This application is for years 10-13 of a MERIT research program focused on gene regulation in sensory neurons of the trigeminal and dorsal root ganglia, which are the gatekeepers for the reception of position, touch, and pain signals throughout the body, conveyed by proprioceptors, mechanoreceptors, and nociceptors, respectively. In these studies we have focused on the roles of the transcription factors Brn3a and Islet1, and have shown these factors to be 'master regulators' of sensory neuron development and gene expression. Mice lacking Brn3a exhibit abnormal sensory axon growth, fail to develop proprioceptors, and have a profound defect in the expression of multiple genes related to pain pathways. Using a conditional knockout strategy, we have shown that Islet1 is required for the differentiation and survival of a majority of nociceptive neurons. Using inducible knockouts, we have shown that transient expression of Islet1 in developing sensory neurons can rescue nociceptor survival, but the expression of receptors and ion channels mediating pain pathways have an ongoing requirement for Islet1. Here we propose three aims focused on understanding the gene regulatory program of pain receptive sensory neurons.
Aim 1. Some nociceptors, plus receptors and channels related to pain pathways, persist in Islet1 null mice, and we hypothesize that these nociceptors are rescued by the expression of the closely related factor Islet2, which is expressed in a subset of Islet1 neurons. To test this hypothesis we will compare sensory ganglia in Islet1/Islet2 compound knockout embryos to those from Islet1, Islet2 and Brn3a single knockouts using microarrays, in situ hybridization, and transgenic axon tracing.
Aim 2. Our recent data indicate that Islet1 has a transient role as a repressor of early developmental genes and also a sustained role as an activator of specific genes related to pain receptor function. To determine the complete set of pain pathway genes dependent on Islet1, we will use transgenic mice expressing an inducible Cre-recombinase from the Islet1 gene locus to delete Islet1 after sensory neurogenesis is complete, and compare these results to our prior experiments in which Islet1 is never expressed in sensory neurons. We will also examine chromatin modifications at the Islet1 target gene loci.
Aim 3. Islet1 and Brn3a are expressed adult sensory ganglia, and we hypothesize that they are required for the maintenance of a correct program of gene expression, particularly for the expression of receptors and channels that mediate pain. To test this hypothesis we will induce excision of conditional alleles of Islet1 and Brn3a in adult mice using tamoxifen-induced Cre-recombinase. We will examine key markers of sensory function using in situ hybridization and immunohistochemistry in the sensory ganglia of these mice, with an emphasis on genes mediating pain pathways, including sensory neuropeptides and members of the Trp, Mrg and NaV gene families.
NARRATIVE This application requests continued funding for a program of basic research focused on the regulation of gene expression in sensory neurons, particularly pain receptor neurons. Management of pain and the prevention of pain-related disability represent some of the greatest unmet challenges in medicine, and these problems impact every medical specialty, from surgery to psychiatry. Veterans are especially susceptable to suffering and disability from pain syndromes because of the high incidence of past trauma from military service. Large cohorts of aging veterans also face degenerative diseases in which inflamatory pain is a major component. The available pharmacological treatments for pain frequently have poor long term effectiveness and significant potential for addiction. Understanding the mechanisms underlying pain and the biology of pain receptor neurons is essential for the development of new approaches to pain treatment. Pain mechanisms can be broadly divided into nociceptive, inflammatory, and neuropathic. Nociception is the primary mechanism for recognizing and avoiding harmful stimuli in the environment, including potentially damaging hot and cold temperatures, noxious chemicals such as acids, and sharp touch. Inflammatory pain results when a pre-existing condition, such as a tissue injury, heightens the sensitivity of the nociceptive pathway through peripheral or spinal mechanisms. Inflammatory pain can be adaptive, leading to the protection of an injured body part, but it is frequently pathological and often results in suffering and disabilty. Neuropathic pain results when pain mechanisms, ususally initiated by trauma or inflammation, 'take on a life of their own' as diseases of the nervous system, uncoupled from ongoing tissue injury. In the last decade enormous progress has been made in defining some of the molecular mediators of the pain response. Recent examples of major breakthroughs in the field include the discovery of the transient receptor potential (Trp) family of liganded ion channels, which include the noxious heat/pepper receptor TrpV1, the mustard oil receptor TrpA1, and the cold/menthol receptor TrpM8. Other examples include specialized sodium channels of the Nav family, with roles in inflammatory pain and in the transmission of pain at low temperatures, and the prokineticin receptor Prokr1, which interacts with TrpV1 to mediate pain responses. These studies show that it may be possible to resolve pain pathways into components that can be targeted through their specific pharmacology or molecular biology. Our research has focused on the role of switching molecules, called transcription factors, which bind to DNA and regulate the expression of the functional proteins that define sensory neurons. In past work, we have shown that the transcription factors Brn3a and Islet1 function as 'master regulators' of sensory neuron development and gene expression. Using 'knockout' mice with targeted mutations in these genes, we have shown that Brn3a and Islet1 are required for pain receptor neurons to correctly connect to their targets in the skin, muscles and spinal cord. The genes regulated by Brn3a and Islet1 are a catalogue of neurotransmitters, receptors and channels already known to be involved in pain pathways, and downstream genes which are not presently known to be involved in these pathways are thus new candidates for mediators of pain responses. This proposal has three specific aims, which together address the overall goal of understanding the regulation of pain pathway genes in mature sensory neurons. First, we will test whether pain receptor neurons are completely dependent on genes of the Islet class by studying mice which lack both Islet1 and the closely related factor Islet2. Second, we will use 'genechips' to assay the entire program of pain-related genes expressed in maturing sensory neurons in Islet1 knockout mice and controls, and we will examine how Islet1 regulates its target genes by modifying gene structure in chromosomes. Third, we will use a system for induced genetic rearrangement to test the function of Brn3a and Islet1 in the sensory ganglia of adult mice. Together these studies will significantly advance our understanding of the molecular regulation of pain receptor pathways, which may form the basis for future pain treatments.
