CaMKII Regulation of Ca2+ Signaling in Rat DRG Neurons

Background: Pain caused by nerve injury accompanies a wide variety of conditions such as trauma, surgical incision and amputation, inflammation, HIV/AIDS and cancer, and is inadequately treated by currently available methods. Primary sensory neurons, including their somata in the dorsal root ganglia (DRG), are critical sites of pathology contributing to neuropathic pain. Elevated cytoplasmic Ca2+ concentration ([Ca 2+] c) that follows neuronal activation critically regulates diverse neuronal functions, including excitability, kinase activity, neurotransmitter release, genetic expression, and apoptotic cell death. Our previous work on rats with painful peripheral nerve injury reveals a reduced [Ca 2+] c signal in primary sensory neurons that leads to neuronal hyperexcitability, but the cause of these changes is unknown. Overall Objective: The currently proposed research will extend these findings, guided by the novel theory that nerve injury causes hyperalgesia by depressing the activity of the Ca2+-calmodulin protein kinase II (CaMKII), a molecular decoder of neuronal frequency information. Various processes regulating [Ca 2+] c are targets for the kinase action of CaMKII. Accordingly, we have defined following aims: Aim 1: To characterize the overall control of the [Ca 2+] c signal by CaMKII. Aim 2: To examine the CaMKII regulation of the specific processes that sequester Ca2+ into the endoplasmic reticulum (ER) and release Ca2+ from these stores, which together shape the [Ca 2+] c signal. Aim 3: To link the findings of Aims 1 and 2 with neuropathic pain by examining Ca2+-regulatory processes sensitive to CaMKII in neurons from animals with neuropathic pain. Study Design: I will use Ca2+ microfluorometry to investigate Ca2+ signaling in neurons dissociated from dorsal root ganglia (DRG) of rats. Specific fluorophores will measure Ca2+ in subcellular compartments during protocols that are designed to reveal the operation of various elements of the signaling mechanism. Selective pharmacologic and molecular agents will be used to inhibit CaMKII activity. Further, highly specific control of CaMKII will be achieved using viral gene transfer for RNA inhibition of CaMKII action, and to deliver a transgene for constitutively active Ca2+-independent recombinant CaMKII. Importance to Public Health: These experiments will provide better understanding of the role of CaMKII in regulating neuronal function, and on how nerve injury causes chronic pain. The DRG is a readily available but underdeveloped site for intervention in neurologic disease. Findings from the studies described in this proposal may be readily translatable into therapies that are anatomically targeted at the peripheral nerve, in order to correct abnormal Ca2+ signaling and thereby relieve neuropathic pain. Relevance to FWF Mission: This grant will support work in a leading anesthesia research department, providing the opportunity for acquisition of valuable new skills and knowledge by a young scientist who will be optimally positioned to make significant contributions upon returning to Austria.