Role of Soluble Adenylyl Cyclase in Neuronal Survival and Regeneration

of a dissertation at the University of Miami. Retinal ganglion cells (RGCs) as well as other CNS neurons do not regenerate after injury and die soon after. While significant progress has been made in understanding the molecular basis for this lack of regenerative ability, this knowledge has not been enough to generate effective therapeutic strategies to promote axonal regeneration or prevent cell death after injury. Multiple signaling pathways are involved in neuronal survival and growth, but particular importance has been attributed to the role of the cyclic adenosine monophosphate (cAMP) pathway and electrical activity in modulating these processes. cAMP works with other signaling pathways, growth factors, and electrical activity to promote neuronal survival and growth. However, the exact mechanism of this coordinated response is poorly understood. It is not known if a novel branch of the cAMP pathway, the soluble adenylyl cyclase (sAC), has any relationship to cAMP-mediated neuroprotection and/or regeneration; or if these effects are related to neuronal electrical activity, or both. To answer this question I investigated the expression and function of the sAC in central nervous system (CNS) RGCs. I found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated (obtained after creating a new electroporation reagent) sAC inhibition dramatically decreased RGC survival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knockout mice or after specifically inhibiting tmAC. I found that cAMP-activated guanine nucleotide exchange factor for Ras-like GTPases (Epac-1 and Epac-2) are expressed intranuclearly in RGCs, and both Epacs are also present in RGC axons and soma. Our preliminary data shows that pharmacological Epac activation (but not tmAC activation) may compensate for the decrease in axon growth caused by pharmacological sAC inhibition, suggesting that Epac may act downstream of sAC to regulate axon growth. To further characterized sAC signaling and function in RGCs, I examine mitochondrial ATP production after sAC inhibition. My preliminary data indicates that pharmacological sAC inhibition impairs ATP production in RGCs. This finding provides one possible explanation for the deleterious effect of sAC inhibition on neuronal growth and survival. I also started examining the cAMP/sAC signaling pathway in retinal macrophages and found abundant …