Intraneuronal inclusions known as Lewy bodies are a hallmark of Parkinson’s disease. Amyloid fibrils formed by the misfolding of alpha-synuclein are the principal component of Lewy Bodies. However, the role of these fibrils and their oligomeric assembly intermediates in neuronal death is poorly understood. This is because alpha-synuclein misfolds into amyloid fibrils in the cytoplasm, whereas experimental studies have typically involved adding fibrils and oligomers to the cell culture medium. Although this enables biophysical characterisation of the fibrils and oligomers before addition to the cells, access to the cytoplasm is limited and can only provide a limited picture of their cellular effects.
This project will use a single molecule nanoinjection platform to deliver alpha-synuclein fibrils and oligomers into the cytoplasm of neurons. Nanoinjection uses quartz needles (≤50nm pore diameter), known as nanopipettes, to deliver molecules into cells. The nanopipettes incorporate electrodes and application of a voltage drives the transport of molecules through the pore. When a protein passes through the nanopipette’s pore there is a corresponding disruption in the ion flow, thus the number of proteins delivered into a cell can be quantified. Thus, this project will not only determine whether alpha-synuclein fibrils and oligomers are toxic in the cytoplasm, but for the first time it will quantify the number of each required to kill a neuron.
Fibrils and oligomers will be made from recombinant α-synuclein, characterised using an array of biophysical techniques, and nanoinjected into neuronal cell lines and primary neurons. To inject cells the nanopipette will be used as a probe for a scanning ion conductance microscope (SICM), which will generate a topographical map of the target cell. This information is used to insert the nanopipette into the cell at a pre-defined depth and location. Application of a voltage will drive the delivery of the fibrils or oligomers into the cell. By monitoring the corresponding disruptions in ion flow, a defined number of oligomers or fibrils will be delivered. The cellular effects of the nanoinjected fibrils and oligomers on cell stress and viability will then be analysed using fluorescence microscopy-based assays.
This PhD will be a collaborative project in the Astbury Centre for Structural Biology and Bragg Centre at the University of Leeds between the research groups of Dr Eric Hewitt (cell biologist), Dr Paolo Actis (nanotechnologist) and Professor Sheena Radford FRS OBE (structural biologist). The project will provide training in interdisciplinary science, with the PhD student developing cutting edge expertise at the interface of the nanotechnology, molecular cell biology and structural biology.
https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/84/dr-eric-hewitt
https://eps.leeds.ac.uk/electronic-engineering/staff/806/dr-paolo-actis
https://biologicalsciences.leeds.ac.uk/biological-sciences/staff/127/professor-sheena-radford
Please direct all queries regarding this project to Dr Eric Hewitt in the first instance: [Email Address Removed]
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle, York and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme and how to apply can be found on our website:
http://www.dimen.org.uk/how-to-apply/application-overview
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