Cholesterol and Amyloid β Modulation of Receptor Tyrosine Kinase Signaling in Neural Cell Models

Abstract

Alzheimer’s disease (AD) is a highly complex condition that results in the gradual yet progressive failures of neuronal signaling, metabolic homeostasis, and glial cell function. Though the exact etiological factor may vary individual to individual, the result is a similar phenotypic convergence of accumulation of amyloid beta (Aβ) plaques and hyperphosphorylated tau tangles. While both Aβ and lipid dysregulation are well established features of AD pathology, the role of membrane composition changes on modulating cellular signaling pathways remains poorly understood. Cholesterol is a critical structural and functional component of the cell membrane, and has emerged as a key regulator protein organization, receptor clustering, and signal transduction. Yet the molecular mechanisms linking receptor mediating signaling to lipid dysregulation in particularly AD relevant neural cell models remains incompletely understood. This thesis employs molecular neuropharmacology approaches to investigate this interaction, of how cholesterol modulation and Aβ exposure can modulate the signaling of receptor tyrosine kinases (RTKs) across various neural cell types. Using a combination of immortalized cell lines and human induced pluripotent stem cell (iPSC) derived models, this work systematically examines how cholesterol enrichment and depletion can influence cellular morphology, viability, and intracellular signaling in the presence and absence of Aβ. In the first part of this thesis, a focused review on the field of AD and relevant literature into RTK, cell membrane, and AD interactions was presented. Along with an overview of methods used in this thesis. In the second part of this thesis, we investigate how cholesterol modulation can alter Aβ induced toxicity, morphology, and RTK signaling pathways in the mouse hippocampal neuronal like HT22 cell model. Using molecular biology techniques such as biochemical assays, cell imaging, flow cytometry, and western blotting, cholesterol enrichment was shown to exacerbate Aβ toxicity and induce morphological changes. Cholesterol enrichment was also found to selectively regulate the RTK, PDGFRα’s activation along with altering downstream MAPK and PLCγ1 activity. Interestingly, receptor phosphorylation levels did not predict downstream signaling outputs, revealing a potential novel finding of cholesterol enrichment regulating a decoupling of RTK activation and intracellular signaling pathways. In the third part of the thesis, we detail the optimization, characterization, and workflow development for generating iPSC-derived neuronal and astrocytic models. Using immunocytochemistry, protein analysis, and molecular biology approaches, this chapter established robust differentiation protocols for the generation of cortical neurons along with star shaped astrocytes. A novel approach for generating GABAergic enriched neuronal networks was also detailed. In the final section of the thesis, we focus on employing a phospho-proteomics approach for analysing the RTK signaling profiles of iPSC-derived neural stem cells (NSCs), cortical neurons, and astrocytes. This analysis revealed, previously unreported, distinct RTK activation signatures unique to each of the neural lineages studied. We also explore cholesterol and Aβ interactions in iPSC-derived astrocyte. Revealing that cholesterol enrichment selectively modulated PDGFRβ activity. Collectively, this thesis advances our understanding of how membrane lipid composition influences RTK signaling dynamics in neural cells, providing mechanistic insight relevant to neurodegenerative disorders.