James Berg - US grants

DESCRIPTION (provided by applicant): Possibly the most effective treatment for epilepsy is not a drug, but a dietary therapy - the ketogenic diet. Though this high fat/low carbohydrate and protein regimen is remarkably effective at reducing and even eliminating seizures, its exact mechanism remains unknown. One possibility that is currently being explored in Gary Yellen's lab is that the diet works via an effect of circulating ketone bodies on neuronal excitability. The specific hypothesis being explored in the Yellen lab is that a reduction in glycolytic ATP caused by the ketone bodies is acutely sensed by the ATP-inhibited potassium channel (KATP channel). Preliminary results show that when acute brain slices are exposed to the increase in ketone bodies seen during the ketogenic diet, there is a depression in spontaneous neuronal electrical activity, an effect that is abolished in the presence of KATP-specific blockers. This study will further test this hypothesis; first by recording from single isolated neurons while changing their metabolic state with ketone bodies, then by using an innovative optical technique, targeted luciferases, to investigate possible fluctuations in sub-cellular Iocalizations of ATP.

DESCRIPTION (provided by applicant): Current from calcium activated chloride channels (CaCCs) has been well described in dorsal root ganglion (DRG) neurons where (due to high intracellular chloride concentration), the nature of CaCC opening is excitatory. In addition, CaCC expression is upregulated in DRG neurons following nerve injury, leading to a possible role in neuropathic pain or regeneration. Our goal is to further understand this channel and the role it plays in neuropathic pain, so that it may someday be used as a therapeutic target. Although CaCC current has been well described, the channel has only recently been identified as TMEM16A, a protein of previously unknown function. We hypothesize that the TMEM16A CaCC plays a role in DRG neuronal physiology. Preliminary studies on neonatal DRG neurons show that a subset of these neurons does indeed display a prominent CaCC current and RT-PCR from DRG tissue shows expression of the TMEM16a CaCC. In experiments on cultured DRG neurons from TMEM16a-/- mice, CaCC current is not observed, making it likely that TMEM16A is responsible for the DRG CaCC current. The initial goal of the projects presented here are to characterize DRG neurons displaying CaCC current using physiology and molecular techniques, then to examine the consequences of the loss of the TMEM16A channel. Following electrophysiological identification of CaCC-positive DRG neurons, single-cell RT-PCR will be performed to determine which cells express TMEM16A. An antibody developed to recognize the TMEM16A protein will be used to determine if there is a distinct subpopulation of DRG neurons that express the channel in the intact ganglia. Studies will then shift to examine the expression of TMEM16A during nerve injury. At a number of time points following sciatic nerve lesion, the expression of TMEM16a will be examined via quantitative RT-PCR. To determine if the distribution of TMEM16A shifts following nerve injury, immunohistochemistry will be performed and compared to the control results obtained previously. Finally, the function of TMEM16A following nerve injury will be investigated. A novel technique will be used wherein TMEM16a-/- neurons are generated in a heterozygous background via somatic recombination (MADM). The ability of these individual neurons to regenerate will be studied using lesion studies as previously described. PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand the calcium activated chloride channel in primary sensory neurons. This ion channel protein is likely upregulated following nerve injury and may play a role in nerve regeneration or neuropathic pain. Using genetic tools and physiology techniques, we hope to elucidate the function of this important protein.