A new negative regulator of autophagy in cellular and organismal homoeostasis

During life, mammals have to keep their bodies and minds in a state of constancy, as much as possible, despite all sorts of challenges. For example, animals are exposed to periods without regular food, with inadvertent exposure to infectious bacteria, and with all sorts of insults that contribute to faster ageing, and the decline of important organs and of cognitive abilities. In the latter examples this includes the accumulation of damaged cellular components, e.g. proteins (including so-called 'unfolded' proteins that have the wrong shape and can stick to each other, forming toxic deposits) and other, larger cellular components, often composed of many hundreds of types of proteins and other molecules (e.g. 'organelles'). Given this, it is remarkable how well mammalian physiology copes with these problems. We refer to maintenance of this "constant state" as homeostasis.

In the applicant's lab, we study a process called autophagy. This is a system for disposing of unwanted components of cells in packets ('garbage bags') called autophagosomes - for example the damaged proteins and organelles referred to above - and other garbage - such as bacteria infecting individual cells (such as occurs, for example, in the lining of the gut on Salmonella infection). Autophagy operates in virtually all bodily tissues. Autophagy famously goes awry in certain diseases, e.g cancer, inflammation, neurodegeneration. However, it has become increasingly clear that autophagy plays a major role in homeostasis, i.e. the maintenance of the normal state of cells in animals, and thus the animal as a whole. Animals require autophagy to fight bacterial infection, prevent ageing-related decline of bodily organs and brain functions. Autophagy also has a neat dual function - the 'garbage' can provide energy and building blocks when it is broken down to keep the cells alive when nutrients are short (as occurs during e.g. fasting). Thus animals also require autophagy to bridge gaps in food intake/usage. However, too much autophagy can also be damaging to cells. To this end, cells control the amount of autophagy carefully, although recent studies show that modestly enhanced autophagy could improve homeostatic outcomes, particularly during ageing.

We propose to provide new information on how this autophagy pathway is controlled. We think we have identified a gene that slows down the autophagy process. By testing this idea, by studying at a very detailed molecular level how this gene (CCPG1) acts in cells, and discovering how it impacts on nutrient/metabolic responses and ageing in mice, we hope to both a) cast new light on mechanisms by which this important pathway of autophagy works in cells and b) discover a new gene that - via its link to autophagy - controls important homeostatic outcomes in adult and ageing animals (relevant to mammalian biology, including livestock and human health). This work may subsequently lead to future research proposal to find ways to manipulate the function of this gene to control autophagy and improve homoeostasis.

We will use standard cell biology technique and biochemical techniques that will allow us to see how the product of the CCPG1 gene associates with and directs the interplay of known molecules that drive the process of autophagy. However, we will also use some cutting-edge microscopy techniques (electron microscopic and so-called 'super-resolution' light microscopic) to gain unprecedented insight into how known molecules in the autophagy pathway are being orchestrated. However, it is difficult to know how relevant effects we see in isolated cells are in a whole animal, where many millions of cells interact in complicated organ systems. So, we have made a mouse deficient in CCPG1. We aim to show that homeostatic functions, particularly during ageing or fasting, are different in these mice.

Autophagy is a trafficking pathway that transports cytosol to the lysosome, in vesicles called autophagosomes. This is vital in animal homeostasis. Cytosol can be degraded to provide energy during periods of fasting. Degrading damaged proteins and organelles defends against cognitive and corporeal decline (ageing). Autophagy can also target infectious cytosolic bacteria.

Progress has been made unpicking the 'core' mechanism of autophagy. However, regulatory pathways are less well defined; particularly specific negative regulators. Other outstanding questions include a) the regulation of a protein complex called ULK (includes the scaffold, FIP200, and ULK protein kinase) and b) understanding how a site for autophagosome generation, the ER-localised omegasome, can specify the composition of autophagosomes. Here we propose a new negative regulator of autophagy, a little-studied single-pass transmembrane protein, specific to vertebrates, called CCPG1. By studying CCPG1 function in the autophagy pathway we will shed light on all above issues AND, conversely, provide insight into the physiological function of this molecule.

Specifically, we will use cell biological and biochemical techniques, including time-resolved correlative light electron microscopy to analyse CCPG1 recruitment from the ER into autophagosomes, and the mechanism thereof, via novel interactions with LC3 and FIP200 that will be separated by mutational analysis. We will show that CCPG1 interacting with FIP200 at autophagic membranes (where these molecules are brought together) participates in a novel mode of regulation of autophagy - the dissociation/inhibition of local ULK complex assemblies. To study this occuring in situ, on autophagic membranes, we will use advanced light microscopic techniques (3D-SIM and FRET acceptor photobleaching). Finally, we will use CCPG1 inhibition (RNAi and a new knockout mouse) to study the physiological role of CCPG1 in fasting, infection and ageing/proteostasis.

Beneficiaries of this work beyond immediate colleagues will include (this is just a summary - see Pathways to Impact for how this will be practically achieved/more tangible details):

1) wider bioscience at University of Edinburgh and Bristol, whose knowledge base will be greatly expanded by the use of specialist facilities there (EM, super-resolution microscopy, LC-MS) to address novel and fundamental biological questions. This will raise awareness of the uses, applicability and practicality of such techniques, supported by specific methodological seminars that will be delivered by the PDRA.

2) human and animal health and quality of life via insight into a process important in homoeostasis, ageing and infection, which, in the medium-long term will be built into models developed by these research communities.

3) internationally, the autophagy, general bioscience and animal and human health research communities, by traditional dissemination of new results that impact on our understanding of autophagy and key aspects of organismal homeostasis, and demonstration of new techniques/approaches (e.g. advanced light microscopy) to carry out such investigations.

4) internationally, the fields of infection research, metabolomics and advanced light microscopy, by demonstration of how these techniques can be brought to bear upon a distinct problem from another field, that of new molecule discovery in a fundamental biological pathway to autophagy - to this end we have selected collaborators in each of these fields who have helped shaped the proposal, in particular the cross-disciplinary elements that the collaborations address, and whose own laboratories and work we expect to mutually benefit from these collaborations.

5) UK economy directly and the finances of the University of Edinburgh, via potential commercialisation of CCPG1 reagents, including novel antisera, which will be licensed for use.

6) the UK biomedical industry via potential investigation of CCPG1 as a target for regulating autophagy, leading potentially to programs to develop new agents.

7) the UK science sectors broadly (academic or industry) which will benefit from exceptionally broad training of the PDRA across multi-disciplinary techniques and in other transferable skills such as writing, public engagement and scientific presentation, and including adoption of the role of visiting worker in another institution by the PDRA (from the prinicipal applicant's lab to the coapplicants' labs), further broadening their horizons and organisational/collaborative skills.

8) public understanding of science, particularly how fundamental biological research leads to improvements in health and quality of life for animals and humans, and how this is supported by research council funding. This will be achieved via the dissemination of the work through media channels and by targeted public engagement/science communication activities.