Development of a probe for prion protein ligand binding and propagation
Background We have developed a fluorescently-labelled version of the prion protein (PrP) which is sensitive to the binding of both biological ligands (e.g. amyloid beta and anti-PrP antibodies) and small molecules (Risse et al., J. Biol Chem 2015 290 17020). These experiments used prion protein expressed in E. coli and therefore devoid of post-translational modification. We have now developed the over-expression and purification of the prion protein in HEK293 cells and plan to determine the effects of the post translational modifications upon ligand binding.
Rotation project Using routine molecular biology techniques point mutations will be introduced into the construct used to over-express PrP. These constructs will then be transfected into HEK293 cells, the resulting protein purified and tested in the established fluorescence polarization assay for ligand binding.
PhD project Once established the labelled fully-modified PrP will provide a valuable tool to monitor the association of PrP with a variety of putative ligands as well as to monitor changes in the PrP species during in vitro amplification processes, giving the student experience in a variety of biophysical and biochemical techniques. In collaboration with Programme 6 the labelling could be extended to include using nanogold to selectively label the PrP, enabling visualisation of its interactions with amyloid beta or with other PrP molecules by electron microscopy.
Programme 8: Characterisation of Aβ and tau strains
Background The two main proteins involved in Alzheimer’s disease, amyloid-β (Aβ) and tau, aggregate in oligomeric and fibrillar forms causing impaired synaptic function, neuroinflammation and neuronal loss, which eventually lead to the full expression of dementia. The precise disease mechanism and the links between these two proteins are still unknown. We are interested in the formation, propagation and spread of Aβ and tau seeds in vivo, and how they influence each other’s pathologies.
Rotation project The project will involve to test the toxicity and aggregation capacity of Aβ and tau seeds from different brain homogenates. We use biosensor cells to quantify tau and Aβ aggregates. The student will learn cell culture techniques, live-imaging, immunostaining and confocal microscopy to characterise the samples. Primary neuronal cultures will be used to determine the toxicity of the seeds.
PhD project The aim of the project is to purify and characterise Aβ and tau seeds present in Alzheimer’s disease brains and mouse models of the disease. Biochemical techniques will be employed to obtain different fractions to be assessed in vitro for toxicity and aggregation by using cell lines and primary cultures. Inoculation of the seeds into Alzheimer’s disease mouse models will provide further confirmation of toxicity and propagation in vivo. Subsequent transmission of the seeds into mice will show if they maintain their specific characteristics and can be classified in strain types.