Effects of Lipid Compounds and Antidepressant Drugs on TRKB Dimerization

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http://urn.fi/URN:NBN:fi:hulib-202112084170
Title: Effects of Lipid Compounds and Antidepressant Drugs on TRKB Dimerization
Author: Seiffert, Nina
Other contributor: Helsingin yliopisto, Bio- ja ympäristötieteellinen tiedekunta
University of Helsinki, Faculty of Biological and Environmental Sciences
Helsingfors universitet, Bio- och miljövetenskapliga fakulteten
Publisher: Helsingin yliopisto
Date: 2021
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202112084170
http://hdl.handle.net/10138/337196
Thesis level: master's thesis
Degree program: Neurotieteen maisteriohjelma
Master 's Programme in Neuroscience
Magisterprogrammet i neurovetenskap
Specialisation: Neurotiede
Neuroscience
Neurovetenskap
Abstract: An increasing number of people are diagnosed with depression. One possible reason for the development of depression is faulty wiring and information processing in certain neural networks (network hypothesis) in the central nervous system. It has been shown that antidepressant drugs (ADs) can induce a juvenile-like plasticity state in the brain (iPlasticity) comparable to the plastic state of critical periods during development. iPlasticity enables the rewiring of neuronal networks in combination with environmental stimuli. At the molecular level, the binding of brain-derived neurotrophic factor (BDNF) to its high-affinity receptor tropomyosin kinase receptor B (TRKB) leads to TRKB dimerization and activation, triggering a downstream signalling cascade promoting brain plasticity. Activation of the TRKB signalling cascade is triggered by neuronal activity as well as AD treatment. Recent findings demonstrate that classical as well as rapid-onset ADs bind directly to the transmembrane domain of TRKB, leading to increased translocation of intracellularly stored TRKB to the plasma membrane and enhanced BDNF binding. Cholesterol, a sterol lipid known to regulate TRKB signalling, has been found to ensure optimal TRKB-BDNF signalling by changing the TRKB dimers’ relative orientation when altering the membrane thickness. A point mutation of TRKB tyrosine 433 to phenylalanine (TRKB.Y433F) has been found to hinder TRKB dimerization. Molecular dynamic simulations reveal that other membrane lipids are likely to participate in AD binding to TRKB. The aim of this thesis was to investigate whether lipid and drug compound treatments affect TRKB dimerization in Neuro2A cells expressing TRKB. Furthermore, we assessed whether the Y433F mutation modulates TRKB dimerization in such treatments. Protein fragment complementation assay (PCA) was used as in vitro protein-protein interaction assay to quantify dimerization of overexpressed TRKB carrying two split luciferase reporter proteins. Additionally, to avoid variability caused by transient transfection and be able to test large compound libraries, the establishment of a stably TRKB-expressing N2A cell line was initiated. The results show that lipid compounds, such as Allopregnanolone, as well as ADs, such as Imipramine and (2R,6R)-Hydroxynorketamine, increased TRKB dimerization in vitro in a dose-dependent manner within 40 minutes. The increase was more pronounced in the TRKB WT-expressing cells. This indicates that the compounds tested here may be directly interacting with TRKB, facilitating dimerization. Moreover, data seem to confirm previous research on the less effective TRKB.Y433F mutation. While stable expression of TRKB carrying one of the luciferase reporter proteins was successfully achieved in a monoclonal cell line, the amount of protein expressed seems to require further optimization before utilising it for PCA. In conclusion, lipid and AD treatments can induce an increase in TRKB dimerization in a dose-dependent fashion. Further investigations are needed to determine where the compounds bind and by which mechanisms they exert their effects on TRKB. Furthermore, the work on the stable cell line will be completed to avoid variability of transient transfection in the future.
Subject: Tropomyosin Receptor Kinase B
Lipids
Antidepressants
Protein-Protein Interaction
Stable Cell Line


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