Research Article: Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Date Published: November 17, 2009

Publisher: Public Library of Science

Author(s): Timothy A. Dunn, Daniel R. Storm, Marla B. Feller, Michael Hendricks.

Abstract: Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.

Partial Text: During development, many regions of the nervous system exhibit spontaneous biochemical and electrical activity. These early forms of activity, which can occur either on a cell-by-cell basis or correlated across cells by early synaptic connections, play a critical role in various developmental processes in the formation of neural circuits [for review, see1,2]. For example, prior to the onset of vision, retinas exhibit highly patterned, spontaneous activity, termed retinal waves [for review, see 3]. At the level of individual retinal ganglion cells (RGCs), retinal waves drive periodic bursts of depolarization, lasting roughly 2–4 seconds, occurring about once per minute [4], [5].

Here we have shown that depolarization-induced increases of PKA activity in developing RGC somas are mediated by a combination of transmembrane and soluble calcium-dependent adenylate cyclases. First, dual imaging of a genetically encoded reporter of PKA activity and a membrane permeant form of the calcium dye fura-2 was used to determine that a threshold calcium increase was necessary to induce a measurable PKA transient. Second, depolarization-induced PKA transients were decreased in amplitude but, surprisingly, persisted in knockout mice lacking the calcium dependent adenylate cyclase AC1 and in double knockout mice lacking both AC1 and AC8. Consistent with this finding, depolarization-induced PKA transients persisted in a subset of RGCs in blockers of all transmembrane ACs. The remaining PKA transients were blocked in soluble AC antagonist, providing the first evidence that a soluble AC is present in developing RGCs. Last, blockade of PDEs increased the basal levels of PKA activity and a calcium dependent PDE modulated the amplitude of depolarization-induced PKA transients. Hence, a variety of calcium-dependent ACs and PDEs are important for translating depolarization into transient activation of the PKA biochemical cascade in RGCs.