Regulation of p75 neurotrophin receptor-mediated neuronal death through post-translational modifications

The neurotrophin family of growth factors has a well characterised role in neural survival and death during development and adulthood. While specific neurotrophins bind to members of the Trk receptor family differentially, they all bind the p75 neurotrophin receptor (NTR). Neuronal death signalling through p75NTR occurs during developmental programmed cell death, and has been implicated in mediating neuronal death in several neurodegenerative conditions, including Alzheimers and motor neuron diseases, and following traumatic injury such as ischaemia. However, p75NTR can also assist in Trk signalling to mediate neuronal survival. The aim of this thesis was to determine a mechanism that resolves the paradox that p75NTR can activate both cell death and, in conjunction with Trk, cell survival signalling pathways. In addition to the interaction of p75NTR with coreceptors, post-translational processing may also determine the signalling outcome. p75NTR undergoes regulated intramembrane proteolysis, in which p75NTR is cleaved extracellularly by a metalloprotease, producing a membrane-associated C-terminal fragment, and subsequently by γ-secretase, producing a membrane-free intracellular fragment. In addition, the Chopper domain, previously shown to be responsible for cell death when attached to the membrane, is palmitoylated, a characteristic that promotes protein receptors to be incorporated into cholesterol-and sphingolipid-rich microdomains within the cell membrane. Thus the hypothesis was that both regulated intramembrane proteolysis and receptor palmitoylation would play a role in the initiation and regulation of p75NTR-mediated neuronal death. The experimental results presented within this thesis demonstrate that regulated intramembrane proteolysis can be ligand activated, and that extracellular cleavage is required for intracellular cleavage to occur. In addition, the results presented provide evidence that γ-secretase intracellular cleavage of p75NTR is promoted when both the C-terminal fragment of p75NTR and active γ-secretase translocate to lipidrich microdomains of the plasma membrane. This translocation and cleavage event are dependent on palmitoylation of p75NTR within the juxtamembrane intracellular domain and dissociation of the C-terminal fragment from activated TrkA. Furthermore, findings presented within this thesis also demonstrate that the inhibition of γ-secretase activity, which leads to the accumulation of the p75NTR Cterminal fragment in lipid-rich microdomains, promotes neuronal death in motor neurons in vitro. In contrast, blocking extracellular cleavage of p75NTR or disrupting membrane cholesterol inhibits the nerve growth factor induced p75NTR-mediated death of cultured motor neurons. Together, the results presented suggest that alterations in the balance of normal regulated intramembrane proteolysis and/or altered membrane lipid integrity due to disrupted cholesterol metabolism, as occurs in neurodegenerative conditions, are key factors in promoting the p75NTR-mediated death signal. These results further enhance our understanding of how neuronal degeneration is mediated. A greater understanding of the molecular mechanisms by which neuronal death is regulated through p75NTR may enable the development of new clinical treatments that improve the prognosis of a range of neurodegenerative conditions. The findings presented point to several possible targets for treatment, including the development of compounds that either specifically regulate the cleavage of p75NTR or maintain a normal balance of cellular membrane cholesterol