FRET-based cGMP Imaging in the Nervous System

Abstract

Impaired cyclic 3’ 5’ guanosine monophosphate (cGMP) signaling has been linked to several neurological disorders, including inaccurate axon formation and pathfinding. It is well known that a cGMP signaling cascade, comprising the C-type natriuretic peptide (CNP), its guanylyl cyclase GC-B and cGMP-dependent protein kinase I (cGKI) is necessary for correct axon bifurcation of dorsal root ganglion neurons (DRGs) in mouse spinal cord. However, the identity of phosphodiesterases (PDEs) that degrade cGMP in DRGs as well as further signaling partners are not well understood. Here, we used Fluorescence resonance energy transfer (FRET)-based cGMP imaging to study cGMP signaling in the nervous system. Therefore, transgenic mice expressing the cGMP sensors cGi500 or mcGi500 globally or in specific tissues were used. We identified PDE2A to be the major enzyme responsible for the degradation of CNP-induced cGMP in DRG neurons. Real-time imaging of DRG somata and growth cones revealed a crosstalk between the CNP/cGMP/cGKI signaling cascade and acetylcholine- or ATP-induced Ca2+. Another appropriate model to investigate neuronal development is the chicken embryo. Transfection of the CMV-cGi500 plasmid into neural crest cells that migrate and became DRG neurons made it possible to perform in ovo real-time cGMP imaging. Imaging experiments and immunostainings revealed a NO/cGMP/cGKI and CNP/cGMP/cGKI pathway in DRGs of chick embryos indicating a relevance of cGMP for DRG axon bifurcation. The NO-dependent soluble guanylyl cyclases, of which two isoforms (NO-GC1 and NO-GC2) are known, are promising drug targets to increase cGMP in the brain. Drug-like small molecules were identified to work synergistically with NO. However, the effects of NO stimulators in the brain are poorly investigated. In this study, we analyzed the impact of two structurally different NO-GC stimulators, IWP-051 and Bay 41-2272, on cGMP signaling in the murine cerebellum, striatum, and hippocampus. FRET-based cGMP imaging revealed that Bay 41-2272 increased DEA/NO-induced cGMP in all three brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP only in cerebellum and striatum but not in the hippocampal CA1 area or primary hippocampal neurons. In-situ hybridization indicated that in murine cerebellum and striatum mRNAs of both NO-GC isoforms are expressed, while in the hippocampal CA1 area only NO-GC2 is expressed. These results suggest an isoform-specific effect of IWP-051 on NO-GC1. Indeed, real-time cGMP imaging of acute brain slices revealed that IWP-051 did not potentiate DEA/NO-induced cGMP in striatum of NO-GC1 knockout mice, while in the striatum of NO-GC2 knockout mice it did. This study showed that NO-GC stimulators enhance cGMP in the brain and should be further investigated for the treatment of brain diseases.