Divide and rule: localised Ca2+ signalling in sensory neurons

In order to perceive and evaluate the environment, animals and humans are equipped with a special part of the nervous system, the somatosensory system. It includes nerves that run through our body and collect information about surrounding conditions (such as temperature and stiffness of objects we touch) and also about our own body's integrity. These nerves contain various molecular sensors that respond to specific external events (like touching a hot object) by producing miniature electrical signals that are then sent to the brain for interpretation. A single nerve usually possesses a range of different sensors to respond to different stimuli, yet the electrical signals produced by different stimuli are very similar. A major puzzle in the field of sensory physiology is how different types of signals are specifically interpreted by a single sensory nerve cell; the overarching aim of this proposal is to attempt to solve this puzzle.

Based on the wealth of our preliminary data and published work from our group and others, we propose the idea that may explain such signal coding: different signalling mechanisms might be 'packed' in specific spots separated in space at certain areas within the nerve cell. Such physical separation of one signalling machinery from another allows nerve cells to use common signalling events and messenger molecules without 'mixing up' the meaning of the message.

We are focusing on one specific aspect: how a nerve responds to local inflammation, a process that often results in pain (such as toothache or arthritic pain) and how these responses are different form nerve's responses to other sensory stimuli.

Our group's work points to one specific structure formed at junctions between the outer barrier ('plasma membrane') of a nerve cell and a cistern-like organelle called the 'endoplasmic reticulum', which is found in the nerve cell itself. Such junctions form tiny spaces ('nanodomains') which are relatively well separated from the rest of the cell interior and contain a slew of sensors responsible for detection of tissue inflammation. Once these sensors are triggered, the cellular machinery activates the sensory nerve cell and ultimately results in the feeling of inflammatory pain. We have already identified some components of this junctional structure and its overall importance for inflammatory pain. However, hardly anything is known about how these junctions are formed and maintained, how the key proteins within the junction are arranged and how these change in response to inflammation. Our project aims to close this gap in our knowledge with an ultimate goal of learning how to disturb this pathway and separate inflammation from pain. This proposal has three specific aims:

1) Identify key components responsible for communication in the sensory nerve's junctional space.
2) Discover how these components interact when a nerve cell responds to inflammation.
3) Develop techniques to study these junctional spaces at the tips of nerve endings.

We have developed a well-rounded and multidisciplinary approach combining cutting-edge methods such as super-resolution microscopy, proteomics, molecular and cell biology approaches and animal behavioural studies. We are confident that this research will bring our understanding of nerve cell signal coding to a new level of insight. Specifically, we will unravel complex molecular machinery responsible for production of inflammatory pain sensation. This new knowledge may shape future methods for pain treatment.

This proposal focuses on intricate molecular mechanisms underlying inflammatory pain. In pain-sensing (nociceptive) sensory neurons, junctions between the plasma membrane (PM) and endoplasmic reticulum (ER) form signalling nanodomains harbouring molecular machinery for generating local Ca2+ signals. These signals excite nociceptors leading to generation of inflammatory pain. We have identified several key molecules involved in such Ca2+ signaling, including excitatory ion channels ANO1 and TRPV1, pro-inflammatory G-protein coupled receptors, inositol 1,4,5-trisphosphate receptors, as well as molecular components of Ca2+ release activated Ca2+ channel, CRAC. We also identified a scaffolding protein, junctophilin-4, as a crucial molecule necessary for junctional signalling integrity. These findings are revealing, yet the molecular architecture and dynamics of the junctional nanodomains and resident signalling complexes are only beginning to emerge. The goal of this proposal is to elucidate, on a nanoscale level, the junctional nanodomain machinery enabling nociceptors to respond to the inflammatory environment and generate the nociceptive input in a reliable, stimulus-specific manner. We have assembled a versatile toolkit, including single-molecule localization approaches (DNA-PAINT, proximity ligation), live imaging, cell and molecular biology approaches and in vivo tests, for comprehensive interrogation of junctional signalling in sensory neurons, covering molecular, cellular and whole organism levels. We will use this knowledge base and methodology to elucidate the role of junctional scaffold proteins in localised Ca2+ signalling in sensory neurons and reveal the dynamics of junctional nanodomains during the inflammatory activation. Finally, we will investigate junctional Ca2+ signalling in situ, at the nerve terminals. The outcome of this research will reveal key principles of intracellular signal transduction and may offer new insights for pain therapeutics.