Local antagonistic interactions between cAMP and cGMP ensure that axon initiation is accompanied by dendrite inhibition

I was fortunately to hear Mu-ming’s talk about the “Development of Neuronal Polarity” last summer. Now, the paper has been published in Science.

Science 29 January 2010:
Vol. 327. no. 5965, pp. 547 – 552
DOI: 10.1126/science.1179735

Local and Long-Range Reciprocal Regulation of cAMP and cGMP in Axon/Dendrite Formation

Maya Shelly 1,* Byung Kook Lim 1,* Laura Cancedda 1,2 Sarah C. Heilshorn 1, Hongfeng Gao 1, Mu-ming Poo 1

Cytosolic cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) often mediate antagonistic cellular actions of extracellular factors, from the regulation of ion channels to cell volume control and axon guidance. We found that localized cAMP and cGMP activities in undifferentiated neurites of cultured hippocampal neurons promote and suppress axon formation, respectively, and exert opposite effects on dendrite formation. Fluorescence resonance energy transfer imaging showed that alterations of the amount of cAMP resulted in opposite changes in the amount of cGMP, and vice versa, through the activation of specific phosphodiesterases and protein kinases. Local elevation of cAMP in one neurite resulted in cAMP reduction in all other neurites of the same neuron. Thus, local and long-range reciprocal regulation of cAMP and cGMP together ensures coordinated development of one axon and multiple dendrites.

1 Division of Neurobiology, Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
2 Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Via Morego 30, Genoa 16163, Italy.
* These authors contributed equally to this work.

Present address: Department of Materials Science and Engineering, Geballe Laboratory for Advanced Materials, Stanford University, Stanford, CA 94305, USA.

To whom correspondence should be addressed. E-mail: mpoo@uclink.berkeley.edu

This is a paper, again, proves the importance of “local autocatalytic activation and long-range inhibition”, which was initially described by Alan Turning in “The Chemical Basis of Morphogenesis“.

Interesting points in the paper are as the following.

• we examined the effect of localized cAMP and cGMP activities in axon/dendrite initiation by plating these neurons on substrates coated with stripes of membrane-permeant fluorescent analogs of cAMP or cGMP [F-cAMP or F-cGMP]
• This retrospective analysis of axon/dendrite initiation was possible because neurite initiation sites on the soma did not move during neuronal polarization
• we plated these neurons on a substrate striped with either the adenylate cyclase (AC) inhibitor SQ-22536 or the protein kinase A (PKA) inhibitor KT5720 or Rp-8-Br-cAMPS, to reduce locally basal cAMP/PKA activity.
• Micro-fabrication and substrate patterning. A silicon wafer was used to generate a template for the poly(dimethylsiloxane) (PDMS) mold and substrates were patterned in parallel stripes of 50 μm width separated by 50 μm gaps. Briefly, PDMS mold was reversibly sealed on poly-L-lysine-coated glass coverslip, and microchannels formed between the PDMS mold and coverslip were used for microfluidic patterning of the following substrates alone or together with fluorescently conjugated BSA (5 μg/ml) as a marker: F-cAMP, F-cGMP, Rp-8-Br-cAMPS, and Rp-8-pCPT-cGMPS (0.2 or 2 μM); KT5720 and KT5823 (2 nM); SQ-22536 and ODQ (0.1 or 1 μM). Solutions were present in the microchannels overnight and allowed to dry. The stripe-coated coverslips were washed extensively before plating of dissociated neurons.
• Preparation of coated glass beads. Glass beads 4.5 – 10 μm in diameter (Polysciences, Inc., Warrington, PA) were coated with forskolin or cGMP-AM for local neurite stimulation, as follows. The beads were washed for 30 min in 70% ethanol, allowed to dry, and were coated with poly-L-lysine solution (0.5 mg/ml) (Sigma, St. Louis, MO) for 4 hr. Following extensive washes with PBS, the beads were allowed to dry. The beads were then incubated with forskolin solution (2 μM) together with fluorescently conjugated BSA (5 μg/ml) and dried to allow adsorption. The dried beads were extensively washed with PBS prior to their use. Cyclic GMP-AM was coated in 30% DMSO / 70% ethanol solution, and allowed to dry. Following extensive washes with ethanol, the beads were dried, and resuspended in PBS.
• one of the three FRET reporters: indicator of cAMP using Epac (ICUE) (30), cGMP energy transfer sensor derived from PDE5A (cGES-DE5) (31), or A-kinase activity reporter (AKAR) (32) (SOM text), which were designed to monitor the amount of cAMP, cGMP, and PKA activity, respectively.
• For the PKA activity reporter, A-kinase activity reporter (32) (AKAR) was constructed by linking enhanced cyan fluorescent protein (CFP), a PKA-specific phosphorylatable peptide sequence, phosphoamino acid binding domain of the protein 14-3-3, and yellow fluorescent protein (YFP) (a gift from Dr. Jin Zhang, Johns Hopkins Univ., Baltimore, MD).
• we inquired whether this antagonism is reflected in the phosphorylation of LKB1 (12), GSK-3b (8, 16), and Akt (8, 15), which are proteins that promote axon formation after their phosphorylation.
• in utero electroporation (34) of constructs expressing enhanced green fluorescent protein (EGFP) and specific siRNAs against either cAMP-selective phosphodiesterase 4D (PDE4D) (12) or soluble guanylate cyclase-b1 subunit (sGC-b1)
• The asymmetry in neurite growth promotion by cAMP elevation may reflect the dominance of a single neurite that first acquired the highest cAMP elevation, together with long-range self–down-regulation of cAMP that suppressed the growth of most other neurites.

By the way, I am really amazed that they could dry the cAMP and cGMP onto the glass, coated and plated neurons on, with culture media for days – completely don’t worry about the diffusion.