Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance
Introduction
The nitric oxide/guanosine 3′,5′-cyclic monophosphate (NO/cGMP) signal transduction pathway has an important role during synaptic plasticity. Nitric oxide synthase (NOS), the enzyme that generates NO, is activated by Ca2+-influx through glutamate receptors of the N-methyl-d-aspartate (NMDA)-type on postsynaptic neurons (Garthwaite, 1991). NO stimulates both post- and presynaptic guanylyl cyclase (GC), leading to the formation of the second messenger cGMP (Boulton et al., 1995, De Vente and Steinbusch, 1992). In turn, cGMP directly opens cyclic nucleotide-gated ion channels (Savchenko et al., 1997) and activates cGMP-dependent protein kinase (PKG), which phosphorylates synaptic proteins (Wang and Robinson, 1997). Inhibitors of NOS, GC, or PKG block long-term potentiation (LTP) of hippocampal synaptic transmission, a physiological correlate of learning and memory processes (Böhme et al., 1991, O’Dell et al., 1991, Schuman and Madison, 1991, Zhuo et al., 1994, Boulton et al., 1995, Lu et al., 1999). Thus, NO may serve as a retrograde signal that closes an important feedback loop during use-dependent synaptic plasticity in the hippocampus (reviewed by Collingridge and Bliss, 1995, Hawkins et al., 1998). While marked reductions in LTP have been demonstrated in knock-out mice that lack both endothelial and neuronal NO synthase (Son et al., 1996), the role of NO as a retrograde messenger in LTP is controversial (discussed in Holscher, 1997, Hawkins et al., 1998). Changes in the strength of the postsynaptic response during LTP as a consequence of the activation of cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent kinase include increased glutamate receptor density and function (reviewed by Lynch, 2004). Several substrate proteins of these kinases are also phosphorylated by PKG which could result in similar changes in postsynaptic function through the NO/cGMP/PKG pathway (Wang and Robinson, 1997, Ahem et al., 2002).
NOS inhibitors impair memory performance in one-trial passive avoidance in chicks (Holscher and Rose, 1993) and spontaneous alternation in mice (Yamada et al., 1996). Local injection of cGMP analogues can ameliorate the deficit induced by NOS-inhibition (Yamada et al., 1996). Intra-hippocampal injections of 8-Br cGMP improved the ability of rats to remember objects (Prickaerts et al., 2002a), as well as retention performance in a passive avoidance task (Bernabeu et al., 1996). Furthermore, cGMP levels in the hippocampus of rats increased transiently during the first 30 min of a passive avoidance task (Bernabeu et al., 1996). These findings support an essential role of cGMP in early memory formation.
An important part of the signal transduction process is the rapid degradation of cGMP by cyclic nucleotide phosphodiesterases (PDEs). At least 21 PDE genes have been identified and subgrouped into 11 families (Soderling and Beavo, 2000, O’Donnell, 2000). PDE4, PDE7 and PDE8 hydrolyse cAMP, while PDE5, PDE6 and PDE9 hydrolyse cGMP; PDE1, PDE2, PDE3, PDE10 and PDE11 can hydrolyse both cAMP and cGMP. The strong expression of PDE2 in neurons of the hippocampus and cortex (Repaske et al., 1993) suggests that this enzyme may control intraneuronal cGMP and cAMP levels in areas that are important for memory formation and storage. A characteristic feature of PDE2 is the positive cooperativity of the substrate cGMP—low levels of cGMP enhance the rate of cAMP hydrolysis by a factor of 5–6 (Martins et al., 1982). Under basal conditions, PDE2 activity in neurons is low, but it is stimulated by the acute increase in cGMP levels following GC activation during signal transduction. Therefore, inhibition of brain PDE2 may selectively increase cGMP and cAMP levels in active synapses and could thus influence synaptic plasticity and memory formation.
We investigated the role of PDE2 in neuronal signal transduction and memory, using the highly potent and selective PDE2 inhibitor 2-(3,4-dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methyllimidazo[5,1-f][1,2,4]triazin-4(3H)-one (Bay 60-7550). To this end, we determined the effects of Bay 60-7550 on cGMP levels in neurons in primary culture and in slices, on hippocampal LTP, and on cognitive function in rats and mice, assessed using an object recognition task, social memory, and a T-maze spontaneous alternation task.
Section snippets
Materials
The novel PDE2 inhibitor 2-(3,4-dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methyl imidazo[5,1-f][1,2,4]triazin-4(3H)-one (Bay 60-7550) (Table 1) and the GC stimulator Bay 41-8543 (Stasch et al., 2002) were synthesized by the Chemistry Department of Pharma Research, Bayer AG (Wuppertal, Germany).
PDE inhibition and selectivity assays
PDE2 was purified from bovine heart, PDE1 from bovine aorta and PDE5 from human platelets (Saenz de Tejada et al., 2001). Human recombinant PDEs were expressed in SF9 cells using
Bay 60-7550—a selective PDE2 inhibitor
Bay 60-7550 inhibited the activity of PDE2 purified from bovine heart with an IC50 value of 2.0±0.7 nM (n=12) and human recombinant PDE2 with an IC50 value of 4.7±1.0 nM (n=7). Bay 60-7550 showed 50-fold selectivity for PDE2 compared to PDE1 and more than 100-fold selectivity compared to PDE5 and the other PDEs tested (PDE3B, PDE4B, PDE7B, PDE8A, PDE9A, PDE10A, PDE11A, see Table 1, Fig. 1A). Bay 60-7550 had an IC50 of >10 μM (less than 50% inhibition at 10 μM) for the following receptors and
Discussion
Owing to its high potency and excellent selectivity profile, Bay 60-7550 is a very useful tool for the study of PDE2-dependent processes. To date, the physiological actions of PDE2 have been characterized with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (Mery and Fischmeister, 1994, Podzuweit et al., 1995), which inhibits PDE2 with an IC50 of ∼1 μM. However, in contrast to Bay 60-7550, EHNA is also a very potent adenosine deaminase inhibitor (IC50∼2 nM), which complicates the interpretation of
Acknowledgements
The LTP studies were conducted in the laboratories of Dr. U.H. Schröder and Professor K. Reymann, Forschungsinstitut Angewandte Neurowissenschaften (FAN), Magdeburg, Germany. A. Sik, Maastricht, is acknowledged for contributions to the object recognition studies. K. Selbach is acknowledged for contributions to the social recognition studies. This work is dedicated to our former colleague, the late Ulrich Niewoehner.
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- 1
Present address: Neuroscience Discovery Research, Eli Lilly and Company Limited, Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK.
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Present address: ID Lelystad, Institute for Animal Science and Health BV, Lelystad 8200 AB, The Netherlands.
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Present address: Merz GmbH, 60318 Frankfurt, Germany.
- 4
Present address: Roche Pharmaceuticals, Palo Alto, CA 94304, USA.
- 5
Present address: Johnson & Johnson PRD, B2340 Beerse, Belgium.
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Present address: Envivo Pharmaceuticals, Cambridge, MA 02139, USA.