Year

2023

Degree Name

Doctor of Philosophy

Department

Graduate School of Medicine

Abstract

Nicotinic acetylcholine receptors (nAChRs) play an essential role the parasympathetic response and have garnered attention as potential therapeutic targets for alleviating chronic visceral and neuropathic pain. Recent investigations have also focused on nAChRs as regulators of immune function through the cholinergic anti-inflammatory pathway. This research aimed to explore the expression and functionality of nAChRs in diverse immune cell lines, particularly focusing on the nAChRs subtypes α7, α9 and/or α10. To assess the expression of α7, α9 and/or α10 subunits, reverse-transcription quantitative polymerase chain-reaction (RT-qPCR) was used. Functional analysis involved evaluating agonistinduced intracellular Ca2+ ([Ca2+]i) signalling events mediated by nAChRs and their pharmacological modulation. Comparison was drawn with more widely expressed and wellcharacterised purinergic receptor (P2R) responses to confirm validity of research. This thesis commences by establishing the theoretical foundation of the cholinergic hypothesis by exploring the role of nAChRs in the progression of neurodegenerative diseases (Chapter 1). The study encompasses the examination of various immune cell lines and their nAChR expression profile under standard culture conditions (Chapter 2). Emphasis was laid into the optimisation of Ca2+ imaging conditions for non-adherent cells (Chapter 3). Chapter 4 investigates the expression and functional profiles of the commonly used cell lines Jurkat T lymphocytes, RPMI 8226 B lymphocytes, THP-1 monocytes and their derived macrophages revealing variable expression of transcripts encoding the α7, α9 and/or α10 nAChR subunits. [Ca2+]i signals mediated by nAChR and purinergic pathways supported the presence of functional nAChRs together with P2Rs in immortalised immune cells. The survey affirms diverse functional traits and subtype distribution among various cell types. Chapter 5 contains a comprehensive evaluation of immortalised human brain microglial cell lines C20 and C06. Close examination of nAChR-mediated [Ca2+]i signals in C20 and C06 microglia disclosed functional evidence of nAChRs in minute subpopulations, unveiling phenotypical heterogeneity in these cell lines. Distinctive [Ca2+]i profiles emerged, indicating representation of multiple nAChR subtypes and their involvement in complex repertoires of intracellular Ca2+ pathways. Through this research, two well-defined nAChR-mediated [Ca2+]i signalling profiles were enriched in the C06 microglial subclones C06-B2 and C06- A4, showcasing robust and homogenous nAChR function. These functionally enriched microglial lines offer valuable resources for exploring the role of nAChRs in human microglia and their significance in neuroprotection; furthermore, they may facilitate the development of therapeutic interventions targeted at mitigating neurodegenerative diseases.

FoR codes (2008)

1116 MEDICAL PHYSIOLOGY, 1107 IMMUNOLOGY, 1109 NEUROSCIENCES

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.