Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

Zhiwen Zhou,Mineo Iseki,Yuji Ikegaya,Ryuta Koyama,Shigeru Matsunaga,Kenji F Tanaka,Norio Matsuki,Masakatsu Watanabe

doi:10.1038/srep19679

Abstract

Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

Highlights

Proper axonal morphogenesis is crucial to the establishment of functional neural circuits[1]
Using photoactivated adenylyl cyclase (PAC) together with pharmacological tools and small interference RNA (siRNA)-mediated knockdown, we found that intracellular cyclic adenosine monophosphate (cAMP) regulates axonal branching and elongation via protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively
We examined the properties of PAC in cultured dentate granule cells (Fig. 1a)

Summary

Introduction

Proper axonal morphogenesis is crucial to the establishment of functional neural circuits[1]. Despite extensive studies into the role of cAMP in axonal growth, little is known about whether and how changes in intracellular cAMP levels regulate axonal morphogenesis. One reason for this gap is that methods to spatiotemporally manipulate intracellular cAMP levels have been unavailable. Most studies have pharmacologically assessed the role of cAMP in axonal morphogenesis via the chronic application of cAMP agonists or antagonists to cultured neurons These methods are insufficient to probe the effects of spatiotemporal cAMP dynamics on axonal morphogenesis. To investigate whether and how intracellular cAMP dynamics regulates axonal elongation and branching, we transfected PAC in primary cultures of the dentate granule cells. Using PAC together with pharmacological tools and siRNA-mediated knockdown, we found that intracellular cAMP regulates axonal branching and elongation via protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively

Methods

Results

Conclusion