Studying BDNF signalling using neurons derived from human embryonic stem cells

Merkouris, Spyridon 2020. Studying BDNF signalling using neurons derived from human embryonic stem cells. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

As a result of a large number of in vitro as well as in vivo experiments with rodents, brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB are now widely appreciated to play major roles in brain function. There is also a growing appreciation that decreased BDNF signalling may be a significant component in a wide range of brain dysfunction in humans based on the discovery of mutations and polymorphisms in the corresponding genes. Yet still very little is known about BDNF expression and TrkB activation in human neurons or the human brain. In order to begin to address these questions, human embryonic stem cells (hESCs) have been used to generate large numbers of neurons suitable for biochemical experiments. In the work reported in the following chapters, two different protocols have been used to grow and differentiate hESCS. In the first, excitatory neurons were generated exhibiting detectable levels of BDNF albeit at low levels and after well over 2 months of culture. Detailed biochemical analyses that could significantly advance the field within a manageable time frame were also hampered by uncontrolled cell division still occurring in excitatory neurons even after extended culture periods. The use of a second, recently published differentiation protocol led to the significantly faster, more reproducible generation of large numbers of TrkB- expressing, mostly inhibitory neurons. A key feature of this robust differentiation protocol was the high proportion of BDNF-responsive cells allowing BDNF-induced phosphorylation to be studied with regard to time course and dose response. This system also allowed comparisons to be made between BDNF, the related factor neurotrophin-4 (NT4) and newly generated TrkB-activating antibodies. These TrkB-activating ligands were compared by extracting RNA from neurons treated for different time periods. One of the TrkB-activating antibody (designated #85) turned out to activate human TrkB at concentrations close to those of the natural ligands and with a strikingly similar pattern of induced mRNA transcripts, especially at early time points. The main conclusion of this work is that a test system based on the generation of BDNF-responsive neurons can be used to develop new reagents able to meaningfully activate TrkB. This work has been published in Merkouris et al 2018 in PNAS (https://www.pnas.org/content/115/30/E7023.long)

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	3 March 2021
Last Modified:	03 Aug 2022 01:42
URI:	https://orca.cardiff.ac.uk/id/eprint/139240

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