| Abstract |
Signaling by the p75 neurotrophin receptor (p75NTR) has been implicated in diverse
neuronal responses, including apoptosis, survival, differentiation, migration, axonal outgrowth,
and myelination. Unlike other neurotrophin receptors, p75NTR has no catalytic activity but rather
acts as a scaffold to assemble a variety of interacting partners that ultimately activate
downstream signaling pathways, such as NF-κB, the Jun N-terminal kinase (JNK) and the RhoA
pathway. Despite being the first receptor identified for the neurotrophin family of neurotrophic
factors, the physiological roles of p75NTR as well as how the different pathways activated by this
receptor are linked to biological outcome have remained elusive. Our goal is to molecularly dissect
the different pathways activated by this receptor, by identifying specific regions/residues in its
intracellular domain involved in the activation of these signaling pathways. To accomplish this, a
variety of mutant variants have been generated by mutating the surface amino acids of the p75NTR
death domain and these variants are examined in various cellular systems in vitro, for their ability
to elicit activation of downstream signaling pathways after stimulation with (pro) neurotrophins.
Primary and main objective of this thesis was to establish an appropriate assay, sensitive enough
to discriminate potential differences between the aforementioned p75NTR mutant variants, on JNK
signaling, as well as on RhoA inactivation leading to apoptosis and to axonal growth respectively.
Our initial experiments were focused on screening the mutants for JNK activation by Western
blotting, but this method was proven to be insensitive. Thus, a new luciferase-based assay was
established and was proven both sensitive and quantitative. This assay was used in a variety of
cell types, including cell lines & primary cells, in order to select the most reliable expression
system for our screening. In addition, new mutant variants of the receptor were generated that
may be able to dissociate the NF-κB and RhoA pathways, which appear (according to our initial
screening) to be either linked or utilizing adjacent aminoacids in the p75NTR death domain.
Uncoupling these pathways will be invaluable for elucidating the physiological roles of p75NTR
signaling in vivo, by investigating the loss-of-function of selected p75NTR mutant variants in knockin
mice.
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p75 neurotrophin receptor : an alanine-scanning mutagenesis approach for dissecting its signaling pathways
