Investigating the TrkB Interactome during Development and in Neurodegeneration
Brain-derived neurotrophic factor (BDNF) is a cardinal regulator of neuronal development, survival and synaptic plasticity, signalling via its major receptor, Tyrosine receptor kinase B (TrkB). In the developing brain, BDNF acts as a guidance cue influencing the direction of the axonal growth cone. In the mature brain, it ensures survival of neurons and synaptic plasticity. Guidance cues remodel the nascent local proteome in the developing nervous system. Recently, guidance cue receptors DCC, Neuropilin-1 and Robo2 were shown to interact with the translation machinery. Both the protein and mRNA interactions of these receptors help them to define which proteins are locally synthesized in response to stimulation. BDNF likewise regulates the local nascent proteome with high spatiotemporal resolution via TrkB activation. While there is extensive data on expression level, trafficking and signalling of TrkB, little is known about its protein and RNA interactions. We hypothesised that like DCC, Neuropilin-1 and Robo-2, TrkB shows similar interaction with the translation machinery. Immunoprecipitation studies for TrkB were performed and protein interactors were analysed by LC-MS/MS, interacting mRNAs were sequenced. A specific set of ribosomal proteins, RNA-binding proteins, rRNAs and mRNAs interacting with TrkB were detected in the embryonic mouse brain. However, in the adult mouse hippocampus, where BDNF-induced local protein synthesis mediates synaptic plasticity, no receptor-ribosome interaction was detected. This suggests a remodelling of the TrkB interactome during development and that BDNF modulates the nascent proteome in adult brain via different mechanisms. To elucidate possible new roles and regulatory mechanisms of TrkB in the mature brain, we screened for novel protein interactions in adult mouse hippocampus and cerebral synaptosomes. The TrkB protein interactome was largely composed of cytoskeleton and transport vesicle-related proteins, as well as transmembrane transporters. At the synapse, in particular, small molecule transporters interacted with TrkB, including ion and glutamate transporting excitatory amino acid transporter 1 (EAAT1). BDNF stimulation interrupts the TrkB-EAAT1 interaction and leads to increased EAAT1-dependent glutamate uptake in primary hippocampal cultures, elucidating a novel crosstalk between neurotrophin receptor and glutamate transporter. The BDNF-TrkB axis is impaired in ageing and in neurodegeneration. We have poor understanding of the processes occurring in these conditions. We hypothesised that the TrkB interactome may vary in aging and neurodegenerative disease as a possible contributor to their impaired signalling. Interestingly, a screen of the interactomes in the mouse brain showed that major TrkB co-precipitating proteins are not affected by ageing. In murine prion disease, which results in extensive neurodegeneration, a TrkB-interactome screen revealed increased association of a distinct set of cell-adhesion proteins with TrkB, including Connexin-43, a gap-junction adhesion molecule which is associated with other neurodegenerative diseases. The identification of novel disease-related interactions may yield future insights into dysregulated BDNF signalling in neurodegeneration. In conclusion, in this thesis, the interactome of TrkB was studied to increase our understanding of BDNF function in health and disease in the mouse brain. Novel interaction partners of the neurotrophin receptor TrkB were identified, during development, in adulthood and ageing, as well as in prion neurodegeneration. Specific interactors identified may, upon further investigation, represent novel mechanistic insights into BDNF signalling and possibly new targets to tackle dysregulated neurotrophic signalling in many pathological conditions.
Development, Glutamate transport, Neurodegeneration, Neurotrophins, Prion disease, Protein-protein interactions, Proteome, Synapse, Translation machinery, TrkB interactome, Tyrosine receptor kinase B
Networked Digital Library of Theses and Dissertations