several different β-hairpin conformations, some of which are building blocks of toxic Aβ oligomers. β-Hairpins of Aβ can adopt a variety of alignments, but the role that β-hairpin alignment plays in the formation and heterogeneity of Aβ oligomers is poorly understood. Chapter 2 details an exploration of the effect of β-hairpin alignment on the oligomerization of Aβ peptides in which we designed and studied two model peptides with two different β-hairpin alignments. Peptides Aβm17–36 and Aβm17–35 mimic two different β-hairpins that Aβ can form, the Aβ17–36 and Aβ17–35 β-hairpins, respectively. In this chapter, I explain how these hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. Our laboratory has synthesized and elucidated the high-resolution structures of a variety of Aβ β-hairpin peptide mimics. These structures have informed the design and synthesis of covalently stabilized β-hairpin oligomer mimics, some of which we have elucidated high-resolution structures of. The insights from these studies and others like it are currently being used to design anti-Aβ antibodies and vaccines to treat AD. Research suggests that antibody therapies designed to target oligomeric Aβ may be more successful at treating AD than antibodies designed to target linear epitopes of Aβ or fibrillar Aβ. Aβ β-hairpins are good epitopes to use in antibody development to selectively target oligomeric Aβ. We recently reported the generation of a polyclonal antibody, pAb2AT-L raised against one of our stabilized Aβ β-hairpin trimer mimics. pAb2AT-L is moderately selective for oligomeric Aβ over monomeric and fibrillar Aβ and stains the diffuse Aβ on the peripheries of Aβ plaques in AD human and mouse brain tissue but does not bind the dense fibrillar plaque cores. Chapter 3 details an investigation into whether pAb2AT-L is neuroprotective against toxic aggregates of Aβ and whether pAb2AT-L can inhibit Aβ aggregation. In this chapter, I detail how pAb2AT-L prevents the toxic effects of Aβ42 on iPSC-derived neurons and HMC3 microglia and inhibits Aβ42 fibrillization at sub-stoichiometric ratios of antibody to Aβ42. Chapter 4 is a guide to working with human iPSC-derived neurons for new-comers to iPSC culture. In this chapter, I review methods for generating, transfecting, and differentiating human iPSCs with an emphasis on neuronal differentiation, and I highlight how human iPSC-derived are crucial for disease-modelling. This chapter also includes detailed protocols that I used to culture and differentiate iPSCs — these are the methods I used to differentiate the neurons described in chapter 3 — and insights I have gained from performing these protocols. Finally, chapter 5 describes the research I performed while working at AbbVie during the summer of 2023 as a bioanalytical intern. The project I completed was quantifying hydrolytic enzymes in human and animal vitreous humors using LC-MS/MS-based targeted proteomics. The vitreous humor is a highly hydrated, viscoelastic, gelatinous fluid that occupies the posterior compartment of the eye between the lens and the retina. Intravitreal injections are a common route of administration for back of the eye diseases but are relatively invasive procedures. To avoid repeating these procedures, biodegradable intravitreal implants have become a popular strategy for achieving sustained drug delivery to the back of the eye. Because these implants are degraded through hydrolysis, hydrolytic enzymes have been hypothesized to play a role in their degradation, but limited information is currently available regarding the abundance of hydrolytic enzymes in the vitreous humor. Quantifying these enzymes is necessary to design accurate models of intravitreal biodegradable implant degradation for drug development. Chapter 5 details the identification and quantification of selected hydrolytic enzymes in the human vitreous humor using liquid chromatography triple quadrupole tandem mass spectrometry.