TOP2ß-Dependent Nuclear DNA Damage Shapes Extracellular Growth Factor Responses via Dynamic AKT Phosphorylation to Control Virus Latency.

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

Repair of topoisomerase 1-induced DNA damage by tyrosyl-DNA phosphodiesterase 2 (TDP2) is dependent on its magnesium binding.

Topoisomerases are enzymes that relax DNA supercoiling during replication and transcription. Camptothecin, a topoisomerase 1 (TOP1) inhibitor, and its analogs trap TOP1 at the 3'-end of DNA as a DNA-bound intermediate, resulting in DNA damage that can kill cells. Drugs with this mechanism of action are widely used to treat cancers. It has previously been shown that tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs TOP1-induced DNA damage generated by camptothecin. In addition, tyrosyl-DNA phosphodiesterase 2 (TDP2) plays critical roles in repairing topoisomerase 2 (TOP2)-induced DNA damage at the 5'-end of DNA and in promoting the repair of TOP1-induced DNA damage in the absence of TDP1. However, the catalytic mechanism by which TDP2 processes TOP1-induced DNA damage has not been elucidated. In this study, we found that a similar catalytic mechanism underlies the repair of TOP1- and TOP2-induced DNA damage by TDP2, with Mg2+-TDP2 binding playing a role in both repair mechanisms. We show chain-terminating nucleoside analogs are incorporated into DNA at the 3'-end and abort DNA replication to kill cells. Furthermore, we found that Mg2+-TDP2 binding also contributes to the repair of incorporated chain-terminating nucleoside analogs. Overall, these findings reveal the role played by Mg2+-TDP2 binding in the repair of both 3'- and 5'-blocking DNA damage.

Inhibition of Phosphodiesterase 2 Ameliorates Post-Traumatic Stress-Induced Alcohol Intake Disorder by Regulating cAMP/cGMP Signaling.

BACKGROUND: Post-traumatic stress disorder (PTSD) is the prevalent psychiatric disorder that induces alcohol use disorders (AUD) such as abnormal alcohol intake and anxiety. However, little is known about whether phosphodiesterase 2 (PDE2)-cAMP/cGMP signaling is involved in PTSD-induced AUD. METHODS: The present study used single-prolonged stress (SPS) to mimic PTSD that induced increases in ethanol intake and preference (2-bottle choice test) and anxiety-like behavior (elevated-plus maze test and novelty suppressed feeding test). PDE2 inhibitor Bay 60-7550 (Bay) was administered to the mice and protein kinase A (PKA) inhibitor H89 and PKG inhibitor KT5823 were micro-injected into dorsolateral striatum (DLS) and central amygdala (CA) of mice to determine whether the effects of Bay on anxiety-like behavior in SPS mice are brain region dependent. RESULTS: PDE2 inhibitor Bay rescued SPS-induced decreases in open arm entries and open arm time exposure in elevated-plus maze test and reversed increased latency to feed in the novelty suppressed feeding test. Moreover, SPS-induced ethanol use disorder was reversed by Bay as evidenced by decreased ethanol intake and preference without changing total fluid intake in the SPS mice after treatment with Bay. However, Bay did not change the ethanol metabolism or sucrose or quinine intake and preference. The locomotor activity was not affected after treatment with Bay. Interestingly, microinjection of PKA or PKG inhibitor H89 or KT5823 into DLS prevented the effects of Bay on alcohol intake and preference and cAMP-response element binding proteins phosphorylation and brain derived neurotrophic factor expression in DLS but not on the anxiety-like behavior in SPS mice. Microinjection of these inhibitors into CA prevented Bay-induced anxiolytic-like effects and cAMP-response element binding proteins phosphorylation and brain derived neurotrophic factor levels in CA but did not affect ethanol intake in SPS mice, indicating that the effects of Bay on different behaviors are brain region dependent. CONCLUSIONS: These findings support the hypothesis that PDE2-cAMP/cGMP signaling may differentially mediate PTSD-induced AUD and anxiety-like behavior.

Discovery of effective phosphodiesterase 2 inhibitors with antioxidant activities for the treatment of Alzheimer's disease.

The multi-target-directed-ligand (MTDL) strategy has been widely applied in the discovery of novel drugs for the treatment of Alzheimer's disease (AD) because of the multifactorial pathological mechanisms of AD. Phosphodiesterase-2 (PDE2) has been identified to be a novel and promising target for AD. However, MTDL combining with the inhibitory activity against PDE2A and other anti-AD factors such as antioxidants has not been developed yet. Herein, a novel series of PDE2 inhibitors with antioxidant capacities were designed, synthesized, and evaluated. Most compounds showed remarkable inhibitory activities against PDE2A as well as antioxidant activities. Compound 6d was selected, which showed good IC50 of 6.1 nM against PDE2A, good antioxidant activity (ORAC (Trolox) = 8.4 eq.) and no cytotoxicity to SH-SY5Y cells. Molecular docking and dynamics simulations were applied for the rational design and explanation of structure-activity relationship (SAR) of lead compounds.

Molecular mechanisms of topoisomerase 2 DNA-protein crosslink resolution.

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.

Introduction to the multi-author-review: emerging advances in the structural chemistry of DNA strand break repair.

DNA strand breaks present a complex challenge for our cells, and the integrity of the DNA damage response machinery is critical for preventing cancer, premature aging, and neurodegenerative syndromes amongst other ailments. This multi-author review issue presents emerging topics relevant to understanding the fundamental structural mechanisms of DNA strand break sensing, signaling, and repair.

4-Benzylideneisoquinoline-1,3(2H,4H)-diones as tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (Top2) mediated DNA damages, including double-strand breaks (DSBs) that underpin the anticancer mechanism of clinical TOP2 poisons such as etoposide (ETP). Inhibition of TDP2 could sensitize cancer cells toward TOP2 poisons by increasing Top2 cleavage complex. We have previously identified isoquinoline-1,3-dione as a selective inhibitor type of TDP2. However, the reported structure-activity relationship (SAR) was limited to simple substitutions on the isoquinoline-1,3-dione core. Herein, we report the extended SAR consisting of the synthesis and testing of a total of 50 analogs featuring N-2 and C-4 modifications. Major SAR observations include the loss of potency upon N-2 substitution, the lack of inhibition with C-4 enamine analogs (subtype 11), or any other C-4 modifications (subtypes 13-15) except for the benzylidene substitution (subtype 12), where eight analogs showed low micromolar potency. The best analog, 12q, inhibited TDP2 with an IC50 of 4.8 µM. Molecular modeling was performed to help understand the observed SAR trends. Overall, these SAR observations which could significantly benefit future work on the design of improved TDP2 inhibitors.

An internal ribosome entry site in the coding region of tyrosyl-DNA phosphodiesterase 2 drives alternative translation start.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a multifunctional protein that has been implicated in a myriad of cellular pathways. Although most well-known for its phosphodiesterase activity removing stalled topoisomerase 2 from DNA, TDP2 has also been shown to interact with both survival and apoptotic mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, it facilitates enterovirus replication and has been genetically linked to neurological disorders ranging from Parkinson's disease to dyslexia. To accurately evaluate TDP2 as a therapeutic target, we need to understand how TDP2 performs such a wide diversity of functions. Here, we use cancer cell lines modified with CRISPR/Cas9 or stably-expressed TDP2-targeted shRNA and transfection of various TDP2 mutants to show that its expression is regulated at the translational level via an internal ribosome entry site (IRES) that initiates translation at codon 54, the second in-frame methionine of the TDP2 coding sequence. We observed that this IRES drives expression of a shorter, N-terminally truncated isoform of TDP2, ΔN-TDP2, which omits a nuclear localization sequence. Additionally, we noted that ΔN-TDP2 retains phosphodiesterase activity and is protective against etoposide-induced cell death, but co-immunoprecipitates with fewer high-molecular-weight ubiquitinated peptide species, suggesting partial loss-of-function of TDP2's ubiquitin-association domain. In summary, our findings suggest the existence of an IRES in the 5' coding sequence of TDP2 that translationally regulates expression of an N-terminally truncated, cytoplasmic isoform of TDP2. These results shed light on the regulation of this multifunctional protein and may inform the design of therapies targeting TDP2 and associated pathways.

Involvement of Phosphodiesterase 2A Activity in the Pathophysiology of Fragile X Syndrome.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in translational regulation of mRNAs that play key roles in synaptic morphology and plasticity. The functional absence of FMRP causes the fragile X syndrome (FXS), the most common form of inherited intellectual disability and the most common monogenic cause of autism. No effective treatment is available for FXS. We recently identified the Phosphodiesterase 2A (Pde2a) mRNA as a prominent target of FMRP. PDE2A enzymatic activity is increased in the brain of Fmr1-KO mice, a recognized model of FXS, leading to decreased levels of cAMP and cGMP. Here, we pharmacologically inhibited PDE2A in Fmr1-KO mice and observed a rescue both of the maturity of dendritic spines and of the exaggerated hippocampal mGluR-dependent long-term depression. Remarkably, PDE2A blockade rescued the social and communicative deficits of both mouse and rat Fmr1-KO animals. Importantly, chronic inhibition of PDE2A in newborn Fmr1-KO mice followed by a washout interval, resulted in the rescue of the altered social behavior observed in adolescent mice. Altogether, these results reveal the key role of PDE2A in the physiopathology of FXS and suggest that its pharmacological inhibition represents a novel therapeutic approach for FXS.

PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis.

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A-/-) is embryonic lethal. Notably, livers of PDE2A-/- embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A-/- liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A-/- livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A-/- embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A-/- embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis.

Dual DNA topoisomerase 1 and tyrosyl-DNA phosphodiesterase 1 inhibition for improved anticancer activity.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of the phosphodiester bond in the DNA-topoisomerase 1 (Top1) covalent complex and repairs some other 3'-end DNA adducts. Currently, Tdp1 functions as an important target in cancer drug design owing to its ability to break down various DNA adducts induced by chemotherapeutics. Tdp1 inhibitors may sensitize tumor cells to the action of Top1 poisons, thereby potentiating their effects. This mini-review summarizes findings from studies reporting the combined inhibition of Top1 and Tdp1. Two different approaches have been considered for developing such drug precursors.

The loss of ß adrenergic receptor mediated release potentiation in a mouse model of fragile X syndrome.

In fragile X syndrome, the absence of Fragile X Mental Retardation Protein (FMRP) is known to alter postsynaptic function, although alterations in presynaptic function also occur. We found that the potentiation of glutamate release induced by the ß adrenergic receptor (ßAR) agonist isoproterenol is absent in cerebrocortical nerve terminals (synaptosomes) from mice lacking FMRP (Fmr1 KO), despite the normal cAMP generation. The glutamate release induced by moderate stimulation of synaptosomes with 5 mM KCl was not potentiated in Fmr1 KO synaptosomes by isoproterenol, nor by stimulating the receptor associated signaling pathway with the adenylyl cyclase activator forskolin or with the Epac activator 8-pCPT. Hence, the impairment in the pathway potentiating release is distal to ßARs. Electron microscopy shows that Fmr1 KO cortical synapses have more docked vesicles than WT synapses, consequently occluding the isoproterenol response through which more SVs approach the active zone (AZ) of the plasma membrane. Weak stimulation of synaptosomes with the Ca2+ ionophore ionomycin recovered the release potentiation driven by forskolin and 8-pCPT but not with isoproterenol, revealing an impairment in the efficiency of receptor generated cAMP to activate the release potentiation pathway. Indeed, inhibiting cyclic nucleotide phosphodiesterase PDE2A with BAY 60-7550 reestablished isoproterenol mediated potentiation in Fmr1 KO synaptosomes. Thus, the lack of ß-AR mediated potentiation of glutamate release appears to be the consequence of an impaired capability of the receptor to mobilize SVs to the AZ and because of a decreased efficiency of cAMP to activate the signaling pathway that enhances neurotransmitter release.

Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure-activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC50 < 50 µM), with 17z showing excellent cell permeability and no cytotoxicity.

Synthesis and In Vitro Evaluation of 8-Pyridinyl-Substituted Benzo[e]imidazo[2,1-c][1,2,4]triazines as Phosphodiesterase 2A Inhibitors.

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1-9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required.

Deficiency of alkaline SMase enhances dextran sulfate sodium-induced colitis in mice with upregulation of autotaxin.

Intestinal alkaline SMase (Alk-SMase) cleaves phosphocholine from SM, platelet-activating factor (PAF), and lysophosphatidylcholine. We recently found that colitis-associated colon cancer was 4- to 5-fold enhanced in Alk-SMase KO mice. Here, we further studied the pathogenesis of colitis induced by dextran sulfate sodium (DSS) in WT and KO mice. Compared with WT mice, KO mice demonstrated greater body weight loss, more severe bloody diarrhea, broader inflammatory cell infiltration, and more serious epithelial injury. Higher levels of PAF and lower levels of interleukin (IL)10 were identified in KO mice 2 days after DSS treatment. A greater and progressive increase of lysophosphatidic acid (LPA) was identified. The change was associated with increased autotaxin expression in both small intestine and colon, which was identified by immunohistochemistry study, Western blot, and sandwich ELISA. The upregulation of autotaxin coincided with an early increase of PAF. IL6 and TNFα were increased in both WT and KO mice. At the later stage (day 8), significant decreases in IL6, IL10, and PAF were identified, and the decreases were greater in KO mice. In conclusion, deficiency of Alk-SMase enhances DSS-induced colitis by mechanisms related to increased autotaxin expression and LPA formation. The early increase of PAF might be a trigger for such reactions.

Proteomics Based Identification of Autotaxin As An Anti-Hepatitis B Virus Factor and a Promoter of Hepatoma Cell Invasion and Migration.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a major cause of cirrhosis and hepatocellular carcinoma. Therefore, we aimed to obtain further information on HBV pathogenesis, and to search for novel putative molecules for anti-HBV therapy. METHODS: We utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify the secretory proteins that are differentially expressed in the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line. Immunohistochemistry (IHC) was employed to assess the clinical relevance of the observations. Small interfering (si)RNA-based silencing transfection methods were carried out to study the function of ENPP2. RESULTS: Totally, 133 unique proteins were identified as differentially expressed in HepG2.2.15 cell line compared with HepG2 cell line. Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 precursor (ENPP2) is one of the most significantly up-regulated secretory proteins associated with HBV replication. This differential expression of ENPP2 was further validated by real-time quantitative RT-PCR, Western Blot and immunohistochemical analysis. To study the function of ENPP2, we knockdown ENPP2 expression in HepG2.2.15 cell line by RNA interference. ENPP2 silencing increased HBV replication approximately 2.3-fold by enhancing, via the type I IFN signaling pathway, HBV cccDNA (covalently closed circular DNA) translation into viral RNA. Moreover, attenuation of ENPP2 expression inhibited both the invasion and migration ability of hepatoma cells in vitro via interacting with the molecules in the tumor microenvironment. CONCLUSION: Our study demonstrates that ENPP2 may be a novel anti-HBV target and indicate that suppression of its expression may inhibit the invasion and migration ability of hepatoma cells.

Effect of TDP2 on the Level of TOP2-DNA Complexes and SUMOylated TOP2-DNA Complexes.

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5'-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5' tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition.

Investigation of an 18F-labelled Imidazopyridotriazine for Molecular Imaging of Cyclic Nucleotide Phosphodiesterase 2A.

Specific radioligands for in vivo visualization and quantification of cyclic nucleotide phosphodiesterase 2A (PDE2A) by positron emission tomography (PET) are increasingly gaining interest in brain research. Herein we describe the synthesis, the 18F-labelling as well as the biological evaluation of our latest PDE2A (radio-)ligand 9-(5-Butoxy-2-fluorophenyl)-2-(2-([18F])fluoroethoxy)-7-methylimidazo[5,1-c]pyrido[2,3-e][1,2,4]triazine (([18F])TA5). It is the most potent PDE2A ligand out of our series of imidazopyridotriazine-based derivatives so far (IC50 hPDE2A = 3.0 nM; IC50 hPDE10A > 1000 nM). Radiolabelling was performed in a one-step procedure starting from the corresponding tosylate precursor. In vitro autoradiography on rat and pig brain slices displayed a homogenous and non-specific binding of the radioligand. Investigation of stability in vivo by reversed-phase HPLC (RP-HPLC) and micellar liquid chromatography (MLC) analyses of plasma and brain samples obtained from mice revealed a high fraction of one main radiometabolite. Hence, we concluded that [18F]TA5 is not appropriate for molecular imaging of PDE2A neither in vitro nor in vivo. Our ongoing work is focusing on further structurally modified compounds with enhanced metabolic stability.

The phosphodiesterase type 2 inhibitor BAY 60-7550 reverses functional impairments induced by brain ischemia by decreasing hippocampal neurodegeneration and enhancing hippocampal neuronal plasticity.

Cognitive and affective impairments are the most characterized consequences following cerebral ischemia. BAY 60-7550, a selective phosphodiesterase type 2 inhibitor (PDE2-I), presents memory-enhancing and anxiolytic-like properties. The behavioral effects of BAY 60-7550 have been associated with its ability to prevent hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) thereby interfering with neuronal plasticity. Here, we hypothesize that PDE2-I treatment could promote functional recovery after brain ischemia. Mice C57Bl/6 were submitted to bilateral common carotid artery occlusion (BCCAO), an experimental model of transient brain ischemia, for 20 min. During 21 days after reperfusion, the animals were tested in a battery of behavioral tests including the elevated zero maze (EZM), object location task (OLT) and forced swim test (FST). The effects of BAY 60-7550 were evaluated on neuronal nuclei (NeuN), caspase-9, cAMP response element-binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. BCCAO increased anxiety levels, impaired hippocampus-dependent cognitive function and induced despair-like behavior in mice. Hippocampal neurodegeneration was evidenced by a decrease in NeuN and increase incaspase-9 protein levels in BCCAO mice. Ischemic mice also showed low BDNF protein levels in the hippocampus. Repeated treatment with BAY 60-7550 attenuated the behavioral impairments induced by BCCAO in mice. Concomitantly, BAY 60-7550 enhanced expression of pCREB and BDNF protein levels in the hippocampus of ischemic mice. The present findings suggest that chronic inhibition of PDE2 provides functional recovery in BCCAO mice possibly by augmenting hippocampal neuronal plasticity.