Periodic Reporting for period 1 - NeuroTrans (NEUROtransmitter TRANSporters: from single molecules to human pathologies)
Reporting period: 2020-02-01 to 2022-01-31
Neurons use electrical signals called action potentials to propagate information along axons and dendrites. In 1936 Otto Loewi received the Nobel Prize in Medicine for the discovery that small molecules (neurotransmitters) are used for inter-neuronal communication. Neuronal signalling requires efficient removal of the released neurotransmitters at synapses, typically achieved through re-uptake by a dedicated transporters, the neurotransmitter:sodium symporter (NSS). Several diseases are associated with improper neurotransmitter clearance (Parkinson’s disease, seizures, depression, or schizophrenia). Recreational drugs like cocaine or ecstasy interfere with transport processes and lead to hallucinations, euphoric stimulation, addiction.
The vision of the ETN NeuroTrans is to (i) gain an improved understanding on how NSS dysfunction contributes to neuropsychiatric diseases and how psychoactive substances interfere with normal NSS function and (ii) to establish a robust framework for comprehending the molecular origin of diseases causing mutations.
The project’s ultimate objective is to develop a solid model that can predict changes in NSS function from fundamental principles and has defined the following scientific objectives:
(i) To obtain kinetic, structural, and mechanics insight into the processes of substrate transport.
(ii) To obtain improved electrophysiological, kinetic and thermodynamic data of substrate binding and transport.
(iii) To identify the molecular perturbations associated with neuropsychiatric disease.
(iv) To identify the mode of action of NPS using new microfluidic devices.
NeuroTrans will establish a highly interdisciplinary doctoral training program by forming an interdisciplinary and intersectorial team of world-leading European researchers. Training will include most important subdisciplines in quantitative biology, including molecular modelling, computer simulations, biophysics, biochemistry, neurobiology, molecular and structural biology and has a strong industry oriented focus, ensuring that the ESRs can become part of Europe’s future generation of innovative leaders.
The vision of the ETN NeuroTrans is to (i) gain an improved understanding on how NSS dysfunction contributes to neuropsychiatric diseases and how psychoactive substances interfere with normal NSS function and (ii) to establish a robust framework for comprehending the molecular origin of diseases causing mutations.
The project’s ultimate objective is to develop a solid model that can predict changes in NSS function from fundamental principles and has defined the following scientific objectives:
(i) To obtain kinetic, structural, and mechanics insight into the processes of substrate transport.
(ii) To obtain improved electrophysiological, kinetic and thermodynamic data of substrate binding and transport.
(iii) To identify the molecular perturbations associated with neuropsychiatric disease.
(iv) To identify the mode of action of NPS using new microfluidic devices.
NeuroTrans will establish a highly interdisciplinary doctoral training program by forming an interdisciplinary and intersectorial team of world-leading European researchers. Training will include most important subdisciplines in quantitative biology, including molecular modelling, computer simulations, biophysics, biochemistry, neurobiology, molecular and structural biology and has a strong industry oriented focus, ensuring that the ESRs can become part of Europe’s future generation of innovative leaders.
The NeuroTrans consortium is using a very broad spectrum of methods and techniques to study the gamma amino acid (GABA) transporters GAT1 and BGT1, the dopamine transporter (DAT), all essential for functioning of the human brain, the bacterial homologues thereof to gain insights in transporter function.
WP1 - Determining transporter structure and gaining insight into complex formation.
The objective of WP1 is to develop a structural understanding of the transporters and their complexes at the molecular level. Several NeuroTrans member labs join forces in an interdisciplinary effort and obtained very promising initial results. First data show that the transporters can be expressed and purified in functional form. Further optimization is imperative to produce sufficient quantities of purified transport for structural studies and is currently explored.
Direct structural measurements of ligand binding using the saturation transfer difference nuclear magnetic resonance (STD NMR) technique show robust signals. Also, experiments carried out using the electron spin resonance (EPR) method and the spectroscopic method Forster Resonance Energy Transfer (FRET) can already record signal and are further optimized.
Transporter models could be obtained, molecular system have been successfully assembled using physiologically relevant membrane environments and are prepared for MD simulations.
WP2 - Characterizing electrophysiological ligand binding and substrate transport with high temporal resolution in intact cells and vesicle preparations using novel technologies.
Substrate transport is an electrogenic event, because moving ions through the membrane. Experimental systems for electrophysiological measurements are being established and preliminary data show currents and capacitive signals. These experiments have revealed an unexpected, but important response to the osmolyte betaine. This finding is of physiological importance for the human brain and is further explored.
A novel methodological approach is being established and optimized, which is based on the Solid Supported Membrane (SSM) technology of beneficiary Nanion.
Homology modelling and initial MD simulations show, that model creation was successful. Production simulations have been started.
WP3 - Dynamic, thermodynamic and mechanistic insights into substrate binding and transport.
WP3 develops a holistic model that integrates all structural, kinetic, and thermodynamic data from several biophysical techniques and biochemical measurements. Experimental procedures have been established using traditional approaches. In parallel, optimizing the thermo-optical detection of NanoTemper devices is ongoing. STD NMR is explored as a second independent approach, which provides structural information beyond measuring kinetic and thermodynamic parameters.
A technological highlight of NeuroTrans is time-resolved electron microscopy (TEM), which is able to directly detect protein conformational changes using X-rays and electron microscopy approaches. Several types of micro- and nano- scale measuring chambers for TEM are being fabricated by photolithography and are tested.
WP4 - Mode of action of novel psychoactive substances (NPS).
WP4 investigates the mode of action of novel psychoactive substances (NPS) and seeks to develop novel instrumentation for improved detection. The pharmacological characterization of NPS is ongoing, using uptake-inhibition and batch release assays. Construction of a new high throughput microfluidic station and an instrument set-up that allows for cell measurements is ongoing. A design compatible with cell cultures has already been achieved.
WP5 - Molecular determinants of novel disease causing mutations.
The goal of WP5 is to characterize the detrimental effect of disease causing DAT mutations that are predominant in patients with neuropsychiatric and neurodegenerative disorders.
Data on the alteration of transporter function caused by the mutation(s) are currently acquired with the goal to characterize their effect.
WP1 - Determining transporter structure and gaining insight into complex formation.
The objective of WP1 is to develop a structural understanding of the transporters and their complexes at the molecular level. Several NeuroTrans member labs join forces in an interdisciplinary effort and obtained very promising initial results. First data show that the transporters can be expressed and purified in functional form. Further optimization is imperative to produce sufficient quantities of purified transport for structural studies and is currently explored.
Direct structural measurements of ligand binding using the saturation transfer difference nuclear magnetic resonance (STD NMR) technique show robust signals. Also, experiments carried out using the electron spin resonance (EPR) method and the spectroscopic method Forster Resonance Energy Transfer (FRET) can already record signal and are further optimized.
Transporter models could be obtained, molecular system have been successfully assembled using physiologically relevant membrane environments and are prepared for MD simulations.
WP2 - Characterizing electrophysiological ligand binding and substrate transport with high temporal resolution in intact cells and vesicle preparations using novel technologies.
Substrate transport is an electrogenic event, because moving ions through the membrane. Experimental systems for electrophysiological measurements are being established and preliminary data show currents and capacitive signals. These experiments have revealed an unexpected, but important response to the osmolyte betaine. This finding is of physiological importance for the human brain and is further explored.
A novel methodological approach is being established and optimized, which is based on the Solid Supported Membrane (SSM) technology of beneficiary Nanion.
Homology modelling and initial MD simulations show, that model creation was successful. Production simulations have been started.
WP3 - Dynamic, thermodynamic and mechanistic insights into substrate binding and transport.
WP3 develops a holistic model that integrates all structural, kinetic, and thermodynamic data from several biophysical techniques and biochemical measurements. Experimental procedures have been established using traditional approaches. In parallel, optimizing the thermo-optical detection of NanoTemper devices is ongoing. STD NMR is explored as a second independent approach, which provides structural information beyond measuring kinetic and thermodynamic parameters.
A technological highlight of NeuroTrans is time-resolved electron microscopy (TEM), which is able to directly detect protein conformational changes using X-rays and electron microscopy approaches. Several types of micro- and nano- scale measuring chambers for TEM are being fabricated by photolithography and are tested.
WP4 - Mode of action of novel psychoactive substances (NPS).
WP4 investigates the mode of action of novel psychoactive substances (NPS) and seeks to develop novel instrumentation for improved detection. The pharmacological characterization of NPS is ongoing, using uptake-inhibition and batch release assays. Construction of a new high throughput microfluidic station and an instrument set-up that allows for cell measurements is ongoing. A design compatible with cell cultures has already been achieved.
WP5 - Molecular determinants of novel disease causing mutations.
The goal of WP5 is to characterize the detrimental effect of disease causing DAT mutations that are predominant in patients with neuropsychiatric and neurodegenerative disorders.
Data on the alteration of transporter function caused by the mutation(s) are currently acquired with the goal to characterize their effect.
NeuroTrans has a strong translational aspect through the development of enabling techniques in biophysics, molecular and structural biology and through industrial partner which establish novel instruments for the use in live cells to quantify kinetics, dynamics and thermodynamic parameters, allowing for exploitation and commercialisation. The NeuroTrans research programme will have major generalizable scientific impact that will be of high relevance to academia, pharmaceutical industry and policy makers and is likely to be applicable to other classes of transporters, but to membrane proteins in general.
The development of novel instruments is expected to strongly boost experimental throughput. With the respect of novel psychoactive substances such as new street drugs, these novel technology is foreseen to significantly decrease the time-lag between initial discovery and complete characterization to provide policy makers and regulatory bodies with information on a useful time-scale.
The development of novel instruments is expected to strongly boost experimental throughput. With the respect of novel psychoactive substances such as new street drugs, these novel technology is foreseen to significantly decrease the time-lag between initial discovery and complete characterization to provide policy makers and regulatory bodies with information on a useful time-scale.