Combining viral and ribosomal mRNA capture technologies to develop a versatile system for neuronal transcriptome profiling

It is very difficult to study the function(s) of specific groups of neurons because the brain contains thousands of different cell types that are juxtaposed and interconnected and vary according to their size, shape, projections and function. Recently a method has been developed that allows all the molecules (messenger RNA) that are being translated into proteins to be in defined in specific neuronal populations. This is a very powerful tool as for the first time the sets of genes that are governing neuronal function (e.g. those controlling the formation of memories) and those that are altered with age and in human neurodegenerative and neuropsychiatric illnesses can be identified. However, this method is dependent on costly transgenic mice lines, and each time a new scientific question is asked a new mouse line needs to be generated. The technique itself is quite complex, and time consuming due to the need to develop and breed (and in many cases cross breed) one or more mouse lines. These considerations severely limit the availability of this technique to researchers and instead a combination of less powerful approaches must be used.
In this study we are combining the expertise of Takeda and the University of Bristol to develop two new methods. These methods use viral vectors instead of transgenic mice (called viral TRAP) and allow neurons to be profiled with a speed, precision and versatility not previously possible with transgenic mice alone. These viral TRAP methods will also result in fewer animals being used by researchers.
The viral TRAP technique will be used by the University of Bristol to identify the genes and proteins a family of RNA binding proteins called scaffold attachment factors (SAF) regulate. In particular this information will be used to understand how SAF proteins govern the processes that regulate memory formation and ageing. The viral TRAP technology will enable Takeda to perform detailed profiling experiments without the need for lengthy, expensive and animal-intensive transgenic programs. Importantly as the viral TRAP technique is portable to other models that Takeda currently use the technology will provide a novel platform that will greatly facilitate both basic biology and future pharmacological response studies. Ultimately, the objective of Takeda is to use this technology to further the understanding of neuronal gene regulation and homeostasis in response to challenge, and to use these insights to identify novel targets for central nervous system disorders.

Our objectives are to combine the complementary expertise of Takeda and Bristol to develop two novel and highly versatile TRAP methods based on (1) a combined lentiviral and transgenic mouse approach (bac-virTRAP) and (2) a stand-alone retrogradely transported lentiviral system (virTRAP). These systems will be used to study scaffold attachment factor (SAF) protein function.

(1) bac-virTRAP - This approach will also use a bac-transgenic mouse line that drives expression of EGFP-l10a transgene expression specifically to dentate gyrus (DG) neurons and the floxed EGFP-l10 mouse line. To investigate SAFB1 function, which is expressed at high levels in the DG, (confirmed by Takeda from previous profiling experiments in this line) we will use VSVG pseudotyped neuron-specific lentiviral vectors to transduce dentate gyrus neurons and deliver synapsin promoter-WPRE-miR30-shRNA cassettes against SAFB1. We will also use viral vector cassettes that simultaneously deliver CRE recombinase and shRNAs and these will be injected into the dentate gyrus of floxed mice. Hence following its injection into the hippocampal dentate gyrus region of loxP-EGFP-l10a mice we will only profile neurons where EGFP-l10 has been activated and SAFB1 knocked down These approaches avoid the need to develop and breed a second (e.g. SAFB1) knockout mouse.

(2) virTRAP - A retrogradely transported rabies hybrid protein pseudotyped lentivirus will be made and used to express EGFP-l10 and a second transgene (an anti-SAFB1 shRNA). Importantly, the retrogradely transported vector TRAP (virTRAP) system can be used in rats and mice and will allow neurons to be defined and profiled according to their anatomically distinct projection patterns. Rat and mouse specific l10a vectors will be made. It is also important to note that these lentiviral vectors will only mediate retrograde transport (most AAV vectors are also transported anterogradely) and will not induce an immune response

Who will benefit from the research?

This research will benefit: (i) local researchers interested in collaborating on the use of the powerful new viral TRAP techniques; (ii) the academic RNA and neuroscience research community; (iii) those in the field of educational science; (iv) the research staff employed on the grant will benefit from training in bioinformatics (v) members of the general public with an interest in brain function and disease; (vi) Industrial partners.
How will they benefit?
(i, ii & iii). The research will be disseminated through peer-reviewed journals within the standard timescale. The research will be presented to the scientific community at national and international conferences. The impact of the research will also be increased once disseminated to our existing collaborators (and upon the establishment of new collaborations): these include collaborations within Bristol (School of Physiology and Pharmacology, School of Clinical Sciences, Frenchay Hospital), Cardiff University Medical School, and our extensive network of industrial collaboration and with University College London. The applicants have experience in presenting the research to the Media, scientific community and wider public audiences including school children through public engagement seminars organised by Bristol Neuroscience. Specific academic beneficiaries have also been described in the appropriate section of this proposal.
(iv) - Staff (Dr Scott) - employed on the project will be trained in using bioinformatics packages to interrogate large transcriptomic data files. Our local collaborators can then also use these techniques. Dr Scott will also communicate our findings to scientific and general audiences through public engagement opportunities; team working and networking.
(v) - The public - findings from this project in the short term will allow us to explore the relationship between the mechanisms governing gene expression and how altered function could lead to the formation of human neurological disease. The brain is a very important organ, commanding special interest from the public, because it holds our memories and governs our behaviour and perceptions. My laboratory gave talks and run workshops at the Bristol Neuroscience festival (Oct 2013) and this great event was extremely popular to a diverse public audience. Other neuroscience activities led by Bristol researchers - e.g. during Brain Awareness Week (a biennial hands-on research festival with a total audience of 4,700) - are equally popular with public audiences, as are public talks on neuroscience topics held regularly by Bristol Neuroscience (BN) http://www.bristol.ac.uk/neuroscience/society/public-past.
(iv) The viral TRAP technology that we develop in the course of this grant will enable Takeda to perform detailed profiling experiments without the need for lengthy, expensive and animal-intensive transgenic programs, thereby shortening timelines to data and greatly reducing animal usage. Importantly the viral TRAP technique will be portable to rats; this is out of reach of Takeda's technology as it currently exists so will provide them with a unique platform to advance basic neurobiology and in vivo pharmacological response studies. Ultimately, the objective of Takeda is to use this technology to further the understanding of neuronal gene regulation and homeostasis in response to challenge, and to use these insights to identify novel targets for central nervous system disorders.