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

对于复杂系统，如突触网络，现实但简化的模型将促进基础和应用研究，旨在加深我们对大脑的理解，并测试新试剂和药物的疗效和副作用。本项目的目的是优化和开发Woolfson实验室开发的hSAF(水凝胶自组装纤维)多肽水凝胶系统，在Henley实验室培养大鼠神经元，并在体外形成定向突触网络。一旦建立，这些排列的培养物将为专门研究突触前或突触后事件提供一个新的极其有用的工具。这将使用Henley实验室中已有的病毒载体来shRNA击倒一个或多个感兴趣的蛋白质，并在适当的情况下，使用分子替换策略来表达shRNA不敏感的WT或突变的荧光团标记蛋白。该项目的主要目标将是开发和使用定制设计的水凝胶，其中包含特定的轴突或树突生长线索，以排列和协调种植到基质中的海马神经元或皮质神经元的生长和突触。最初，这些将是线性的，但目标是利用伍尔夫森实验室正在开发的技术开发一种环状支架。这将为封闭和定向的神经元网络提供支架，类似于单个自闭式培养神经元，但更有用的是该网络将由可识别和排列的神经元组成，每个神经元都可以单独操作。成功的概念验证实验已经表明，PC12细胞在和hSAF中生长良好，并修饰了纤维连接蛋白RGDS基序，介导细胞与细胞外基质的附着。随着项目的发展，我们打算探索其他基序来实现hSAF的功能；例如，在水凝胶的特定区域掺入BDNF或netrin可以促进和吸引轴突生长和突触形成。

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.