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.
In this application we propose to establish a novel neurogenetic mouse model that will serve as a molecular genetic tool to study the mechanisms of neuronal function and phenotypic identity between distinct subtypes of somatosensory neurons under normal and pathological conditions.
|Lippoldt, Erika K; Ongun, Serra; Kusaka, Geoffrey K et al. (2016) Inflammatory and neuropathic cold allodynia are selectively mediated by the neurotrophic factor receptor GFR?3. Proc Natl Acad Sci U S A 113:4506-11|
|Palkar, Radhika; Lippoldt, Erika K; McKemy, David D (2015) The molecular and cellular basis of thermosensation in mammals. Curr Opin Neurobiol 34:14-9|
|Knowlton, Wendy M; Palkar, Radhika; Lippoldt, Erika K et al. (2013) A sensory-labeled line for cold: TRPM8-expressing sensory neurons define the cellular basis for cold, cold pain, and cooling-mediated analgesia. J Neurosci 33:2837-48|
|Lippoldt, Erika K; Elmes, Russell R; McCoy, Daniel D et al. (2013) Artemin, a glial cell line-derived neurotrophic factor family member, induces TRPM8-dependent cold pain. J Neurosci 33:12543-52|
|McKemy, David D (2013) The molecular and cellular basis of cold sensation. ACS Chem Neurosci 4:238-47|