Transgenic approaches to sensory neuron signalling

There have been remarkable advances in understanding genetics recently. We know the sequence of the entire human genome, and understand in broad terms how cells make RNA molecules and proteins which underlie normal development and the functioning of adult life-forms. It has become clear, consistent with our ideas about evolution, that many of the molecular systems that function in human physiology also exist in simpler organisms. Mice are particularly interesting, because they can be modified genetically and the consequences of changing patterns of gene expression can then be examined at many levels - in terms of cellular function, organ function and even behaviour. This has stimulated an ambitious international attempt to modify the expression of all mouse genes and examine the consequences. The information obtained is extremely useful for understanding the basic physiology of mammals, and therefore also has enormous significance for understanding health and disease in man. In our own lab we identified a protein that sends electrical signals to the central nervous system, signaling the existence of painful stimuli. When we deleted the gene so that no protein was made, the mice were essentially pain free. After this work, a number of groups homed in on the gene that encodes a very similar protein in humans with inherited pain disorders. They found that mutations in the gene that made the protein over-active resulted in chronic pain syndromes in humans. They also found that a small number of people that no longer made a functional version of this protein were pain free. This is important in deciding what molecules to target for the production of new pain-killing drugs. Estimates suggest that 40 million people in the developed world suffer chronic pain for which present drugs are unsuitable or ineffective. Thus our mouse genetic analysis has helped identify a protein which may be very useful for the development of pain-killing drugs of a new class to those that exist at present. Our present proposal exploits new technology refined over the past decade that enables us to delete genes in specific tissues or cell types. We know that there are many different sorts of sensory neurons that innervate the skin, muscle and viscera, and signal events such as touch, muscle position or painful stimuli to the brain. We can kill different sorts of sensory neurons defined by the proteins they make, and see what the functions of these specialized sensory neurons are. We can also identify the proteins that are responsible for responding to touch, temperature changes or painful stimuli by deleting individual genes in these cells. We can also examine how temperature or pressure receptors in the skin may signal indirectly to the sensory neurons by stopping protein expression in skin cells. Similar techniques can be used to examine the molecules that underlie electrical signaling to the central nervous system. Some cells assumed to be involved with immune responses have now been shown to be able to contribute to pain states. We will also examine how they do this, by deleting genes that encode soluble messengers that are candidates for this action. The present project thus assembles a team of expert geneticists, scientist who measure electrical signaling in the nervous system, and experts in assessing mouse behaviour to learn more about the specialized function of different sorts of sensory neurons, how they signal to the central nervous system, and the importance of other cell types with which they may interact to sense the external environment. We are confident that our studies will identify components of the pain detection system that may be important for drug development in the future. In addition we will learn far more about the functioning of sensory neurons and their interactions with other cells.

Deleting genes in specific tissues of transgenic mice using the Cre-loxP system is proving informative. We can exploit this system to kill subsets of sensory neurons which express different cell surface markers and thus analyse their specialised functions in vivo. In addition, we can delete candidate genes that underlie sensory transduction of thermal or mechanical stimuli, and carry out electrophysiological and behavioural analyses at the level of the intact mouse. This kind of study allows us to obtain information that would be almost impossible to obtain pharmacologically, given the broad pattern of expression of many receptors and channels that are likely to have a number of distinct physiological roles. We have examined the role in pain pathways of a number of voltage-gated sodium channels that are activated downstream of primary transducing receptors; we will complete this analysis by gene deletion studies of the remaining candidates (Nav1.1, 1.2 and 1.6). Cre-loxP technology also enables us to analyse the contribution of pre- and post-synaptic receptors in regulating sensory signals to the central nervous system, by ablating channels just at the pre-synaptic site. Some sensory signalling of thermal or mechanical stimuli may occur in the skin, which indirectly signals to sensory neurons. By deleting transducing channels in keratinocytes alone, we can assess the relative significance of this form of sensory transduction compared to direct activation of sensory neurons. In addition we can analyse sensory neuron-microglial interactions, recently invoked as a critical factor in chronic pain states. We are able to kill microglia, delete P2X receptors expressed by these cells, as well as ablate signalling molecules released from sensory neurons that may activate microglia. This project will thus provide insights into touch and damage sensing mechanisms, and cell-cell interactions that play an important role in sensory transduction and pain pathways.