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Elucidating DAGL function in neural stem cells

Reference	BB/D007887/1
Principal Investigator / Supervisor	Professor Patrick Doherty
Co-Investigators / Co-Supervisors	Dr Emma-Jane Williams
Institution	King's College London
Department	Wolfson Centre for Age Related Diseases
Funding type	Research
Value (£)	274,029
Status	Completed
Type	Research Grant
Start date	01/04/2006
End date	28/02/2010
Duration	47 months

Abstract

We have recently cloned and characterised the first sn-1-specific diacylglycerol lipases (DAGL alpha and DAGL beta). When present in cells, the enzymes allow for a novel-signalling axis that can couple the FGFR to the endocannabinoid pathway to promote axonal growth. These enzymes hydrolyse DAG in response to growth factor signalling, and in doing so generate 2-arachidonlyglycerol (2-AG), the most abundant endocannabinoid in the brain. As well as acting as a full agonist at the two know cannabinoid receptors (CB1 and CB2), 2-AG is a precursor for several other highly active lipid-based second messengers (e.g. arachidonic acid and its derivatives). We have now discovered that both enzymes are highly regulated in terms of their expression not only between cells, but also in their compartmentalisation within cells. For example, during development both enzymes are in neuronal axons, but in the adult they become restricted to neuronal dendrites. A surprising observation, that forms the basis of this proposal, is our finding that both enzymes are expressed by neural stem cells (NSCs) in the adult brain, but not in the differentiated progeny of the NSCs. Furthermore, we have found that pharmacological inhibition of the enzymes is associated with a dramatic inhibition of neurogenesis in the adult brain. Our hypothesis is that DAGL activity controls fundamental aspects of neural stem cell biology by orchestrating the nature of the post-receptor second messenger response to growth factors. More specifically, we postulate that when DAGLs are present receptors for the growth factors will couple to pathways that maintain the stem cell in a viable, but undifferentiated state; whereas when they are absent, the same receptors will couple to pathways that promote cell proliferation, differentiation and/or linage restriction. The hypothesis is underpinned by strong preliminary data and will be rigorously tested by addressing a series of fundamentally important lead questions.Each question uses well-established methodologies and is formulated in a manner to give unambiguous data. The lead questions are: - (1) To what extent does DAGL activity control intracellular signalling in NSCs? This will be determined inhibiting DAGL activity in cultured NSCs followed by quantitative western blotting to measure the phosphorylation status of ~ 80 sites on effector molecules whose function is regulated by phosphorylation. (2) How does DAGL signalling regulate intracellular signalling in NSCs? We will select the key phosphorylation events regulated by the DAGLs and use standard pharmacological approaches, together with siRNA, to identify the upstream and downstream components of the signalling pathway. (3) Does DAGL activity influence the survival, proliferation and/or differentiation of NSCs? We will use standard methods (cell survival, proliferation and differentiation assays) to determine the effects of loss of DAGL function on cultured NSCs. (4) What impact does loss of DAGLa and DAGLb have on neurogenesis in vivo? We will quantify neurogenesis in post-natal and adult brains of knockout mice that no longer express DAGLa , DAGLb or the CB1 cannabinoid receptor. Most of the tissues have already been banked for this aspect of the study.

Summary

If our brain gets damaged it is not very good at repairing itself. For many years' people believed that our brain could not repair itself because it was unable to make new neurons to replace damaged neurons. However, about 10 years ago, scientists discovered that the brain can make new neurons and that it probably does so throughout our life. So, the big question now is if the brain can make new neurons, how come it still cannot repair itself? Perhaps the adult brain is not as good as making new neurons as it might be, and if we were able to help it to do this, then we might be able to promote brain repair. If this were to be the case, we might be able to develop new treatments for people who suffer a stroke, or people who have diseases like Parkinson's disease. The cells that make the new neurons are called neural stem cells (NSCs). We know very little about how they are controlled, and in particular what keeps them alive and what determines how they make new neurons. However, there is evidence that 'growth factors' are important. These molecules bind to receptors on the NSC and tell the cell what to do. The involves the NSC making 'second-messengers' in response to the growth factor, and it is the nature of the second messenger response that will determine whether the NSC stay's alive, dies, or divides to make two 'daughter cells'. Importantly, when the NSC cell divides asymmetrically only one of the daughters is identical to the 'mother', in other words it is born as a new NSC. In contrast, the other daughter does not inherit the same features as the mother, and it is born as a different type of cell. This new type of cell is no longer a NSC, and it in turn cannot make any new NSCs. This is because it has started to 'differentiate' in order to take on the job of making the new neurons. In order to understanding what controls the 'life-cycle' of the NSC, we need to know what the differences are between the NSC and the 'differentiated' daughter cell. In particular, we need to know if they differ in their ability to mount what scientists call 'the post-receptor second messenger response' after they bind the same growth factor. Very recently, we have found that the NSCs contain important enzymes called DAG lipases, and that these enzymes do not appear to be in the differentiated daughter cell. This is an exciting observation as the DAG lipases are enzymes that can control the post-receptor second messenger response. These enzymes will allow the NSC and its daughter to make a completely different set of messengers from each other even when the two cells bind the same growth factor(s). This in turn might hold the key to understanding why these cells are different from each other. We think that the presence of the DAG lipases is required to keep the NSC alive and our experiments over the next couple of years will tell us if we are right or wrong. Most of the important questions are best addressed using NSCs that can be grown outside of an animal in a simple culture dish. The work supported by this grant will allow us to determine to what extent the DAG lipase activity determines the type of second messenger that the cells make. We will also identify the important messengers that the DAG lipases make and determine how these messengers influence the survival, proliferation and/or differentiation of NSCs. Finally, we will examine intact mouse brains to see if what we learn from the study of the cultured NCS is pertinent to the living animal. At the end of the study we hope to understand exactly how the DAG lipases control NSC function, and by doing so start to think about strategies aimed at promoting the formation of new neurons in the adult brain.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Ageing, Neuroscience and Behaviour, Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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