Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

4-23-2026

Graduate Advisor

Michael R.Nichols

Committee

Michael R.Nichols

Cynthia Dupureur

Chung F. Wong

Wendy Olivas

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia worldwide. While Amyloid-β (Aβ) aggregation is a defining pathological feature, increasing evidence highlights chronic neuroinflammation as a critical contributor to disease progression. Soluble Aβ assemblies can act as damage associated molecular patterns (DAMPs) including activation of the NLRP3 inflammasome, which links amyloid pathology to sustained inflammatory responses in the brain (Heneka et al., 2015; Nakanishi et al., 2018). However, the molecular mechanisms by which specific Aβ conformations influence inflammasome assembly remain incompletely understood.This study aimed to investigate whether distinct aggregation states of amyloid β42 (Aβ42) are internalized in HEK293T cells and whether intracellular Aβ directly or proximally associates with inflammasome components. First, the internalization efficiency of different Aβ42 conformations was evaluated, with particular focus on protofibrillar species, which have been shown to exhibit enhanced cellular internalization compared to monomers and fibrils (Gouwens et al., 2016). Establishing these uptake dynamics provides a foundation for examining intracellular interactions. Second, the expression and subcellular localization of the inflammasome components NLRP3, ASC, and procaspase-1 were characterized under single and co-expression conditions. This approach enables assessment of inflammasome assembly and spatial organization in a controlled system, building on established mammalian reconstitution models (Makoni et al., 2021). Third, this work examined whether internalized Aβ associates with inflammasome proteins within cells. While Aβ is known to activate innate immune pathways (Heneka et al., 2013; Venegas et al., 2017), it remains unclear whether this occurs exclusively through indirect cellular stress mechanisms or involves direct or proximal interactions with inflammasome components. Evidence that ASC specks can bind Aβ and promote its aggregation (Venegas et al., 2017), suggests the potential for bidirectional interactions between amyloid assemblies and inflammasome machinery. We hypothesized that specific Aβ42 conformations, particularly protofibrils, directly or proximally interact with NLRP3 inflammasome proteins. By integrating Aβ structural biology, cellular uptake, and inflammasome reconstitution approaches, this study established a framework to assess direct interactions between Aβ42 and NLRP3 inflammasome proteins. Focusing on the spatial colocalization of Aβ with NLRP3, ASC, and procaspase-1, this work provided a mechanistic foundation for understanding how amyloid assemblies may physically engage inflammasome proteins. These findings laid the groundwork for future studies aimed at determining whether such interactions contribute to inflammasome activation and downstream inflammatory signaling in Alzheimer’s disease.

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