Junctional multiprotein signaling complexes in sensory neurons

In order to perceive and evaluate the environment mammals are equipped with peripheral nerves (peripheral somatosensory system). These nerves run through our body and collect information about rigidity, warmth and chemical composition of the surrounding milieu and also about our own body's integrity. These nerves are equipped with various molecular sensors that respond to specific external stimuli, transforming these into the uniform electrical impulses ('action potentials') that are then sent to the brain for interpretation. Single somatosensory nerve often expresses a variety of different sensors or sensory mechanisms that respond to distinct stimuli, yet the output signals produced by a nerve are very similar. A major conundrum in the field is how different types of signals are specifically interpreted by a single sensory nerve cells; the main aim of this proposal is to shed light on this question.

Based on the wealth of preliminary data and published work from our group and others we hypothesize that one mechanism for such intracellular signal specificity lies in the assembly of different intracellular signaling mechanisms into distinct, physically associated protein complexes. Such physical separation of one signaling machinery from another allows them to use common signaling events and messenger molecules without 'mixing up' the meaning of the message. We will focus on one such multiprotein complex which is responsible for body's detection of tissue inflammation (i.e. inflammatory pain). We have already established that there are intricate multiprotein signaling complexes in some sensory nerves that bring together receptors for chemical mediators of inflammation and some signaling proteins that are targeted by these receptors. However, hardly anything is known about the overall constitution of these complexes, functional arrangements of their components, relationships with other signalling mechanisms, whether these complexes are dynamic or static or whether these can be manipulated for therapeutic benefits. Our project aims to answer these intriguing questions. We have three specific aims: 1) to reveal molecular composition of the inflammatory multiprotein signaling complexes in sensory nerves; 2) to elucidate functional significance of these complexes; 3) to develop strategies for manipulations with complex integrity for scientific and therapeutic purposes.

We developed a comprehensive and multidisciplinary approach in which fidelity, specificity and localization of neuronal communication mechanisms will be elucidated in their complexity. This approach combines cutting-edge methods such as Nobel Prize winning super-resolution microscopy, proteomics, molecular and structural biology approaches and in vivo studies. We are confident that this research will bring our understanding of mammalian sensory systems and, particularly, of inflammatory pain mechanisms, to a new level of insight. Importantly, our findings may shape new approaches for analgesic drug development and pain management.

Mammalian somatosensory neurons combine functions of versatile detectors and communication devices; they express molecular sensors for a great variety of stimuli, such as touch, temperature, tissue damage and various chemical mediators of inflammation. In the latter case these mediators often act by triggering intracellular signaling cascades. A central conundrum in intracellular signaling is that a handful of intracellular signalling molecules orchestrate thousands of diverse actions, some of which even producing opposite results in neuronal function. Therefore, robust mechanisms mediating precise coupling of specific intracellular signals to particular outputs must be in place. Our central hypothesis is that such specificity arises from spatially-restricted intracellular signalling in multi-protein complexes. In this project we aim to build upon our recent findings on the specificity of intracellular signaling in neurons to elucidate and mechanistically explain spatially restricted intracellular signalling produced by bradykinin receptors in 'pain' (nociceptive) sensory neurons. Our prime focus will be on the, as yet little understood, junctional multiprotein signaling complexes (JMSC) assembled at junctions between the plasma membrane (PM) and the endoplasmic reticulum (ER). We have the following specific aims: 1) to reveal molecular composition of the JMSC in sensory neurons; 2) To elucidate functional significance of JMSC; 3) to develop strategies for manipulation with the JMSC's integrity for scientific and therapeutic purposes. Our experimental strategy combines cutting-edge imaging, proteomics, molecular and structural biology approaches and in vivo studies; this powerful toolkit will likely to deliver a qualitative leap in our understanding of inflammatory G protein coupled receptor signaling in sensory neurons.

The research project outlined in this proposal seeks to build a mechanistic understanding of signalling complexes of mammalian peripheral somatosensory neurons and to reveal principles of specificity of somatosensory signaling on a nanoscale level. Moreover, the project may identify new targets for pain management and therefore it aligns with BBSRC's Strategic research priority "bioscience for health" and its key priorities "Generate new knowledge of the biological mechanisms of development and the maintenance of health across the lifecourse" and "Develop and apply new tools in areas such as chemical biology, high resolution structural analysis, 'omics, biomarkers and bioimaging, high throughput and comparative genomics and modelling". The project also aligns with the following strategic priorities: "Animal health", "Technology development to the biosciences" and "Systems approach to biosciences". Thus, the results of this study will have impact by directly addressing BBSRC's priority areas and helping to fulfill the BBSRC mission.

At a fundamental level and in the short-term, our research will be of interest and benefit to other researchers, including physiologists, biochemists, cell biologists and neurobiologists with an interest in mechanisms of intracellular signalling, sensory physiology and pain. We believe our work will lead to the further elucidation of such mechanisms. We hope to develop tools that can be used to disrupt specific macromolecular complexes and these tools can potentially be used in further research and for therapeutic benefits. We will aim to exploit any such opportunities ourselves, but such findings will also appeal, in the short to mid-term, to other academics and pharmaceutical companies.

In the mid- to long-term, we anticipate that any therapeutically useful approaches arising (e.g. based on the use of competitor peptides for disruption of junctional multiprotein signaling complexes) will inform rational drug design aimed at disrupting intracellular signaling cascades in sensory nerves leading to inflammatory pain and 'nerurogenic' inflammation.

In the long term, our research will be of primary importance to patients suffering from pain. The British Pain Society estimates that some 10 million individuals in the UK suffer pain on a daily basis, resulting in a major impact on life quality and the ability to function normally without distress. Many chronic pain conditions, such as arthritis, have inflammatory origin and are associated with ageing. Any form of practical pain relief arising from our studies will therefore have significant impact on this large fraction of the population in terms of improving quality of life and their ability to contribute to society through practical activity, including working. Development of new therapies arising from fundamental scientific research focused on specific molecular targets in sensory nerves is a long-term project, involving years of effort in the research laboratory, drug development industry and clinical trial arena. Given the widespread prevalence of pain within the population, and its global economic, social and cultural impact, such investment must be considered worthwhile.

Additional impact will be delivered through the provision of skilled people to the workforce. The postdoctoral fellow and technician will receive training in cutting edge methods and approaches (superresolution microscopy, in situ proteomics, electrophysiology, imaging, behavioural approaches etc.); they will be able to apply these skills in their future carriers. Additionally, a number of undergraduate students (4-5 per year) will be able to participate in cutting-edge research during their final-year research projects in applicants' laboratories.