Periodic Reporting for period 1 - STrkB (The structural biology of TrkB-BDNF signalling)
Reporting period: 2017-07-01 to 2019-06-30
The molecular events underlying the formation and function of neuronal circuits during brain development are strictly coordinated and controlled. Neurotrophin family factors are important players in this process and act primarily through interactions with tropomyosin-related kinase (Trk) receptors, promoting neuronal survival and synaptogenesis. Furthermore, the mature brain is not a “static” organ, undergoing constant morphological and functional changes at the level of neuronal synapses.
Among the major neurotrophins, the brain-derived neurotrophic factor (BDNF) molecules act by binding to the TrkB receptor, a type-I membrane protein. This leads to TrkB dimerization, auto-phosphorylation and initiates downstream signalling events. However, the molecular architecture of this complex and the mechanism of signal propagation across the plasma membrane remained unknown.
BDNF expression levels in the brain are diminished in neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. It is proposed that onset of disease occurs before the characteristic morphological changes readily observable in the brain, starting at the level of synaptic dysfunction. Therefore, future approaches for the treatment of neurodegenerative diseases are proposed to include “synaptic repair”, which targets pathophysiology before pathogenesis. Due to its functions, therapies exploiting the use of BDNF and functional mimetics have been proposed, yet clinical trials performed to date led to inconclusive results due to its lack of stability and solubility.
To understanding the mechanism of BDNF-dependent TrkB signalling, and provide a platform for novel therapies targeting this pathway, the overall objective of this project was to define in structural and mechanistic terms the steps leading to TrkB activation upon BDNF binding. Using a combination of X-ray crystallography, single-particle cryo-electron microscopy and complementary biophysical techniques, this project provided the first insights into the organization of the BDNF-TrkB complex in the extracellular region.
Among the major neurotrophins, the brain-derived neurotrophic factor (BDNF) molecules act by binding to the TrkB receptor, a type-I membrane protein. This leads to TrkB dimerization, auto-phosphorylation and initiates downstream signalling events. However, the molecular architecture of this complex and the mechanism of signal propagation across the plasma membrane remained unknown.
BDNF expression levels in the brain are diminished in neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. It is proposed that onset of disease occurs before the characteristic morphological changes readily observable in the brain, starting at the level of synaptic dysfunction. Therefore, future approaches for the treatment of neurodegenerative diseases are proposed to include “synaptic repair”, which targets pathophysiology before pathogenesis. Due to its functions, therapies exploiting the use of BDNF and functional mimetics have been proposed, yet clinical trials performed to date led to inconclusive results due to its lack of stability and solubility.
To understanding the mechanism of BDNF-dependent TrkB signalling, and provide a platform for novel therapies targeting this pathway, the overall objective of this project was to define in structural and mechanistic terms the steps leading to TrkB activation upon BDNF binding. Using a combination of X-ray crystallography, single-particle cryo-electron microscopy and complementary biophysical techniques, this project provided the first insights into the organization of the BDNF-TrkB complex in the extracellular region.
The structural investigation of the TrkB-BDNF complex was particularly challenging. A large number of constructs were designed and screened and crystallization trials were performed in order to obtain well-diffracting crystals. A library of high-affinity single chain antibodies (known as nanobodies) was raised against the TrkB receptor. Nanobodies were found to be very helpful crystallization chaperones, allowing the solution of high-resolution crystal structures for the extracellular part of the TrkB-BDNF complex. Studies to evaluate the functional impact of nanobodies, specifically their potential as modulators of TrkB-BDNF signalling, are currently ongoing.
This work led to the first high-resolution structure of BDNF in complex with the extracellular region of TrkB. Once the ongoing efforts aimed at understanding the mechanism of TrkB signal transduction are completed, this structural information will be used to design novel BDNF mimetic molecules which will be evaluated in experimental models for neurodegenerative disorders.