Author: Dusan Matusica
Matusica, Dusan, 2008 Regulation of p75NTR Trafficking by Neurotrophins in the NSC-34 Motor Neuron Cell Line, Flinders University, School of Medicine
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Neurotrophins are a family of growth factors necessary for the development and maintenance of the nervous system. They produce their effects through receptor mediated signaling mechanisms that are highly regulated by sophisticated intracellular transport networks. The impairment of intracellular trafficking of neurotrophins in motor neurons has been identified as one possible factor in the development of motor neuron diseases, but remains inadequately studied. Aided by advances in imaging technology and the development of more powerful and sensitive detection tools for in-vitro studies, the dynamics of intracellular transport of neurotrophins are beginning to be unraveled. However, a primary limiting factor in the study of neurotrophin-transport dynamics in motor neurons has been the lack of alternative and easily available in-vitro systems able to substitute the often difficult and costly primary motor neuron cultures. The aim of this project was to develop a suitable motor neuron model using the NSC-34 cell line for the study of receptor mediated trafficking events through endosomal transport pathways. Successful evaluation and characterization of NSC-34 cells for motor neuron specific markers would result in the investigation of the p75 neurotrophin receptor (p75NTR) trafficking pathways in the presence of exogenous neurotrophins, with a variety of confocal imaging techniques. Chapter 3 describes the optimisation of NSC-34 cell culture conditions through media modification and the development of a suitable growth substrate matrix, which significantly improved cell adhesion, differentiation and the ability to culture the cells for extended time periods in serum free conditions. Quantitative measurements of cell proliferation, culture viability, cell-body size and neurite length are described to highlight the increased value of the cell line for long-term culture and experiments examining a broad range of issues relevant to motor neurons. In Chapter 4, multiple experimental approaches were used to extensively screen the NSC-34 cell line for the presence of motor neuron-specific markers, neurotrophin receptors and proteins involved in regulation of endosomal transport. This characterization established the presence of a developing motor neuron-like neurotrophin receptor profile (p75NTR, TrkB and TrkC), a genetic marker of developing motor neurons, cholinergic markers, proteins regulating transport within the endosomal pathway, and additional proteins previously shown to directly interact with neurotrophin receptors, including sortilin, and the lipid raft associated ganglioside GT1b. Furthermore, evidence is provided that NSC-34 cells undergo apoptosis in response to exogenous nerve growth factor (NGF) or neurotrophin-3 (NT-3), but not brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4). In addition characterization of mouse specific p75NTR antibodies is presented to establish their suitability for internalization studies without altering the binding of exogenous neurotrophins to the receptor. Subsequent confocal microscopy examination focusing on p75NTR trafficking in Chapter 5 revealed that internalization and intracellular transport of this receptor is regulated by exogenous neurotrophins at the cell surface where ligand binding and internalization occur, and in endosomal compartments where the bulk of receptors and ligands are targeted to their specific destinations. Evidence is provided showing that p75NTR internalization is altered in the presence of NGF, NT-3, or NT-4, but not BDNF, and the receptor is diverted into non-clathrin mediated endosomal pathways in response to NGF but not BDNF. Immunofluorescence confocal microscopy suggests that p75NTR recycles to the plasma membrane in a Rab4 GTPase dependent manner in the absence of neurotrophins. Addition of neurotrophins diverted p75NTR from the recycling Rab4 positive pathway, into EEA-1 positive sorting endosomes in the presence of NGF or NT-3, or lysosomal degradation in the presence of BDNF or NT-4. This study clearly demonstrates the suitability of the NSC-34 cell line as an alternate in-vitro system for the study of motor neuron biology, particularly the study of neurotrophin receptor trafficking. Taken together the results represented in this study suggest for the first time, that the fate of the p75NTR receptor depends on which neurotrophin is bound. These findings have important implications for understanding the dynamic mechanisms of action of p75NTR in normal neuronal function, and may also offer further insight into the potential role of neurotrophins in the treatment of neurodegenerative diseases.
Keywords: Neurotrophins,P75NTR,endocytosis,intracellular trafficking,motor neuron,NSC-34
Subject: Neuroscience thesis
Thesis type: Doctor of Philosophy
Completed: 2008
School: School of Medicine
Supervisor: Prof. Robert A. Rush