Dissertation

Proteomic Profiling of Neuronal and Microglial Cells Using TurboID in Inflammatory and Homeostatic States for Extension Into In Vivo Systems

Sunna, Sydney Nicole

2023

Overview

The brain is a cellularly complex organ possessing glia, neurons, and vascular cells. Each cell type supports distinct physiological roles in homeostatic states to orchestrate higher-order cognitive processes. Likewise, each cell type expresses unique proteomic profiles capable of emerging physiological phenotypes with distinct vulnerabilities in neurodegenerative disease. Alzheimer’s disease (AD) is the most common neurodegenerative disease, and ongoing systems-level analyses continue to highlight the importance of cellular complexity with disease progression. Bulk brain analyses provide a broad picture of global molecular transformations occurring in the brain that correlate with disease pathology and other traits. However, these bulk methods cannot directly resolve molecular changes occurring in distinct brain cell types. Cellular isolation upstream of mass-spectrometry poses important challenges ranging from contamination from other cell types, reliance on well-validated surface markers which can alter in disease states, and the inability to purify adult neurons. The recent development of proximity-based biotin ligases including TurboID, have made it possible to label and purify cellular proteomes in living cells and animals without the need for cellular isolation. This thesis includes the foundational in vitro studies validating the use of TurboID-based proximity labeling to resolve proteomic differences between two brain cell types (neurons and microglia) under both homeostatic and neuroinflammatory contexts. Additionally, these studies interrogated the proteomic breadth captured by cytosolic TurboID-mediated biotinylation, the impact of TurboID expression on homeostatic phenotypes, the cellular-distinction of proteins labeled by cytosolic TurboID, and the propensity of agnostically-directed cytosolic TurboID to label proteins of disease relevance in homeostatic and neuroinflammatory conditions. Our proteomic analyses demonstrate that cytosolic TurboID and streptavidin-based affinity purification capture >50% of microglial and neuroblastoma proteomes. Cytosolic expression of TurboID minimally impacted cellular proteome abundances, and did not significantly impact cellular respiration or microglial cytokine release profiles with inflammatory challenge. TurboID-NES captured proteins of relevance to neurodegeneration in both microglia and neuroblastoma cell lines, and successfully captured a portion of microglial proteomic changes in response to inflammatory challenge. These in vitro experiments laid the foundation for the generation of novel Rosa26TurboID/wt/Camk2a-Cre mice capable of labeling excitatory neuronal proteomes in living mice. This thesis includes foundational experiments validating the genetic strategy underlying the Rosa26TurboID/wt/Camk2a-Cre mice, as well as experiments assessing the impact of inflammation specifically on the proteomes of excitatory neurons, which are not apparent at the bulk proteome level. Using TurboID as a discovery tool, our findings show that neuroinflammation is associated with an increase in glutamatergic post-synaptic proteins in Camk2a neurons which may be indicative of neuronal hyper-excitability.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Bioinformatics / Cellular biology / Molecular biology / Pathology
• ISBN: 9798381681901