Targeting circadian mechanism for novel chronic pain treatments

Chronic pain is a huge burden for society. Treatments are not good enough and difficult to develop. Sensory information, such as touch and pain, is carried by specialised nerves called peripheral sensory neurons.

We have powerful in-built mechanisms for regulating our perception of pain. This includes clocks present in our body which helps us align to the 24 hour day and night cycle. It is quite well know that many pain conditions worsen or improve at particular times of the day. Understanding why pain can flare up and settle down in this way might offer new ways to treat and manage chronic pain. It may also help patients understand when their pain condition is likely to exacerbate and to then develop better coping strategies and allow better timing of medical pain treatments.

We have discovered in mice that peripheral nerves, including pain nerves, have an internal clock. We now intend to investigate the clock in human peripheral sensory nerves. This is possible through a technology called induced pluripotent stem cells (iPSC). iPSC are made by taking a person's blood or skin cell and turning it into a stem cell by activating specific genes. Thenm by growing the stem cells in particular conditions, they can be turned into pain nerve cells. In this project we are also able to obtain pain nerves from people who have recently died and agreed to donate their organs. We will use iPSC-pain nerves and human pain nerves from organ donors to examine the clock. We will investigate how the clock can change the way in which pain nerves function - being more excitable for example at particular times of the day.

The mechanisms we discovery may be important for people suffering chronic pain. We will first investigate in human pain nerve cells which properties are determined by the internal clock. We will then examine the molecules that may be important in this process. One of the most important type of molecules for nerves are called ion channels. These allow salts such as sodium and potassium to cross in and out of the cell. Nerves use ion channels to set how excitable they are and to send signals. We think the clock is regulating the way in which ion channels are behaving. By exploring this in detail, we believe we can find new ways to treat pain.

Physiological pain and chronic pain disorders show circadian variation which is challenging for patients to manage. Using mouse models we have shown that this circadian variation arises from an autonomous molecular clock in peripheral sensory neurons. The clock establishes diurnal differences in neurophysiological properties of nociceptors, including large differences in voltage gated sodium channel (VGSC) conductances. Using a small molecule, KL001, to prevent degradation of Cry1/2 (the repressor arm of the clock), we find pain responses are ameliorated. In this project we will investigate in detail circadian mechanisms in human nociceptors, the effector mechanisms of the clock and potential novel drug targets. We will first engineer inducible clock gene deletions in induced pluripotent stem cells (iPSC), which allow us to investigate the respective roles of the clock activators and repressors in determining neuronal properties and ionic conductances. We will differentiate iPSC into mature nociceptors using our establised protocols and perform patch-clamp recording, calcium imaging and multi-well multi-electrode arrays (MEA) assays in iPSC lines with and without clock genes. We will validate circadian nociceptor physiology in human nociceptors from deceased organ donors. We will next take advantage of comprehensive human sensory ganglia molecular dataset (snRNAseq, splice isoforms, ATAseq, proteomics/phosphoproteomics) to understand how the clock exerts its effects - which we anticipate will be through VGSC. We will then investigate iPSC lines from chronic pain patients to explore in detail the intersection of circadian biology with molecular determinants of chronic pain. To progress toward novel treatments, we will use the iPSC chronic pain models to validate targets within the clock and downstream of the clock. Our project represent a key step to progressing the basic research alrady completed towards meaningful new therapies for chronic pain.