Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Susan Meakin

Abstract

Introduction: Brain-derived neurotrophic factor (BDNF) facilitates neuronal differentiation by activation of the TrkB receptor-tyrosine-kinase. BDNF/TrkB also modulates the activity of the excitatory N-methyl-D-aspartate neurotransmitter receptor (NMDAR), thereby also regulating neural plasticity and LTP/LTD, two forms of synaptic plasticity that contribute to the storage of information inside the brain. How TrkB cross-talks to modulate NMDAR function is not known, although our working model involves the RasGrf1 signaling molecule, a nucleotide exchange factor for Ras/Rac-GTPases, which interacts with both receptors. In response to TrkB activation, Ras-Grf1 is tyrosine phosphorylated and mediates neurite outgrowth in PC12 cells. RasGrf1 also binds constitutively to the NMDAR to stimulate long-term-depression (LTD) in primary neurons. We hypothesize that TrkB activation of Ras-Grf1 will increase neuronal outgrowth in TrkB expressing PC12 derived cells (TrkB-B5) and BDNF will uncouple RasGrf1 from the NMDAR thereby facilitating a decrease in LTD and an increase in long-term-potentiation (LTP) in primary neurons.

Method: PC12 (TrkB-B5) cells were transfected with appropriate plasmids, treated with BDNF (1ng/ml) and the percentage of neurite outgrowth was determined. Brain tissue slices from P30 mice were stimulated with either BDNF (100 ng/ml), NMDA (100 µM) or co-stimulated with both. Slices were then lysed and the protein interactions were assayed by immunoprecipitation and western blotting.

Results: We found that RasGrf1 expression significantly (P-value 1048 and Tyr1062 appeared to be crucial sites of tyrosine phosphorylation on RasGrf1 for neurotrophin mediated neurite outgrowth in cell culture, tyrosine phosphorylation of RasGrf1 could not be detected in neural tissue slices in response to BDNF. Furthermore, we found a direct interaction between RasGrf1 and TrkB receptor in response to BDNF treatment in slice cultures. In addition, BDNF stimulated the tyrosine phosphorylation of the NR2B subunit of the NMDA receptor at residue Tyr1472, which facilitates receptor retention at the cell surface, and also stimulated a dissociation of RasGrf1 from the NMDA receptor.

Conclusion: My results strongly suggest that BDNF stimulation changes NMDAR signaling via TrkB activation and that this is mediated by RasGrf1. Future studies using RasGrf1 knockout mice will further address BDNF dependent changes in the activation of individual signalling molecules, and hippocampal culture studies from wild-type/RasGrf1 knock-out mice will address whether changes in BDNF-induced neuronal dendritic growth and spine formation are dependent on RasGrf1.


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