Elsevier

Brain Research

Volume 1518, 26 June 2013, Pages 1-8
Brain Research

Research Report
Neuronal expression of soluble adenylyl cyclase in the mammalian brain

https://doi.org/10.1016/j.brainres.2013.04.027Get rights and content

Highlights

  • We describe a second sAC knockout model (sAC-C2 KO).

  • Confirm sAC expression in astrocytes.

  • Show sAC labeling in neurons of WT mice but not in C1KO or C2KO mice.

  • Show sAC is expressed in cerebellum, hippocampus, and visual cortex.

  • Show sAC is expressed in axon terminals and dendritic spines.

Abstract

Cyclic 3′,5′-adenosine monophosphate (cAMP) is a critical and ubiquitous second messenger involved in a multitude of signaling pathways. Soluble adenylyl cyclase (sAC) is a novel source of cAMP subject to unique localization and regulation. It was originally discovered in mammalian testis and found to be activated by bicarbonate and calcium. sAC has been implicated in diverse processes, including astrocyte-neuron metabolic coupling and axonal outgrowth of neurons. However, despite these functional studies, demonstration of sAC protein expression outside of testis has been controversial. Recently, we showed sAC immunoreactivity in astrocytes, but the question of neuronal expression of sAC remained. We now describe the generation of a second sAC knockout mouse model (C2KO) designed to more definitively address questions of sAC expression, and we demonstrate conclusively using immune-electron microscopy that sAC is expressed in neuronal profiles in the central nervous system.

Introduction

In mammals, signaling by the archetypal second messenger cAMP is determined by the balance between its synthesis by adenylyl cyclases and its degradation by phosphodiesterases (PDEs). The most widely studied source of cAMP in mammals is a family of G protein regulated, transmembrane adenylyl cyclases (tmACs). These enzymes contain two transmembrane domains and are localized at the plasma membrane. In 1999, a second source of cAMP was discovered that lacks transmembrane domains called “soluble adenylyl cyclase” (sAC) (Buck et al., 1999). Alternative splicing generates at least 2 sAC isoforms with distinct forms of regulation (Buck et al., 1999, Jaiswal and Conti, 2001). Plus, an alternative start site has been proposed to generate even greater isoform diversity (Fig. 1A) (Farrell et al., 2008). sAC is activated by HCO3 (Chen et al., 2000) and Ca2+ (Litvin et al., 2003), and it can be localized anywhere within the cell, even within organelles (Zippin et al., 2003, Zippin et al., 2004, Acin-Perez et al., 2009). sAC was found to be involved in the regulation of sperm capacitation and hyperactivated motility, bicarbonate-sensing in the eye, CO2-sensing in lung cilia and mitochondria, and pH sensing in epididymis and kidney (reviewed in (Tresguerres et al., 2011)).

Roles for sAC have also been identified in the developing brain (Wu et al., 2006), in axonal outgrowth (Stessin et al., 2006, Corredor et al., 2012) and in astrocyte-neuron metabolic coupling (Choi et al., 2012). While these and other reports demonstrated sAC mRNA expression in the brain (Sinclair et al., 2000, Geng et al., 2005, Farrell et al., 2008, Moore et al., 2008), confirming sAC protein expression has been controversial. We reported sAC protein expression in rat dorsal root ganglia and spinal cord neurons by immunofluorescence and in total rat brain by immunoprecipitation (Wu et al., 2006). However, when antibodies were used to examine sAC expression in mice, supposed sAC signals did not disappear in homozygous Sacytm1Lex knockout (C1KO) mice (Farrell et al., 2008, Corredor et al., 2012). We also showed that sAC was downstream from the neuronal guidance cue netrin-1 (Wu et al., 2006), but a different study contradicted this conclusion (Moore et al., 2008). An independent study showed sAC to be necessary for retinal ganglion cell survival and axon growth (Corredor et al., 2012); however, they were unable to show definitive sAC expression. They suggested this could be due to the proposal by Farrell et al. that neurons express C2-only isoforms derived from an alternate start site (Fig. 1A) (Farrell et al., 2008). Because of these contradicting reports, sAC expression in brain, and specifically neurons, has been questioned.

In this report, we describe generation of a sAC-C2 knockout mouse strain (C2KO), which we use to definitively demonstrate sAC expression in neurons of the cerebellum, hippocampus, and visual cortex, regions of the brain with proposed functions for sAC or cAMP biology. In these regions, we demonstrate sAC-immunoreactivity in wild type mice that is absent in equivalent regions of C1KO and C2KO mice.

Section snippets

Results

Despite reports showing sAC's roles in multiple systems, definitive detection of sAC in mouse somatic tissues has proven difficult. The previous gold standard for sAC detection utilized the C1KO mouse, which interrupted a sequence in the C1 catalytic domain (Fig. 1A) (Esposito et al., 2004). Using this standard, sAC activity (Esposito et al., 2004, Hess et al., 2005, Xie et al., 2006) and expression of sACt and sACfl isoforms (Hess et al., 2005, Farrell et al., 2008) had been definitively

Discussion

The C2KO mouse strain is a new tool that allows for definitive investigation of sAC expression, especially with the possibility of C2-only isoforms (Farrell et al., 2008). Studies with this new strain showed that non-specific bands might interfere with attempts to detect sAC expression via immunoprecipitation. However, utilizing both C1KO and C2KO mice, we confirm expression of sAC in brain, specifically in astrocytes, and we definitively demonstrate for the first time its expression in

Experimental procedures

All animal work was performed with approval from the Institutional Animal Care and Use Committee of Weill Cornell Medical College (IACUC) and conforms to NIH guidelines for the Care and Use of Laboratory Animals.

Acknowledgments

Grant support: NIH MSTP Grant GM007739 (JC), T32 DA007274 (JM), DA08259, HL096571 and HL098351 (TAM), GM62328, HD059913, and NS55255 (to JB and LRL).

References (33)

  • F. Xie et al.

    Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

    Dev. Biol.

    (2006)
  • M. Zaccolo

    Spatial control of cAMP signalling in health and disease

    Curr. Opin. Pharmacol.

    (2011)
  • T. Braun et al.

    Development of a Mn-2+-sensitive, “soluble” adenylate cyclase in rat testis

    Proc. Nat. Acad. Sci. USA

    (1975)
  • J. Buck et al.

    Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals

    Proc. Nat. Acad. Sci. USA

    (1999)
  • L. Cancedda et al.

    Patterned vision causes CRE-mediated gene expression in the visual cortex through PKA and ERK

    J. Neurosci.: Off. J. Soc. Neurosci.

    (2003)
  • Y. Chen et al.

    Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor

    Science

    (2000)
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