Publication Date

2012-10-18

Availability

Open access

Embargo Period

2012-10-18

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2011-11-08

First Committee Member

Jeffrey L. Goldberg

Second Committee Member

Brian Noga

Third Committee Member

Ian Hentall

Fourth Committee Member

Patrick Wood

Fifth Committee Member

Jochen Buck

Abstract

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 sAC expression in these cells, which are notably responsive to cAMP. Given the known pro-regenerative interactions between macrophages and RGCs, these observations support the use of retinal macrophages as a tool to study sAC and/or to exploit potential therapeutic applications. In conclusion, these data identify a previously unknown sAC-mediated cAMP signaling pathway regulating RGC survival and axon growth, where sAC is necessary for RGC survival and growth. Possible mechanisms for how sAC promotes growth and survival are proposed, including its role in mitochondrial function (e.g., participating in ATP synthesis), and its collaboration with tmAcs (e. g. where sAC is necessary for axon growth and AC1/AC8 are important for synaptic plasticity) and electrical activity (e.g. where electrical activity-mediated increase in intracellular calcium synergizes with bicarbonate for optimal sAC activation, increased intracellular cAMP and improved survival). This work provides new knowledge about the importance of cAMP signaling in CNS neurons and opens a field of investigation oriented to explore new neuroprotective or regenerative strategies based on sAC modulation.

Keywords

Neuroprotection; Neuronal regeneration; signaling pathways; Electrical stimulation; Retinal ganglion cells; cAMP

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