Extracellular vesicles - Characterising the structure-function relationships of extracellular vesicles in cell communication within the neurovascular

"The nature of cell communication in a multicellular organism may result in health or disease. Within the central nervous system (CNS), it is not fully understood how the mix of cells (neuronal, non-neuronal and immune cells) communicate, nor the language that is used. The change in this communication in age-associated conditions is also poorly understood.
Over recent years, extracellular vesicles (EV) have been identified as a novel intercellular communication mechanism1. These membrane bags, released from cells during health, disease and cell death, carry many factors to recipient cells to elicit responses (desirable and non-desirable). There are paradoxical data to suggest EV may be both neuroprotective and neurotoxic2. This highlights the need for detailed EV analysis from each of the CNS cell types so we may understand EV uptake, function and how they change in age-associated conditions.
Preliminary work reveals that EV carry a large range of factors that may underpin their activity, including a family of active enzymes which may help to control inflammation and repair responses2. This project seeks to identify and characterise the key factors associated with EV, that mediate their function.
The aims of this project are:
to generate cells of the neurovascular unit from induced pluripotent stem cells and assess EV release in mono- and co-culture
to assess the ability of EV to induce responses on other cells of the neurovascular unit (e.g. inflammatory responses)
to define the molecular mediators of function through detailed MS analysis
to define EV surface proteins mediating intercellular communication
to investigate EV-associated enzyme activity that controls inflammation.
identify key proteins present on the surface of EV that mediate EV function in interacting with macrophages and modulating the innate immune response. This will work from a large dataset of mass-spec results to identify lead molecules for further study.
to define the function of prioritised proteins in EV activity. Using a variety of cell biological and molecular approaches, this will assess proteins as key ligands for EV binding, uptake or active function.

Techniques that will be utilised during the project:
Analysis of extensive proteomics data: to identify lead targets for further analysis using systems biology approaches.
Cell culture: Isolation of primary cells from human peripheral blood; Tissue culture of a range of cell lines and primary cells. Induced pluripotent stem cell culture to derive functional neuronal networks, astrocytes and microglia.
Vesicle analyses: isolation and analysis of EV structure and function using tunable resistive pulse sensing and flow cytometry. Analysis of immune-modulating capability using chemoattraction assays and qPCR.
Imaging and analysis: Flow cytometry, light and fluorescence microscopy and automated cell imaging.
Cell assays: Induction and analysis of cell death, binding and phagocytosis assays.
Mass spectrometry: for the analysis of EV proteome.

References:
1. Van Niel, G., D'Angelo, G., & Raposo (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews: Molecular Cell Biology: DOI: 10.1038/nrm.2017.125
2. Shi, M., Sheng, L., Stewart, T., Zabetian, C.P. & Zhang, J. (2019). New Windows into the brain: CNS-derived extracellular vesicles. Progress in Neurobiology: DOI: 10.1016/j.pneurobio.2019.01.005
3. Devitt, A., Griffiths, H.R. & Milic I. (2018). Communicating with the dead: lipids, lipid mediators and extracellular vesicles. Biochemical Society Transactions: DOI: 10.1042/BST2016477
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