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

Primary Supervisor: Professor Andrew Devitt, Biomedical & Biological Sciences

Secondary Supervisors: Dr Eric Hill; Dr Ivana Milic

PhD project title: Extracellular vesicles – Characterising the structure-function relationships of extracellular vesicles in cell communication within the neurovascular unit.

University of Registration: Aston University

Project outline:

The nature of cell communication within multicellular organisms may result in health or disease. Within the central nervous system (CNS), we do not understand fully how the complex mix of cells (neuronal, non-neuronal and immune cells) communicate, nor the language that is used. Nor do we understand fully how this is changed in age-associated conditions.

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. Overall, this highlights the need for detailed EV analysis from each of the CNS cell types so we may understand EV uptake, function and crucially, how they change in age-associated conditions.

Our 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 PhD programme will seek to identify and characterise the key factors associated with EV, that mediate their function. This studentship will join an active BBSRC-funded team focused on EV structure-function studies.

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, further ‘response’ EV production
  • to define the molecular mediators of function through detailed MS analysis to define
  • EV surface proteins mediating intercellular communication
  • 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.

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

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology

Techniques that will be undertaken during the project:

  • Analysis and interrogation 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 and astrocytes and microglia.  
  • Vesicle analyses: isolation and analysis of EV structure and function using qNano tunable resistive pulse sensing, flow cytometry, electron microscopy, analysis of immune-modulating capability in vitro using chemoattraction assays and qPCR.
  • Imaging and analysis: Flow cytometry, light and fluorescence microscopy including real-time microscopy and confocal microscopy, automated cell imaging for functional studies
  • Cell assays: Induction and analysis of cell death, Binding and phagocytosis assays
  • Mass spectrometry: for the analysis of EV proteome

Contact: Professor Andrew Devitt, Aston University