Abstract
Regulated cell death (RCD) is a fundamental biological process that shapes the developing nervous system and contributes to neurodegenerative disease. While intrinsic apoptosis has long been considered the dominant mechanism governing neuronal loss, emerging evidence suggests that additional pathways, including extrinsic apoptosis, necroptosis, and other forms of inflammatory cell death, may play critical and context-dependent roles. However, the cell-type specificity, spatial organization, and relative contributions of these pathways in both development and disease remain incompletely understood.
In this thesis, I use high-dimensional single-cell and spatial proteomic approaches to systematically investigate RCD programs across two biological contexts: telencephalic development and Alzheimer’s disease (AD). In Chapter 2, I employ single-cell mass cytometry (CyTOF) to profile the developing mouse telencephalon in wild-type and genetic models lacking key regulators of extrinsic apoptosis and necroptosis. I demonstrate that combined deletion of Caspase-8 and RIPK3 results in increased cellular abundance and selective expansion of specific populations, including intermediate progenitors and endothelial cells, revealing previously underappreciated, cell type–specific roles for these pathways in regulating developmental cell number. These findings challenge the prevailing paradigm that intrinsic apoptosis alone governs developmental cell elimination.
In Chapter 3, I extend this framework to neurodegeneration by adapting a multiplexed imaging mass cytometry (IMC) pipeline to map cell identity and pathological burden in situ. Using a 26-plex antibody panel applied to two complementary AD mouse models (5xFAD and PS19), I generate a spatially resolved proteomic atlas at whole-hemisphere scale and at higher resolution within disease-vulnerable regions including the hippocampus, isocortex, and piriform cortex. This approach enables simultaneous quantification of amyloid-beta and tau pathology alongside cell-type composition, providing insight into region-specific vulnerability and the spatial coordination of neurodegenerative processes. While the panel was designed to capture the broad cellular and pathological landscape of AD, attempts to include cell death markers were constrained by technical challenges encountered during antibody validation and metal conjugation, which precluded their reliable incorporation into the current 26-plex panel. The direct spatial mapping of apoptotic, necroptotic, and pyroptotic activity within the AD tissue context therefore remains an important direction for future work.
Together, this work advances our understanding of CNS biology across two contexts. Chapter 2 employs mass cytometry to characterise, at single-cell resolution, how extrinsic apoptosis and necroptosis remodel distinct cell populations during CNS development. Chapter 3 employs IMC to generate a spatially resolved proteomic atlas of the AD brain at both whole-hemisphere and region-specific resolution, revealing how cell-type composition and pathological burden are distributed across spatial scales in two complementary AD mouse models. Individually, each chapter provides new biological insight into its respective context; collectively, they demonstrate the power of mass spectrometry-based proteomic approaches, applied at different scales and resolutions, for investigating cellular states in the healthy and diseased brain and opening new avenues for understanding the proteomic basis of neurological disease.
