Uncovering the role of the ESCRT machinery in neuron pruning

Like a computer or any other complex electronic device our brain needs to be accurately 'wired together' to function properly. Current thinking suggests that disruptions in wiring may underlie a number of psychiatric disorders, so to understand how wiring in the nervous system goes wrong is very important for aiding therapeutic approaches for such disorders. As such this work is likely to contribute to the healthcare sector and thus to our society.
As the nervous system forms, nerve cells grow to make connections with one another and as such are like the wires of an electrical device. When nerve cells grow, they do so in an exuberant manner, often generating many extension branches. Some of these branches enter the wrong territory or are redundant and need to be removed. We call this removal of branches 'pruning'. Pruning of excess or redundant branches is important for adjusting the nervous system so that it can work with precision. In many cases pruning happens by branches being cut off, hence the term 'pruning'. Although we know a great deal about how the nervous system develops, our understanding of how branches are physically cut away remains a mystery.
We have been using the fruitfly Drosophila to try and answer this question. We use flies because they share most of their genes with humans, so what we learn in the fly can be easily translated to humans. At the same time, fruitflies have a relatively simple nervous system that is more accessible and easier to study than more complex nervous systems as found in mouse. Flies are perfect for applying genetics to investigate their biology. For example, fruitflies grow quickly so one can do genetic screens; ie. breaking genes, which is a powerful way uncovering how things work.
In this project we are focusing on pruning in the nervous system. Here, the fly provides a great opportunity, as many of its nerve cells are recycled during metamorphosis, when a maggot turns into a fly. Pruning is important for the recycling process. We study how nerve cells undergo pruning by using fluorescent 'glowing' genes from a jellyfish, which allows us to see nerve cells inside the living animal.
Using these glowing nerve cells and fly genetics we found that a family of proteins called the Endosomal Sorting Complexes Required for Transport (ESCRT) are important for pruning. These ESCRT proteins assemble and together form a machine that cuts cell membranes. Because cells are really a large system of membranes containing different components, this group of proteins is important and used for many different processes in different parts of cells e.g. they are also used when cells divide in two. To schedule these proteins for each specific task, they have very specific adaptors that direct them to work in the right place at the right time. We think that something like the cutting that occurs when a cell divides is also happening during nerve cell branch pruning. We are the first people to have any evidence to suggest that the ESCRT machinery is involved in cutting nerve cell branches. Because this observation gives us a new clue as to the genes and mechanisms that regulate pruning in the nervous system, this is a very important observation.
We would now like to confirm these observations and extend them. We want to know which of the ESCRT family members are important for pruning and which are not; how these machines assemble, whether they are actually needed directly at the place where the branch is cut and how the cutting process is controlled by the adaptors.
We hope our work will resolve part of this mystery and that in future the new insights that our work can help up understand what happens when wiring goes wrong in human disorders of the nervous system, and, by extension what therapeutic approaches could be developed to correct such defects or lessen their impact on mental health and well being.

Pruning is fundamental for the construction of functional neuronal circuits, and its disruption has been implicated in a number of connectivity disorders. The phenomenon of large-scale pruning, where relatively long neuron branches are removed, is found throughout the nervous system. Branches are eliminated not by a distal to proximal retraction event but are deleted by a local degeneration.

Current data suggest that this type of developmental degeneration is controlled by an active program of auto-destruction. This proposal seeks to identify the molecular mechanisms that orchestrate branch cutting in this type of pruning.

Our work exploits the metamorphic remodeling of sensory neurons in Drosophila to reveal the mechanisms that control pruning. We have found that the Endosomal Sorting Complexes Required for Transport (ESCRT) family of proteins are required for neuron pruning. The ESCRT machinery has been found to be essential for a range of processes in many different cellular locale e.g. multi-vesicular body genesis, cytokinetic abscission and viral budding. The function of these proteins is conserved from yeast to man. This is the first time the ESCRT machinery has been shown to play a role in pruning.

The outcome of this work will be the identification of a specific ESCRT pruning module, whose function appears conserved in different neurons within Drosophila. It will test its requirement within the dendritic compartment where branch severing takes place. Live imaging studies will also give key insights into the dynamic recruitment of the 'ESCRT pruning module'. We will also assess, at a detailed molecular level, the interactions between the ESCRT-III scission machinery and the adaptor protein Mop. Together, this project will provide a real 'step-change' in our understanding of how branches are cut during neuron pruning.

Who might benefit from this research and what might they benefit from?

Academia;
In recent years the biology of neurite auto-destruction has become a growth research area in Neuroscience (i.e. International Workshop on Molecular Mechanisms of Axon Degeneration). Labs who study this problem both nationally and internationally will use our techniques, tools and ideas. We also expect the techniques that we bring forward will encourage labs that primarily use vertebrate models to transfer certain questions to Drosophila because of the benefits that this model system offers. As such our work fully complies with the 3Rs remit. As ESCRT biology touches nearly every aspect of the development and physiology colleagues working on this pathway in Drosophila will benefit from the reagents we make. Any DNA constructs generated will be deposited with the non-profit distribution service Addgene to aid dissemination and fly stocks will be made available to the Drosophila research community.
I am currently collaborating with three individuals within my department, one of which is funded by the BBSRC, and have ongoing international collaborations with Dr Yusuke Toyama at NUS/MBI Singapore and with DrJames Truman Janelia Farm HHMI, VA USA, all these collaborations will benefit indirectly from this support.

Public Sector;
We are actively talking to clinicians studying connectivity disorders. I have been in regular contact with Dr Marco Catani (Neuroanatomy and Tractography Laboratory, Institute of Psychiatry). His lab studies anatomical and functional aspects of the simian and human brain connectivity). We have been openly discussed our respect research programmes and goals within the field.

Business/Industry;
We have established contact with Brainwave-Discovery Limited, who develop fly models for use in the pharmaceutical industry. In collaboration with Brainwave-Discovery Limited we have the opportunity to test drug compounds to identify those, which will regulate ESCRT-dependent neuronal pruning.

Third Sector;
I have established a dialogue with Dr Chris Manning, a former GP who is promoting the dissemination of basic research findings in neuroscience to a audience of GPs through the charity College of Medicine (http://www.collegeofmedicine.org.uk/). In Spring 2014 I will present our current research and provide an overview of connectivity disorders to this group.

Public outreach;
I am active in public engagement and take my role as a 'ambassador for science ' seriously. This year my lab spent a weekend in the Science Museum, London, explaining to the public how we use Drosophila to study the nervous system. We demonstrated state-of-the-art optogenetic techniques and used hands-on experiments mutant flies to explain how synaptic transmission works (these mutants become paralyzed when warmed up in your hand). I am currently involved in the Science Uncovered project at the Natural History Museum and will take every opportunity to continue this dialogue with the public in the coming years.

Art/Culture.
With the artist Andrew Carnie we hope to develop an ambitious large-scale synchronized video projection installation exploring ideas of how the dendritic form in our bodies mirrors forms in the nature. Funding will be sought through an Art Council bid.
Alongside this I have established project with Shobana Jeyasingh (http://www.shobanajeyasingh.co.uk/). Shobana is acclaimed for her pioneering work in choreography. Our collaboration explores how discipline-specific perspectives and language can allow powerful reinterpretations of dance and performance.

Schools;
This last year I have developed teaching tools for Key Stage 2, Sc2 Life processes and living things. I have test-run this with two different classes of 10 year olds, teaching cell biology and lifecycles using Drosophila. In 2014 I will take this into schools in the South London area that are struggling to motivate students in science topics.