PaRTiS: Peripheral RNA Translation in Sensitisation

Our bodies have a natural response to pain, which helps us avoid harmful things. When we touch something sharp, we feel pain and quickly move our hand away. This is because special nerve cells in our body detect the painful event and send a message to our brain to make us feel pain. Pain is an unpleasant feeling, but it's important because it tells us that something is wrong, and we need to take action to protect ourselves.

When we get an injury, like a cut or a bruise, our body sends extra blood to the area to help it heal. This can cause the area to become swollen and painful, even after we've removed the entity that caused the injury. This is called sensitization, and it is a natural way for our body to protect the injured area while it is healing. However, sensitisation can sometimes last for a long time, even after the injury has healed, leading to maladaptive responses and chronic pain.

We don't fully understand how our body co-ordinates sensitization, or what make the sensitisation last longer, but we do know that there are many different proteins involved. Some of these proteins can make the nerve cells in our body more sensitive to pain. We want to understand how these proteins work and where they are located in the nerve cells, so that we can find ways to stop chronic pain from happening.

Nerve cells have a very complex structure, with elongated projections that can reach up to a meter and extend to all tissues in the body, understanding their function requires the study of their different morphological parts independently. In fact, the very endings of these nerve cell have the capacity produce proteins according to their functional needs. We have used modern approaches in tissue culture and sequencing to identify some potential targets (mRNA) that might be locally synthetised in the nerve endings to produce proteins involved in sensitization. This project aims to test them to see if they really do play a role and unravel their molecular mechanisms.
To do this, we are using a simple model system - fruit flies.

Although fruit flies might seem very different to mammals like us, their nerves cell are very similar to human nerves in structure and function- they use similar molecular mechanisms and display many key pain signalling proteins in common to those in humans. Drosophila has been proven as a valid model to study many processes including pain sensation ( nociception) and sensitisation. Drosophila larvae respond to pain by rolling away from the noxious stimulus, and this behaviour is modulated by inflammation. Moreover the Drosophila equivalent of our skin is transparent so we can mark and visualise our protein of interest.

In this project we will use established and state-of-the-art tools to visualise nerve cell responses to painful stimuli and investigate the role of our identified targets in sensitization. We have established a behavioural assay to study the effects of the novel targets on the fly larvae's rolling behaviour. We can compare how the larvae react to pain in the presence and absence of the target modulators to identify their role in sensitisation. This will help us identify new proteins involved in sensitization.
Additionally, starting from the target with a known role in sensitisation and continuing with the one that we will discover, we will study how they work and how they affect the cells. We will then test our findings in mammalian cells to confirm their effectiveness.

What we learn will help us understand how sensory nerves behave under normal and altered circumstances (sensitisation), and allow the design of new medicines to stop chronic pain form happening.
By working directly on peripheral sensory nerves - that lie outside the brain - better pain-relieving drugs (analgesics) could be created that lack the addictive and psychoactive effects of centrally-acting agents.

Sensory neurons are responsible for sensing and adaptively responding to noxious stimulation. After noxious stimulation, sensory neurons normally return to a basal/inactive level. Conditions like inflammation impair the homeostatic restoration of this equilibrium, leading to hypersensitivity that, if persistent, can lead to chronic pain.

The cellular dynamics that control neurons undergoing sensitisation are poorly understood. Local translation of mRNA at the nerve endings is emerging as a mechanism underlying sensitisation. Missing is a comprehensive atlas of genes underlying sensitisation, their localisation to, and signalling dynamics within, nerve endings during inflammation.

Using next-generation RNA-seq we identified a pool of terminally-localised mRNA in mouse sensory neurons enriched by inflammatory priming. Our proof-of-concept studies validated two genes' ability to mediated neuronal sensitisation.

We will test the hypothesis that inflammation evokes translation of locally enriched mRNA in sensory nerve terminals to mediate neuronal sensitisation, employing a simple, powerful, and ethical in vivo model: Drosophila melanogaster larvae. Both mammals and Drosophila employ conserved channels and receptors to mediate pain processes and sensitisation.

This project leverages cutting-edge genetic and imaging methods to study nociceptive molecular signalling within sensory nerve endings of Drosophila larvae, establishing a new experimental platform to visualise nocifensive behaviour (Obj1), the underpinning subcellular local protein translation (Obj2), and local calcium dynamics (Obj 3) evoked by a noxious stimulus within sensory neuron termini. We will back-translate the newly identified genes for local translational modulation in nociception into a murine model (Obj4).

We will identify novel genes that mediate sensitisation via a local transcriptions at nerve endings, future potential draggable targets in preventing chronic pain.