Utilising De Novo-Designed A-Helical Peptides As A Modular Cytoscaffold For Primary Neurons And Sensor For Small-Molecules

Realistic, but simplified models for complex systems, such as synaptic networks, would facilitate both basic and applied research geared at developing our understanding of the brain, and testing the efficacy and side effects of new reagents and drugs. The aim of this project is optimize and exploit the hSAF (hydrogelating self-assembling fibers) peptide hydrogel system developed in the Woolfson lab to grow cultured rat neurons and form directionally orientated synaptic networks in vitro in the Henley lab.
Once established, these aligned cultures will provide a new and extremely useful tool for investigating specifically pre- or postsynaptic events. This will be achieved using viral vectors already available in Henley's lab to shRNA knock-down one or more protein of interest and, where appropriate, use molecular replacement strategies to express shRNA- insensitive WT or mutated fluorophore-tagged protein.
A major goal for the project will be to develop and use custom designed hydrogels incorporating specific axonal or dendritic growth cues to align and coordinate the growth and synapses of hippocampal or cortical neurons seeded into the matrix. Initially, these will be linear but the aim is to develop a torus-shaped scaffold using techniques in development in the Woolfson lab. This will provide the scaffold for a closed and orientated neuronal network analogous to single autaptic cultured neurons, but much more useful because the network will comprise identifiable and aligned neurons, each of which can be individually manipulated.
Successful proof-of-concept experiments have already shown that PC12 cells grow well in and hSAFs decorated with the fibronectin RGDS motif that mediates cell attachment to the extracellular matrix. As the project develops, we intend to explore of other motifs to functionalise the hSAFs; for example, incorporation BDNF or netrin within specific regions of the hydrogel promote and attract neurite outgrowth and synapse formation.