Periodic Reporting for period 1 - DiBDEV (Differentiation and characterisation of brain-derived extracellular vesicles from the peripheral blood for understanding the neuropathophysiological mechanisms of migraine.)
Periodo di rendicontazione: 2022-06-13 al 2024-06-12
Migraine is a neurological condition with symptoms such as severe headache which lasts up to 72 hours. 15-25% of adults aged 18-65 experience migraines and accordingly migraine ranks sixth in the diseases that cause the most years lived with disability globally. The mechanisms triggering migraine in a healthy brain are poorly understood but it is thought that the extracellular vesicles released by cells in the brain might help us to understand the molecular events that occur during migraine attacks.
Extracellular vesicles are very small parcels of molecules (including protein and RNA) enclosed in a membrane that are released by cells into their environment. These extracellular vesicles act like a cellular postal service, carrying molecular messages from the original cell through the body’s fluids, then delivering the messages to their target cell. Therefore, if we could intercept and capture these molecular messages in the extracellular vesicles, we could read what the cells are saying to each other and learn why they are behaving in a particular way (e.g. to cause migraine). It is thought that some of the extracellular vesicles from brain cells enter the bloodstream, creating an opportunity in that if we could capture these brain-derived extracellular vesicles from the blood outside the brain (e.g. taken in a standard clinical blood test), we could find out what the cells in the brain are saying to each other and what is happening at the molecular level. This is important because current methods to study the brain during migraine rely on animal models (which may not translate to humans) or imaging techniques, which do not have the resolution to tell us what is happening with molecules in the brain.
One principal challenge of capturing brain-derived extracellular vesicles in the blood is that they are very rare – most extracellular vesicles in the blood actually come from other cells in the body which are also sending out messages. What’s more, all these extracellular vesicles look very similar, so it is difficult to tell which come from the brain or elsewhere. With this crowded environment in the blood, it is still possible to pick out brain-derived extracellular vesicles by targeting specific molecules on the vesicle surface which belong to brain cells but are not found in other parts of the body. However, the body is very complex and there are very few molecules unique to the brain, with most also being found in other areas. Therefore, by using multiple molecules to identify brain-derived extracellular vesicles, we can be more confident that they have come from the brain and not elsewhere, and thus that the brain is the origin of the messages they extracellular vesicles carry.
The main aim of this project is to capture extracellular vesicles (EVs) originating from individual brain cell types (neurons, astrocytes and microglia) from the peripheral blood so that brain-derived EVs (BDEVs) of migraine patients can be analysed. From this, we could gain a better understanding of the cellular and molecular processes during migraine attacks, with possible treatment derivations, as well as if there is a role played by extracellular vesicles in migraine pathology.
Extracellular vesicles are very small parcels of molecules (including protein and RNA) enclosed in a membrane that are released by cells into their environment. These extracellular vesicles act like a cellular postal service, carrying molecular messages from the original cell through the body’s fluids, then delivering the messages to their target cell. Therefore, if we could intercept and capture these molecular messages in the extracellular vesicles, we could read what the cells are saying to each other and learn why they are behaving in a particular way (e.g. to cause migraine). It is thought that some of the extracellular vesicles from brain cells enter the bloodstream, creating an opportunity in that if we could capture these brain-derived extracellular vesicles from the blood outside the brain (e.g. taken in a standard clinical blood test), we could find out what the cells in the brain are saying to each other and what is happening at the molecular level. This is important because current methods to study the brain during migraine rely on animal models (which may not translate to humans) or imaging techniques, which do not have the resolution to tell us what is happening with molecules in the brain.
One principal challenge of capturing brain-derived extracellular vesicles in the blood is that they are very rare – most extracellular vesicles in the blood actually come from other cells in the body which are also sending out messages. What’s more, all these extracellular vesicles look very similar, so it is difficult to tell which come from the brain or elsewhere. With this crowded environment in the blood, it is still possible to pick out brain-derived extracellular vesicles by targeting specific molecules on the vesicle surface which belong to brain cells but are not found in other parts of the body. However, the body is very complex and there are very few molecules unique to the brain, with most also being found in other areas. Therefore, by using multiple molecules to identify brain-derived extracellular vesicles, we can be more confident that they have come from the brain and not elsewhere, and thus that the brain is the origin of the messages they extracellular vesicles carry.
The main aim of this project is to capture extracellular vesicles (EVs) originating from individual brain cell types (neurons, astrocytes and microglia) from the peripheral blood so that brain-derived EVs (BDEVs) of migraine patients can be analysed. From this, we could gain a better understanding of the cellular and molecular processes during migraine attacks, with possible treatment derivations, as well as if there is a role played by extracellular vesicles in migraine pathology.
The plan for this project was to first start by isolating extracellular vesicles (EVs) from different cell types directly from brain tissue to establish the process for capture of brain cell-derived EVs before moving on to blood samples. At the start of this project, time was first spent by comparing different methods to flush out blood (and blood-borne EVs) from the brain tissue of mice before isolating the specific brain cell-derived EVs. This found that using an anticoagulant in the solutions and cooling down the solutions before flushing out the tissue could improve the removal of blood and reduce cellular stress. Then the first aim of this project was to test different methods for EV isolation from brain tissues. For this, the previously published protocol for nickel-based isolation (NBI) of EVs was compared to a commercial EV precipitation reagent using the host lab’s own protocol. This showed that each method differentially enriched for different EV and neuronal proteins, and NBI of EVs may result in less contamination by non-EV proteins.
Steps were then taken to validate an approach to enrich for neuronal EVs by removing non-neuronal EVs from the total brain EV pool. As part of this, an assay was performed to digest proteins outside EVs (whilst the proteins inside EVs were protected by the EV membrane) to identify which neuronal markers belonged to the EVs themselves and which existed as proteins outside the EVs. Attempts were also made to capture neuronal and astrocytic EVs.
The same approach was then taken to blood plasma, where NBI and the commercial EV precipitation reagent were compared again, with the NBI method modified with additional washes during the EV isolation process to help to remove non-EV blood proteins. Optimisations of the method to enrich for neuronal EVs by removing non-neuronal EVs from the total blood EV pool were also performed, which revealed several underappreciated technical challenges of using this approach to enrich for rare EV populations in blood plasma. Like with the brain, attempts were also made to capture neuronal and astrocytic EVs from blood plasma.
A process was also set up for consenting patients then collecting and processing patient blood samples for EV isolation.
Steps were then taken to validate an approach to enrich for neuronal EVs by removing non-neuronal EVs from the total brain EV pool. As part of this, an assay was performed to digest proteins outside EVs (whilst the proteins inside EVs were protected by the EV membrane) to identify which neuronal markers belonged to the EVs themselves and which existed as proteins outside the EVs. Attempts were also made to capture neuronal and astrocytic EVs.
The same approach was then taken to blood plasma, where NBI and the commercial EV precipitation reagent were compared again, with the NBI method modified with additional washes during the EV isolation process to help to remove non-EV blood proteins. Optimisations of the method to enrich for neuronal EVs by removing non-neuronal EVs from the total blood EV pool were also performed, which revealed several underappreciated technical challenges of using this approach to enrich for rare EV populations in blood plasma. Like with the brain, attempts were also made to capture neuronal and astrocytic EVs from blood plasma.
A process was also set up for consenting patients then collecting and processing patient blood samples for EV isolation.
The results beyond the state of the art from this project include an improved method to flush blood from brain tissue before brain-derived extracellular vesicle (EV) studies and showing how different EV isolation methods can change the molecular composition of the resulting EV preparations; thus, influencing the results of downstream analysis. Results also include a new method to enrich for neuronal EV populations by removing non-neuronal EVs, and an assay to verify the neuronal markers enriched using this approach. Underappreciated technical factors were identified that can affect how this approach is applied with blood plasma derived samples. Applying the new approach to neuronal EV isolation in this project by removing the majority of non-neuronal EVs requires further research with blood samples from migraine patients to isolate brain-derived neuronal EVs for analysis to identify the molecular drivers of migraine.