A platform for high throughput, cell type-restricted in vivo knockdown of pre- or postsynaptic gene expression

One of the most challenging problems in science is to understand how the molecules expressed by nerve cells in the brain enable thoughts and actions to take place. Addressing this challenge is of fundamental importance for understanding how brains work. It will also underpin future development of therapies for neurological and psychiatric disorders, and of biologically inspired computing technologies. However, while cells in the brain are very much like those in other organs, figuring out how molecules in cells contribute to brain functions is exceptionally challenging because of the brain's great complexity. Conventionally one could study the function of a molecule by finding drugs that bind to it, or by engineering animals from which the molecule is deleted. However, many brain functions involve signals being passed between different types of cell that are found nearby one another. With conventional approaches it is usually difficult and very time consuming to figure out which cell type mediates a molecules effects. Moreover, with these approaches a single model animal can usually only be used to study one molecular target.

To address these issues we propose to develop a system for relatively low cost and efficient investigation of the role of any given molecule in signalling between a particular population of neurons and identified neurons with which they interact. This system will use viruses to introduce two types of genetic sequence into neurons. The first encodes short interfering RNAs. These are short genetic sequences that contain recognition sites that enable them to "knockdown" targeted molecules. They can be designed to knockdown expression of almost any molecule of interest. The second type of genetic sequence encodes proteins that act either as fluorescent labels or as light-sensitive neuronal activators. These proteins can be used to identify and control infected neurons. The particular power of our system comes from a novel approach we will use to control the cells in which these two types of genetic sequence are expressed. This system makes expression of both types of sequence require the presence of marker molecules called Cre and Flp. By introducing viruses into animals in which Cre and Flp label cell populations of interest, we can target expression of the virally delivered proteins and interfering RNAs to these populations.

We propose to validate this new approach by developing new tools aimed at studying neural circuits in a brain area called the entorhinal cortex. This region is important for spatial cognition. Focussing on this region allows us to take advantage of well established experimental assays, while generating tools that should enable us to address previously very challenging questions. Our first aim will be to generate and characterise tools that knockdown expression of particular ion channels in only a single type of cell. Our second aim will be to make tools that knockdown expression of receptors at either the up- or downstream side of a connection between two distinct populations of neurons. The tools will also allow the upstream neurons to be activated specifically with light and the downstream neurons to be identified by their fluorescence in order to guide subsequent electrical recordings. Our third aim will be to carry out preliminary work to extend our approach to investigation of multiple molecular targets in parallel in the same animal.

On completion of the project we aim to have introduced and validated a new toolset for high throughput and low cost investigation of the roles of signalling molecules at connections between defined neuronal populations. The platforms that we aim to establish will be of general utility for fundamental and applied research into molecular mechanisms of signalling between cell populations. Applications include investigation of mechanisms of cognitive function in the young and ageing brain, and development of novel models for drug development.

Identification of molecular mechanisms is critical to progress in biological research and its extension to industrial applications. Current approaches to manipulating gene function in genetically defined cell populations are expensive, time consuming and require large numbers of animals. Tools for viral delivery to genetically defined neuronal populations of siRNAs co-expressed with markers or optogenetic activators are not yet available. Thus, we aim to develop tools for rapid, low cost and specific in vivo molecular manipulation of interactions between genetically defined, spatially intermingled cell populations.

We will first develop new constructs and viral vectors for knockdown, using siRNAs, of genes in cell populations defined by expression of Cre. We will establish proof of principle with experiments evaluating in vivo knockdown of HCN1 channels. We will next extend this system to enable investigation of interactions between two genetically defined neuronal populations. We will develop vectors for knockdown of genes in cell populations defined by expression of Flp. We will combine these vectors with optogenetic activation of presynaptic neurones defined by expression of Cre. To demonstrate the utility of this approach we will test the contribution of pre- and post-synaptic GluN1 to synaptic responses of interacting populations of glutamatergic neurons and interneurons. Finally, we will aim to establish a proof of principle for multiplexed manipulation and evaluation of target molecule functions.

On completion of the proposed work we expect to have developed well validated tools for gene knockdown in defined cell populations in vivo and to have established exemplar pipelines for application of these methods to fundamental and applied neuroscience questions. The platforms that we aim to establish will be of general utility for fundamental and applied research into molecular mechanisms of signalling between genetically defined cell populations.

The proposed work will establish new tools with the potential to transform our understanding of brain function and approaches to drug development. Applications include fundamental neuroscience research, applied investigation of lifelong health and ageing, and development of new models for drug discovery. Potential beneficiaries range from technology and pharmaceutical industries in the commercial private sector, through researchers in applied fields, in particular those oriented towards lifelong health solutions, to the general public as a whole. We describe below benefits in each area of impact. In some cases the critical path to impact may be direct, for example by immediate commercial application of our research outputs. In other cases it will be through application of the newly developed tools to applied research either in industry or academia. In our separate Pathways to Impact statement we describe diverse activities that we will employ to facilitate impact in each area. Impact ion our immediate research area is outlined elsewhere in the proposal.

1. It is widely recognised that the drug discovery process is severely hindered by a lack of basic understanding of the cellular and molecular organisation of brain areas important to healthy neurological and mental function. Biotechnology and pharmaceutical industries will therefore benefit from the new knowledge and research tools generated by the project. For example, entorhinal cortex function, GluNs, HCN channels and mechanisms controlling the balance of excitation and inhibition in the brain are all of potential commercial interest because of known roles in many disorders.

2. Alterations in neural circuits are believed to be central to cognitive changes that accompany ageing, but our lack of understanding of these circuits at a molecular level is a substantial obstacle to establishing which aspects of the ageing process one should focus in order to promote healthy ageing. Our validation experiments focus on the entorhinal cortex, which is one of the brain areas believed to be most important for cognitive changes that accompany ageing. Our new tools and results of our validation experiments will therefore be of benefit to a wide range of specialists and organisations with an interest in developing strategies to promote healthy ageing.

3. The proposed project will also contribute to UK capacity building in synthetic biology and in systems biology. This has been identified by the BBSRC as a strategic priority of long-term benefit to the UK. The proposed work will provide training for the postdoctoral research associates employed to work on the project and for PhD, Masters and undergraduate students who will have the opportunity to work on experimental systems that develop from the project. The University of Edinburgh is particularly well placed for the project to contribute to postgraduate training, with several successful PhD and Masters programs, both within the host School (Biomedical Sciences) and within the School of Informatics.

5. Understanding the brain is widely recognised as one of the most important challenges in modern science and public demand for knowledge of how our brains work is reflected in the high media profile given discoveries in neuroscience research. Because of their relevance to human cognition and ageing, the results of the proposed study therefore have the potential to contribute to public engagement with systems biology and research into the brain.