Peripheral axonal injury changes the expression of hundreds of genes in dorsal root ganglion neurons, including many that may contribute to neuropathic pain by increasing the excitability of the injured neurons and by altering their synaptic transmission and connectivity in the dorsal horn of the spinal cord. We hypothesize that the regulation of """"""""pain-contributing"""""""" genes is orchestrated by induction of transcription factors that act as """"""""master switches"""""""" to control coordinated changes in expression of programs of diverse genes. More specifically, we propose that activating transcription factor 3 (ATF3) is one of the master regulators of the pain phenotype after peripheral nerve injury. To study the nature and regulation of the transcriptional component of neuropathic pain and the particular involvement of ATF3 we propose to: 1. Test if ATF3 expression in DRG neurons is necessary after peripheral nerve injury, and sufficient in non-injured states, to alter pain-related behavior. To do this we will investigate the effect on pain sensitivity in the spared nerve injury neuropathic pain model of both knockdown of ATF3 expression, by AAV8 delivery of shRNA to DRG neurons, and of conditional ATF3 knockout limited to adult DRG neurons, using an inducible Brn3a Cre driver. We will also examine if forced expression of ATF3 in postnatal non-injured DRG neurons increases pain-related sensitivity, using transgenic mice with the ATF3 gene under control of a neuron-specific thy1.2 regulatory sequence. 2. Establish if ATF3 contributes to the pain phenotype after nerve injury by altering the expression of pain- and growth-related genes. This will involve comparing microarray expression profiles of DRGs from intact and nerve-injured ATF3 overexpressing and knockout mice with wild types, and identification of those genes to which ATF3 binds in injured DRGs using chromatin immunoprecipitation. Validation of regulation by ATF3 will be by luciferase reporter assay and the role of candidate genes in neuropathic pain evaluated. 3. Determine if central sprouting of A-fiber afferents into the superficial laminae of the dorsal horn occurs after peripheral nerve injury and is driven by ATF3 expression. To do this we will use a transgenic mouse engineered to express an axonally transported GFP fusion protein only in large myelinated DRG neurons (using a Parv-Cre driver) in order to label the central terminals of these A-fibers. We find in pilot studies that peripheral nerve injury results in the sprouting of A-fiber central terminals into lamina II outer and will now both extend this and test if ATF3 contributes, using knockdown and knockout strategies.
Peripheral nerve injury can result in severe intractable pain resistant to most forms of therapy (peripheral neuropathic pain). These studies are designed to determine how changes in gene expression in sensory neurons after nerve damage are orchestrated and act to produce long-term changes in the function and structure of the nervous system, with a view to identifying novel approaches to prevent these changes, and thereby the evolution of chronic neuropathic pain.
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