PDE9A inhibition rescues amyloid beta-induced deficits in synaptic plasticity and cognition

Neurobiology of Aging xxx (2014) 1e7

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PDE9A inhibition rescues amyloid beta-induced deficits in synaptic plasticity and cognition Katja S. Kroker a, *,1, Chantal Mathis b,1, Anelise Marti a, Jean-Christophe Cassel b, Holger Rosenbrock a, 2, Cornelia Dorner-Ciossek a, 2 a b

Department of CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany Laboratoire de Neurosciences Cognitives et Adaptatives, CNRS, Université de Strasbourg UMR 7364, GDR CNRS 2905, Strasbourg, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2013 Received in revised form 14 March 2014 Accepted 20 March 2014

The cyclic nucleotide cGMP is an important intracellular messenger for synaptic plasticity and memory function in rodents. Therefore, inhibition of cGMP degrading phosphodiesterases, like PDE9A, has gained interest as potential target for treatment of cognition deficits in indications like Alzheimer’s disease (AD). In fact, PDE9A inhibition results in increased hippocampal long-term potentiation and exhibits procognitive effects in rodents. To date, however, no evidence has been published linking PDE9A inhibition to the pathologic hallmarks of AD such as amyloid beta (Ab) deposition. Therefore, we investigated the role of PDE9A inhibition in an AD relevant context by testing its effects on Ab-related deficits in synaptic plasticity and cognition. The PDE9A inhibitor BAY 73-6691 was found to restore long-term potentiation impaired by Ab42 oligomers. Furthermore, we demonstrated that BAY 73-6691 enhanced cGMP levels in the hippocampus of APP transgenic tg2576 mice and improved memory performance of these mice. Altogether, our results support the hypothesis that inhibition of PDE9A could be a beneficial approach for the treatment of memory impairment in AD patients. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: PDE9 LTP cGMP Alzheimer’s disease BAY 73-6691 Amyloid beta Object location task tg2576 Hippocampus Cognition

1. Introduction In the past decades, the cyclic nucleotide cGMP has been recognized as a key intracellular secondary messenger mediating signal transduction and synaptic plasticity in the brain (Blokland et al., 2006; Prickaerts et al., 2004; Rutten et al., 2007; Son et al., 1998). Its synthesis is catalyzed by 2 isoforms of guanylate cyclases, which are activated by nitric oxide and convert GTP to cGMP (Christopherson et al., 1999; Garthwaite, 2008). After its conversion, cGMP activates an intracellular signaling cascade including the targets PKG and the transcription factor CREB (Ko and Kelly, 1999; Lu et al., 1999), which is a key mediator of synaptic plasticity related to learning and memory (Blokland et al., 2006; Prickaerts et al., 2004; Rutten et al., 2007; Son et al., 1998). The phosphodiesterases 5, 6, and 9 selectively degrade cGMP (Blokland et al., 2012;

* Corresponding author at: Department of CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, Birkendorfer Strasse 65, 88397 Biberach, Germany. Tel.: þ49 7351 5492216; fax: þ49 7351 5498928. E-mail address: [email protected] (K.S. Kroker). 1 These authors equally contributed to the work. 2 These authors equally contributed to the work. 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.023

 ska and Strosznajder, 2010; Menniti et al., 2006), Domek-qopacin with PDE9A having the highest affinity for cGMP (Km z 170 nM; Fisher et al., 1998). PDE9A has gained special interest because of its expression in cognition-relevant brain regions such as the cortex and the hippocampus (Andreeva et al., 2001; Lakics et al., 2010; van Staveren et al., 2002). Recent studies showed that PDE9A inhibitors can increase cGMP levels in the rodent brain (Verhoest et al., 2009) and in the cerebrospinal fluid of both rodents and humans (Hutson et al., 2011; Nicholas et al., 2009). In rat hippocampal slices, it was demonstrated that PDE9A inhibition leads to facilitation of longterm potentiation (LTP) (Kroker et al., 2012; van der Staay et al., 2008), a widely used cellular model of memory formation (Bliss and Collingridge, 1993). Furthermore, in rodents the PDE9A inhibitors BAY 73-6691 (van der Staay et al., 2008) and PF-04447943 (Hutson et al., 2011) were found to enhance memory in various memory tasks. Such positive effects have led to the hypothesis that inhibition of PDE9A could be a beneficial approach for the treatment of memory dysfunction (Blokland et al., 2012; Menniti et al., 2006; Reneerkens et al., 2009; Schmidt, 2010), which is the cognitive signature of Alzheimer’s disease (AD) (Blennow et al., 2006; Francis et al., 1999; Kar et al., 2004). The histopathological hallmarks of AD comprise intracellular neurofibrillary tangles of tau

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protein and extracellular senile plaques of amyloid beta (Ab) peptides (Alzheimer’s Association, 2012; Querfurth and LaFerla, 2010). So far, no data have been published linking PDE9A inhibition to Ab, which is believed to be the putative cause of the AD pathophysiology because of its synaptotoxic features (Ferreira and Klein, 2011; Haass and Selkoe, 2007; Klein, 2013). Therefore, using well-established models of amyloid pathology, we determined the effects of PDE9A inhibition on both Ab-impaired synaptic plasticity and Ab-induced cognitive deficits. In particular, we assessed, in rat hippocampal slices, the restoration by the PDE9A inhibitor BAY 73-6691 of Ab42 oligomer-impaired LTP. Using the tg2576 mouse model of amyloidosis, we then investigated the effect of BAY 73-6691 on cGMP levels in the hippocampus and on memory performance in a hippocampus-dependent object location task. 2. Methods 2.1. Animals Procedures involving animals and their care were conducted in conformity with institutional and European Union guidelines (EEC Council Directive 86/609) and were approved by the Ethical Committee of the responsible regional council (Tübingen) and the Strasbourg regional ethical committee (CREMEAS authorization number: AL02/14/06/12). LTP experiments were performed in male Wistar rats (Janvier, Le Genest Saint Isle, France) aged 6 to 7 weeks. Tg2576 mice express the transgene coding for the 695 amino acid human APP isoform containing the double “Swedish” mutation (APPswe, Lys 670Asn, and Met 671Leu; Hsiao et al., 1996), which leads to increased production of Ab. The object location task and brain cGMP levels were measured in tg2576 mice and their corresponding wild-type litter mate (Bayer Pharma AG, Wuppertal, Germany) when aged 10 months. 2.2. Preparation of brain slices, recording, drug application, data acquisition, and analysis 2.2.1. Preparation of brain slices Hippocampal rat slices were prepared according to Kroker et al. (2011a). Briefly, rats were anaesthetized with isoflurane and killed by decapitation. Brains were quickly removed and transverse hippocampal brain slices (400 mm) were prepared. Slices were allowed to recover in a holding chamber containing artificial cerebrospinal fluid (bubbled with 95% O2 and 5% CO2) for at least 1 hour. Then, they were transferred to integrated brain slice chambers and continuously superfused (at a flow rate of 2.5 mL min1). Before doing any electrophysiological recordings, the slices were allowed to equilibrate for at least 30 minutes. 2.2.2. Slice recording, data acquisition, and analysis Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region by stimulation of the Schaffer collateralecommissural fibers in the stratum radiatum according to Kroker et al. (2011a). Briefly, glass electrodes were placed in the apical dendritic layer and the amplitudes of fEPSPs were used as the parameter of interest. To generate fEPSPs at a constant subthreshold stimulus, the stimulus strength of the pulses was adjusted to 20%e 30% of the fEPSP maximum, and this voltage was used for the experiment. During baseline recording each slice was stimulated every 30 seconds for at least 1 hour. LTP was induced by repeated strong high frequency stimulation consisting of 100 pulses at the frequency of 100 Hz, repeated 2 times in 5-minutes intervals (Kroker et al., 2011b; Lu et al., 1999). A modular electrophysiology system, supplied by npi electronic GmbH (Tamm, Germany), conducted the

low noise recordings of extracellular signals. AC-coupled signals were amplified 1000-fold and internally filtered with a 5 kHz lowpass filter as well as a 3 Hz high-pass filter. For data acquisition and analysis the software Notocord was used. Data are shown as mean percentage (SEM) of the baseline fEPSP amplitude. Data were analyzed using 1-way analysis of variance (ANOVA) (post hoc test: Bonferroni test) to compare multiple conditions. In all cases a pvalue  0.05 was considered significant. Histograms show the mean amplitude (SEM) of fEPSPs measured between 115 and 125 minutes after LTP stimulation according to Jia et al. (2010) and Kroker et al. (2011b). 2.2.3. Compounds, Ab oligomer preparation, and application The following drugs were used: ODQ and KT 5823 were purchased from Sigma-Aldrich Corporation (St Louis, USA). The compound BAY 73-6691 (1-(2-Chlorophenyl)-6-[(2R)-3,3,3trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one) was synthesized as a racemate in the Department of Medicinal Chemistry of Boehringer Ingelheim Pharma GmbH & Co KG. The compounds were prepared as stock solutions (ODQ and BAY 73-6691 were prepared in DMSO [the final concentration of DMSO was 0.1%]) and diluted in artificial cerebrospinal fluid immediately before application. Ab42 oligomers (Ab derived diffusible ligands) were prepared according to Lambert et al. (1998) with slight modifications (Moreth et al., 2013). Briefly, Ab42 (Bachem, Germany) was solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol by sonication (10 minutes) to a final concentration of 1 mM. Then, it was flash-frozen in liquid nitrogen and lyophilized. The lyophilized Ab42 was solubilized in 10 mM NaOH and brought up in ice-cold Ham’s F12 medium (wt/oz phenol red) to a final concentration of 100 mM (pH 7.4 at 4  C for at least 14 hours). The samples were centrifuged at 15,000 g for 10 minutes at 4  C. The supernatant containing Ab42 oligomers (Ab derived diffusible ligands) was then used immediately. All compounds and the Ab42 oligomers were applied 30 minutes before LTP stimulation and remained in the superfusion buffer for 60 minutes. 2.3. Mouse brain cGMP measurements Measurements of cGMP accumulation in brain tissue following drug administration were performed as previously described (Schmidt et al., 2008). Tg2576 mice were killed by focused microwave irradiation of the brain, 60 minutes after oral administration of vehicle (0.5% natrosol in water containing 0.01% Tween-80) or various doses of BAY 73-6691 (n ¼ 8 animals per group for all groups). The whole hippocampus was isolated, homogenized, and homogenates were centrifuged in 0.5 N HCl. Supernatant concentrations of cyclic nucleotides were measured using enzyme immunoassay kits according to the manufacturer’s protocol (Direct cGMP EIA kit, Enzo Life Science GmbH, Germany). Data were analyzed using a 1-way ANOVA followed by Dunnett post hoc analysis to compare drug doses to vehicle. 2.4. Object location memory task Object location tasks are based on the spontaneous tendency of mice to explore preferentially displaced objects (moved to a new location) when the spatial configuration of several familiar objects is modified. The apparatus consisted of a Plexiglas open field (52  52  40 cm). The objects (size: 10e17 cm) were made of metal, glass, or plastic. The object location task was performed according to Yassine et al. (2013). Briefly, the mice were first habituated to the open-field and the presence of objects with a daily 10-minute session for 2 days. The next day, the mice were

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allowed to explore for a 10-minute acquisition trial the spatial configuration of 3 new objects. After 4 minutes spent in their home cage, the mice explored a new set of identical objects (duplicates) for a 5-minute retention trial with 1 object moved to a new position. Object exploration was defined as the nose pointing toward the object within 1 centimeter. Open-field and objects were wiped with 70% ethanol between trials. Object location memory task was performed 60 minutes after oral administration of vehicle (0.5% natrosol in water containing 0.01% Tween-80) or various doses of BAY 73-6691 (n ¼ 9e12 animals per group). The effect of the treatment on object location memory was evaluated by comparing the time spent on the displaced object to the mean time spent on the 2 non-displaced objects for the 5 experimental groups. Statistical analyses used an analysis of variance with repeated measures on the factor object (repeatedmeasures ANOVA; Statistica 8.0, Statsoft, Inc, Tulsa, OK, USA) followed, when appropriate, by a post hoc analysis using the NewmaneKeuls (NK) test.

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3. Results 3.1. Effect of inhibiting PDE9A by BAY 73-6691 on Ab42 oligomer impaired LTP Fig. 1 shows the effect of BAY 73-6691 (1 mM) on LTP impaired by Ab42 oligomers (20 nM). These concentrations were chosen based on previously published data on the individual effects of BAY 73-6691 and Ab42 oligomers on hippocampal LTP (Kroker et al., 2012, 2013). The impairment induced by Ab42 oligomers on LTP was fully compensated for by BAY 73-6691 (Fig. 1A). Neither 20 nM Ab42 oligomers (Fig. 1A), nor 1 mM BAY 73-6691 (Supplementary Fig. S1B), or a combination of both (Fig. 1A) affected basal fEPSPs. To test whether this effect of BAY 73-6691 was attributable to an increase of the NO/cGMP pathway, the effect of the soluble guanylyl cyclase inhibitor ODQ was analyzed. Five micromoles of ODQ, a concentration shown to be sufficient to significantly impair LTP (Lu et al., 1999) was used. As shown in Fig. 1B, the addition of

A

B

C

Fig. 1. BAY 73-6691 reverses LTP being impaired by Ab42 oligomers via the NO-pathway and activation of PKG. The potential of the PDE9A inhibitor BAY 73-6691 to reverse LTP that had been impaired by Ab42 oligomers and the underlying pathway is analyzed. All drugs are applied 30 minutes before LTP stimulation (arrows) and remain for 60 minutes (horizontal bar). Neither Ab42 oligomers nor any compound or combination affects basal fEPSPs. Individual fEPSPs before (1) and after LTP stimulation (2) are shown (vertical bar: 0.5 mV, horizontal bar: 10 ms). *** p < 0.001, 1-way ANOVA (post hoc test: Bonferroni test). Error bars represent  SEM. (A) BAY 73-6691 (1 mM) completely reverses the Ab42 oligomer (20 nM)-mediated impairment of LTP (control: n ¼ 10; 20 nM Ab42: n ¼ 8; Ab42 þ BAY 73-6691: n ¼ 5). (B) To determine whether the restoration of Ab42 oligomermediated impairment of LTP by BAY 73-6691 is based on the NO pathway, a combination of Ab42 oligomers (20 nM), BAY 73-6691 (1 mM), and the soluble guanylyl cyclase inhibitor ODQ (5 mM) is tested. The reversing effect of BAY 73-6691 is completely abolished (control: n ¼ 10; Ab42 þ BAY 73-6691: n ¼ 5; Ab42 þ ODQ: n ¼ 5; Ab42 þ BAY 73-6691 þ ODQ: n ¼ 5). (C) To analyze whether the reversal effect of BAY 73-6691 regarding LTP that had been impaired by Ab42 oligomers is based on the activation of PKG, a combination of Ab42 oligomers (20 nM), BAY 73-6691 (1 mM) and the PKG-inhibitor KT 5823 (2 mM) is determined. The reversing effect of BAY 73-6691 is completely abolished (control: n ¼ 10; Ab42 þ BAY 73-6691: n ¼ 5; Ab42 þ KT 5823: n ¼ 5; Ab42 þ BAY 73-6691 þ KT 5823: n ¼ 5). Abbreviations: Ab, amyloid beta; ANOVA, analysis of variance; fEPSPs, field excitatory postsynaptic potentials; LTP, long-term potentiation; SEM, standard error of the mean.

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5 mM ODQ to 1 mM BAY 73-6691 obliterated the BAY 73-6691mediated enhancement of LTP. Furthermore, to analyze whether BAY 73-6691 produced its effect through the activation of PKG, the PKG-inhibitor KT 5823 was used at a concentration of 2 mM, which is known to be sufficient to significantly impair protein-synthesis dependent LTP (Lu et al., 1999). As shown in Fig. 1C, the addition of 2 mM KT 5823 to 1 mM BAY 73-6691 attenuated the BAY 73-6691mediated enhancement of LTP. Basal fEPSPs were not affected by the mixture of BAY 73-6691 and ODQ or BAY 73-6691 and KT 5823 (Fig. 1B and C; Supplementary Fig. S2). These results indicate that the effect BAY 73-6691 on impaired LTP is linked to facilitation of the NO/cGMP/PKG pathway. 3.2. Inhibition of PDE9A elevates the level of cGMP Oral administration of BAY 73-6691 (1e125 mg/kg per os) induced a significant increase of tissue concentrations of cGMP in the hippocampus of tg2576 mice at the highest dose tested (Fig. 2). This result demonstrates in principle target (PDE9) engagement in the brain, that is, the compound is able to block the PDE9 enzyme in animals. 3.3. Object location memory task BAY 73-6691 administration significantly improved performance for object location memory in tg2576 mice (F [4,44] ¼ 4.60, p ¼ 0.003, Fig. 3A for NK-test comparisons). Remarkably, tg2576 mice treated with 5 mg/kg BAY 73-6691 reached performances similar to those of wild-type mice. The compound had no effect on the total time spent exploring the 3 objects during the acquisition trial (F [4,44] ¼ 1.07, p ¼ 0.38; no significance with NK test, Fig. 3B). 4. Discussion To date, no evidence has been published linking PDE9A inhibition to the underlying pathologic hallmarks of AD. Thus, the present study is the first one having investigated the role of PDE9A inhibition with particular focus on the deficits associated with AD pathogenesis. For all experiments, the potent and specific PDE9A inhibitor BAY 73-6691 was used. This compound selectively inhibits human and murine PDE9A activity in vitro and shows only moderate activity on other phosphodiesterases (Wunder et al., 2005). The hippocampus was the region of interest for all experiments

Fig. 2. PDE9A inhibition increases the level of cGMP in the hippocampus in tg2576 mice. Inhibition of PDE9A by BAY 73-6691 produces a significant increase of cGMP in the hippocampus of tg2576 mice, 60 minutes post oral administration of BAY 73-6691 as compared with vehicle (n ¼ 8 animals per treatment group). ** p < 0.01, 1-way ANOVA followed by Dunnett post hoc. Error bars represent  SEM. Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean.

performed in this study, because it is known to play a crucial role in memory formation in humans and animals (Assini et al., 2009; Deshmukh and Knierim, 2013; Piekema et al., 2006; Pihlajamäki et al., 2004; Scoville and Milner, 1957; Squire, 1992). Moreover, it is one of the first regions of the brain to be affected in AD patients causing the first cognitive symptoms (Burgess et al., 2002; Frisoni et al., 2008), for example, deficits in forming memory for object locations in space (Fleischman and Gabrieli, 1999; Kessels et al., 2010; Lee et al., 2003; MacDuffie et al., 2012). As a first step of our approach, we assessed the effect of the PDE9A inhibitor BAY 73-6691 on LTP in rat hippocampal slices exposed to Ab42 oligomers. LTP in hippocampal slices is a widely used cellular model of memory formation (Bliss and Collingridge, 1993), which can be impaired by Ab42 oligomers (S1A; Kroker et al., 2013; Nomura et al., 2012; Selkoe, 2008; Walsh and Selkoe, 2007). Moreover, the PDE9A inhibitor BAY 73-6691 was shown to enhance LTP (S1B; Kroker et al., 2012; van der Staay et al., 2008). However, although Ab42 oligomers and a PDE9A inhibitor were tested individually in different studies (Supplementary Fig. S1; Kroker et al., 2012, 2013; Nomura et al., 2012; Selkoe, 2008; van der Staay et al., 2008; Walsh and Selkoe, 2007), a combined approach of Ab42 oligomers together with PDE9A inhibition has not been performed so far. For this purpose, we first recorded LTP in the CA1 region of hippocampal rat slices and impaired it with Ab42 oligomers. Subsequently, we

Fig. 3. BAY 73-6691 improves object location memory in tg2576 mice without affecting total exploration time. (A) BAY 73-6691 improves object location memory in tg2576 mice. BAY 73-6691 (0.2, 1, or 5 mg/kg, per os) or vehicle was administered 60 minutes before the acquisition trial. Memory performance is evaluated for each group by comparing the time spent exploring the displaced object to the mean time spent exploring the non-displaced objects during the 5-minute retention trial. * p < 0.05, ** p < 0.01, and *** p < 0.001, NK test. Error bars represent  SEM. (B) BAY 73-6691 administration has no effect on object exploration during the acquisition trial. BAY 73-6691 (0.2, 1, or 5 mg/kg, per os) or vehicle is administered 60 minutes before the 10-minute acquisition trial. Data are expressed as the total time spent exploring the 3 objects. Abbreviations: NK, NewmaneKeuls test; SEM, standard error of the mean.

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were able to demonstrate that PDE9A inhibition by BAY 73-6691 restored LTP in rat hippocampal slices impaired by Ab42 oligomers (Fig. 1). Furthermore, we were able to show that the rescue requires the activity of soluble guanylyl cyclase and protein kinase PKG indicating that the effect is based on the NO/cGMP/PKG pathway (Fig. 2; Supplementary Fig. S2). Here, all LTP experiments were performed in hippocampal slices obtained from rats because of previous experience with BAY 73-6691 and Ab42 oligomers in this experimental model (Kroker et al., 2012, 2013). This enabled us to use defined and validated drug concentrations known to be efficient regarding LTP and hence to obtain comparable data. Moreover, we analyzed the potential of BAY 73-6691 to improve LTP exclusively impaired by Ab42 oligomers and no other Ab aggregates, for example, dimers or fibrils. The reason for choosing this experimental design was based on the fact that Ab42 oligomers are currently believed to be the putative cause of AD pathophysiology because of their synaptotoxic features (Ferreira and Klein, 2011; Haass and Selkoe, 2007; Klein, 2013). For example, Ab42 oligomers were shown to cause abnormal dendritic spine morphology, changes in synaptic receptor composition, and synaptic vesicle trafficking as well as spine loss (Lacor et al., 2007; Nimmrich and Ebert, 2009; Viola et al., 2008). Moreover, it was reported that acute injection of Ab42 oligomers into the hippocampus of rats and mice causes impairments in learning and memory (Freir et al., 2011; Reed et al., 2011; Youssef et al., 2008). Furthermore, recent data supports the Ab42 oligomer hypothesis by demonstrating that the presence of Ab42 oligomers and plaques but not amyloid plaques alone are responsible for the demented state of the patient (Esparza et al., 2013). We investigated whether the PDE9 inhibitor BAY 73-6691 is able to increase cGMP levels in the hippocampus and to enhance hippocampal dependent memory performance in tg2576. These mice show increased amounts of Ab oligomers but no overt Ab plaque deposits: Ab oligomers appear already at an age of approximately 6 months whereas amyloid plaques formation only begins by the age of 10e12 months (Kawarabayashi et al., 2001, 2004). Yassine et al. (2013) recently demonstrated that the memory impairment of tg2576 mice in the object location task appears within the same age range of Ab oligomers appearance, that is, 6e7 months. Before testing the effect of PDE9A inhibition on these Ab-related memory deficits, we verified that BAY 73e6691 has the potential to increase cGMP levels in the hippocampus of tg2576 mice. So far, the increasing effect of PDE9A inhibition on cGMP levels was only shown in wild-type rodent brains by using PF-04447943 (Hutson et al., 2011; Kleiman et al., 2012). It is noteworthy that this kind of in vivo and/or ex vivo assay is not suitable for predicting effective doses in behavioral cognition tasks, for example, due to dilution effects by tissue homogenization and assay processing. Nevertheless, this assay can be used to demonstrate target engagement in principle, that is, PDE9 inhibition in brain by the compound (Kleiman et al., 2012). Here, we demonstrated that BAY 73-6691 administered at a dose of 125 mg/kg indeed increased cGMP levels in the hippocampus of tg2576 mice (Fig. 3). Next, we addressed the question whether BAY 73-6691 had an effect on hippocampal-dependent learning and memory performances in tg2576 mice by using the object location memory task. This task is based on the spontaneous tendency of mice to explore preferentially displaced objects (moved to a new location) when the spatial configuration of several familiar objects is modified. Since the efficacy of PDE9A inhibitors regarding cognition was only demonstrated in wild-type rodents so far (Hutson et al., 2011; Kleiman et al., 2012; van der Staay et al., 2008), our study demonstrates for the first time that BAY 73-6691 can improve object location memory performance of tg2576 mice starting at a dose of 0.2 mg/kg in an object location task (Fig. 3A). The highest dose tested here, 5 mg/kg, completely reversed the profound deficit displayed by tg2576 mice

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in this task. There is no significant effect of the compound on the general exploration activity of the objects (Fig. 3B), which suggests that there are no side effects on exploratory behavior of the mice and that the positive effect is specific to memory. The effective doses of BAY 73-6691 in this object location task are comparable with those found to be active doses in social and object recognition tasks (van der Staay et al., 2008). However, cGMP levels in the hippocampus were increased only at the dose of 125 mg/kg and not at the 1 or 5 mg/kg dose, which improved memory performance. As stated previously, the reason for this discrepancy might be the unsuitability of the cGMP assay in tissue homogenates to predict effective doses in behavioral cognition tasks; instead this read-out is considered to serve exclusively the purpose to proof target engagement in principle (Kleiman et al., 2012). To conclude, this study demonstrates that PDE9A inhibition can reverse deficits caused by Ab in synaptic plasticity and cognition. In particular, we found that the PDE9A inhibitor BAY 73-6691 reverses LTP in rat hippocampal slices that had been impaired by Ab42 oligomers. Furthermore, we showed that BAY 73-6691 enhanced cGMP levels in the hippocampus of the tg2576 transgenic mouse model of AD and improved memory performance in a hippocampus-dependent task. To date, PDE9A inhibition was only shown to increase cGMP levels in the brain, enhance hippocampal LTP and improve cognition in wild-type rodents. Here, we provide evidence that PDE9A inhibition improves synaptic plasticity and memory performance in animals with an amyloid pathology. Therefore, the results presented in this study suggest that inhibition of PDE9A might be a powerful approach for the treatment of cognitive dysfunction in AD patients. Disclosure statement There are no actual or potential conflicts of interest. Acknowledgements The authors wish to thank Carole Strittmatter and Nancy Kötteritzsch for perfoming the object location memory test and the cGMP measurements, respectively. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2014.03.023. References Alzheimer’s Association, 2012. Alzheimer’s disease facts and figures. Alzheimers Dement. 8, 131e168. Andreeva, S.G., Dikkes, P., Epstein, P.M., Rosenberg, P.A., 2001. Expression of cGMPspecific phosphodiesterase 9A mRNA in the rat brain. J. Neurosci. 21, 9068e9076. Assini, F.L., Duzzioni, M., Takahashi, R.N., 2009. Object location memory in mice: pharmacological validation and further evidence of hippocampal CA1 participation. Behav. Brain Res. 204, 206e211. Blennow, K., de Leon, M.J., Zetterberg, H., 2006. Alzheimer’s disease. Lancet 368, 387e403. Bliss, T.V.P., Collingridge, G.L., 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31e39. Blokland, A., Menniti, F.S., Prickaerts, J., 2012. PDE inhibition and cognition enhancement. Expert Opin. Ther. Pat 22, 349e354. Blokland, A., Schreiber, R., Prickaerts, J., 2006. Improving memory: a role for phosphodiesterases. Curr. Pharm. Des. 12, 2511e2523. Burgess, N., Maguire, E.A., O’Keefe, J., 2002. The human hippocampus and spatial and episodic memory. Neuron 35, 625e641. Christopherson, K.S., Hillier, B.J., Lim, W.A., Bredt, D.S., 1999. PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain. J. Biol. Chem. 274, 27467e27473.

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