David D. McKemy - US grants

DESCRIPTION (provided by applicant): In mammals, the detection of stimuli of a chemical, mechanical, or thermal nature occurs through a complex network of primary and higher-order sensory neurons that make up our somatosensory system. A significant advance in our understanding of somatosensation at the molecular level came from the identification of members of the transient receptor potential (TRP) channel family as the primary detectors of thermal stimuli in peripheral nerves. These "thermosensors" are sensitive over distinct temperature ranges and, in most cases, define discrete populations of sensory neurons. However, how signals evoked by thermal stimuli are interpreted and processed at levels higher than the peripheral afferents, as well as the architecture of the neural networks used to communicate these stimuli is still unclear. The aims of this application are to develop genetic model systems that will enable the mapping of somatosensory neural networks involved in the detection of specific stimulus modalities. These exploratory studies will focus on the recently cloned cold and menthol receptor, CMR1 or TRPM8. We intend to use the TRPM8 transcriptional promoter to specifically express a transneuronal tracer in TRPM8+ peripheral afferent nerves in order to map the neural networks and synaptic connections used by these neurons to communicate temperature centrally. After this animal model has been established, we will then assess for changes in spinal organization of TRPM8 neural networks that may result after the induction of inflammatory or neuropathic pain. We will also assess the physiological relevance of this population of sensory nerves by developing a mouse model in which these cells can be conditionally ablated. The experienced gained from these exploratory studies will be used for future investigations of other thermosensors and will expand our understanding of how we detect and discriminate sensory stimuli.

DESCRIPTION (provided by applicant): In the peripheral nervous system, somatosensory neurons report a wide range of temperatures, from noxious heat to noxious cold. We know that distinct subsets of nerve fibers will respond to specific temperature thresholds, presumably based upon the thermosensory molecules they express. Moreover, these nerves are key players in the detection of painful, tissue damaging stimuli. We have recently begun to grasp how these nerve fibers detect temperature due to the identification of molecules that respond to distinct thermal stimuli. These molecules, members of the transient receptor potential (TRP) channel family, were identified by their sensitivity to compounds, such as capsaicin and menthol, which mimic distinct psychophysical sensations. Taken together, these thermosensors can detect the broad range of temperatures we perceive and provides a molecular explanation for how temperature is detected. Nonetheless, our understanding of the cellular transduction mechanisms that mediate and regulate these thermosensors remains limited. Therefore, we are analyzing the sensory afferents expressing the cold and menthol receptor, TRPM8, to further our characterization of the transduction mechanisms mediating cold sensation. To this end, we have generated transgenic mice that express a fluorescent label specifically in TRPM8-positive sensory neurons that is detectable in vivo. With this animal model, we are investigating the peripheral projections of cold-sensitive afferents, the mechanisms of cold adaptation in native cells, and the in vivo role of these neurons in somatosensation.

DESCRIPTION (provided by applicant): There are a limited number of robust animal models that can be used to assay phenotypic changes associated with acute and chronic pain. This is true not only for animal behaviors, but also with respect to mechanistic details of changes that occur in relevant tissues at the site of injury, including nociceptive nerve fibers. For a more in-depth understanding of how inflammation leads to pain and hyperexcitability, it will be informative to know how this type of pathology leads to molecular, cellular, and anatomical changes in specific cohorts of afferent neurons that mediate such sensitization. At the molecular level, members of the TRP family of ion channels play essential roles in the formation of both acute and persistent pain in vivo, particularly the irritant receptor channel TRPA1. TRPA1 is critical for the development of inflammatory hypersensitivity to both mechanical and thermal stimuli, implicating the afferent neurons expressing TRPA1 as being of equal importance in inflammatory signaling. Here we propose to address the property of these neurons, but not necessarily the channel itself, with the generation of two mouse models with which we will examine how their phenotype and morphology changes with inflammatory hypersensitivity, and through cell ablation, will determine their necessity in acute and inflammatory signaling. We have initiated the creation of transgenic mice in which neurons that express TRPA1 are labeled with a fluorescent axonal tracer which we will use to establish the neurochemical phenotype and morphology of the peripheral and central terminations of TRPA1 neurons in vivo. These analyses will be done under normal conditions and in the context of mouse models of inflammatory injury. Next, using this newly developed transgenic strategy, we will target TRPA1 neurons for conditional ablation and determine their role in acute and persistent pain in vivo. As whole, these exploratory studies will tease out the role of this specific neuronal population in somatosensory signaling and the formation of inflammatory hypersensitivity, as well as develop novel animal models that selectively target TRPA1 neurons for future studies into their physiology and function.

Summary Traditional neuronal gene expression profiling normally employs some method of isolating acutely dissociated primary neurons, a strategy that can introduce undesirable trauma to the cell during the isolation procedures, as well as requires a large sample size in order to generate sufficient starting material. These limitations are of particular concern for functionally-distinct sensory afferents in the peripheral nervous system (PNS) as they are poorly represented cell-types within sensory ganglia whose gene expression phenotype is exquisitely sensitive to any form of perturbation. To overcome these limitations we propose to use the translating ribosome affinity purification (TRAP) technique to identify translating mRNAs in genetically targeted somatosensory afferents, an approach that has yet to be used in the PNS. TRAP involves the expression of a tagged ribosomal protein such that actively translating mRNAs can be isolated by immunoaffinity purification. By targeting specific cell populations, gene expression profiling can be performed without subjecting cells to invasive isolation techniques. Here we propose two Aims in which transgenic mice will be generated that target a modality- specific neuronal cohort for translational profiling under normal conditions, followed by a determination of how this profile changes under pathological conditions characterized by painful hypersensitivity. Using the R21 mechanism, we will target the small subset of sensory neurons that express TRPM8, a cold-gated ion channel and the principal sensor of cold temperatures in vivo. TRPM8-null mice are deficient in a wide array of cold responses, from those perceived as pleasantly cool to painfully cold, and lack injury-induced cold hypersensitivity. We hypothesize that this latter phenotype is partly due to altered gene expression within this cohort, as has been shown to occur in the general population under a range of pathological conditions, a posit we will directly test in our studies. Thus, the completion of this exploratory proposal will establish novel molecular genetic methodologies in the PNS that can be used in any genetically tractable neuronal subtype, allowing gene expression profiling between functionally distinct neurons and assessment of molecular phenotypes within sub-populations.

DESCRIPTION (provided by applicant): The menthol receptor TRPM8 is considered the principal cold sensor in mammalian sensory neurons as animals lacking TRPM8 function are deficient in cold and cold pain behaviors. However some residual cold sensitivity remains, indicating the possible presence of TRPM8-independent cold transduction mechanisms. Other cell types, such as those expressing TRPV1 and TRPA1 channels, are critical for somatosensory signaling, and have been implicated in certain aspects of cold sensation. Genetic approaches to determine the role of these channels and cell-types are complicated by the fact that the resulting phenotypes are investigated either many days after manipulation, or in developmentally disparate backgrounds. An elegant approach has recently been devised that targets cell impermeant sodium (Na+) channel blockers to only primary sensory neurons mediating pain (nociceptors). Specifically, when stimulated with agonists for nociceptor-specific TRPV1 and TRPA1 channels, large molecules such as the charged lidocaine derivative QX-314 permeate through these channels, thereby selectively blocking nerve conduction in just these neuronal populations. Previous reports failed to find large molecule entry through TRPM8 channels, suggesting that TRPM8 neurons cannot be targeted in this manner. Our underlying hypothesis, supported by our novel preliminary data, is that large molecules permeate cells through TRPM8 when the channel is activated by potent agonists, and we propose to determine if cold and cold pain can be ameliorated by selectively blocking nerve conduction of these and other neuronal populations. Aim 1 will determine the mechanisms whereby TRPM8 channels permeate large cations in vitro and be used as a means to target Na+-channel blockers to TRPM8 neurons. Aim 2 will determine if targeting Na+-channel blockers in TRPM8 neurons alters cold sensation in mice, and determine if other primary sensory neurons also contribute to cold. Aim 3 will extend these behavioral analyses to determine if chronic cold pain induced by injury can be ameliorated by targeting Na+-channel blockers to TRPM8, TRPV1, or TRPA1 neurons. With these studies we will determine if cold and cold pain can be specifically inhibited by the selective entry of large, cell impermeant anesthetics, as well as use this novel approach to further define the cellular basis for cold and cold pain, including identifying TRPM8-independent neuronal populations that contribute to this somatosensory modality.

Project Summary The detection of external stimuli such as temperature is critical for survival, yet inappropriate responses to temperature do have a significant negative impact on overall health. The sensations and the physiological effects of cold are distinct among somatosensory modalities in that cold provides a pleasant, soothing sensation at mild temperatures, but is also agonizing as temperatures decrease. Remarkably, how this one somatosensory modality mediates this diverse range of physiological effects is not known. The menthol receptor TRPM8 is considered the principal cold sensor in mammalian sensory neurons, but the irritant receptor TRPA1 have also been associated with cold pain. Mice lacking TRPM8 channels retain some limited cold sensitivity, but we find that ablation of TRPM8-expressing neurons in mice abolishes essentially all acute cold and cold pain behaviors, results implying TRPM8-independent cold transduction mechanisms in TRPM8+ neurons. TRPA1 channels appear to serve no role in acute cold, but likely contribute to injury-induced cold pain. TRPM8 and TRPA1 are not co-expressed, yet the interplay between TRPA1 and TRPM8 after injury has not been examined, nor have the molecular and cellular mechanisms leading to injury-induced sensitization. Recently, we found that the glial cell-line derived neurotrophic factor-like (GDNF) ligand artemin is a mediator of TRPM8-dependent cold pain, and that the artemin receptor GFR?3 is required for pathological cold allodynia. However, the molecular and cellular mechanisms whereby this pathway leads to cold allodynia is not known. Here we propose to use a combination of molecular, cellular, behavioral, and pharmacological approaches to fill in these gaps in our knowledge of the mechanisms underlying cold sensation. First, we will determine the role of TRPM8 and TRPA1 channels in cold allodynia. Second, we define the cellular basis for artemin-induced cold hypersensitivity. Third, we will test the necessity of the classical GFR? co-receptor Ret in cold allodynia and determine if other candidate co-receptors mediate this form of pathology. Lastly, we will generate transgenic mice in which genetically defined subpopulations of TRPM8 neurons are conditionally ablated in vivo to determine if innocuous cool, noxious cold, and analgesia are mediated cell autonomously. At the conclusion of these studies, we will have defined a signal transduction pathway leading to cold pain, and if these stimuli are transmitted via distinct neural circuits to mediate the range of behavioral and physiological responses to cold temperatures.

Migraine is one of the most debilitating disorders in humans, affecting greater than 15% of the population with minimal therapeutic avenues for migraine sufferers. The challenge in treatment is that migraine involves many sensory pathways, including irregular excitability in central and peripheral nociceptive neurons. To address this complex disorder, several groups have recently performed genome-wide associated studies (GWAS) to ascertain candidate molecules mediating migraine pathology, identifying the cold and menthol receptor TRPM8 as a migraine susceptibility gene. Interestingly, these studies identified mutations only in noncoding regions of TRPM8, therefore making it likely that these mutations alter expression levels and not channel function. While it is not known what role, if any, the channel has in migraine, TRPM8 serves an important role in pathological cold pain, suggesting there may be a corollary between cold and migraine pain. Recently, we found that the glial cell line-derived neurotrophic factor (GDNF) family ligand artemin and its receptor, GDNF family receptor alpha 3 (GFRa3), are the principle mediators of TRPM8-dependent cold pain, of note as each has also been linked to migraine. These results have led us to the hypothesis that TRPM8 channels and afferents, via altered signaling due to artemin interacting with GFRa3, are a component of the underlying mechanisms of migraine. To test this, we propose to use this exploratory mechanism to (1) determine if TRPM8 channels or neurons are involved in migraine-like pain behaviors in mice, then (2) similarly ask if artemin and GFRa3 are required for migraine pathogenesis. At the completion of this exploratory study, we will have determined if either TRPM8 or artemin/GFRa3 signaling serve a role in migraine. Moreover, the proposed experiments will determine if either of the signaling pathways under consideration are relevant for migraine, potentially serving as the foundation for future investigations.