A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species.

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A , GATA2 , and DPYSL3 , are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

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Casein kinase I (CKI) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC 50 ϭ 7.7 Ϯ 2.2 nM) and selective (Ͼ30-fold with respect to 42 additional kinases) inhibitor of CKI in isolated enzyme preparations; in transfected whole cell assays, it caused a concentrationrelated redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (Ϫ1.97 Ϯ 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose-and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these druginduced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t 1/2 ϭ 0.46 Ϯ 0.04 h). Together, these results suggest that inhibition of CKI yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.Circadian behavior is mediated by a timed sequence of intracellular events, genomic in nature, occurring in mammals within so-called pacemaker cells of the suprachiasmatic nucleus (SCN) (Antle and Silver, 2005). Here, rhythmicity relies on a common theme of precisely regulated gene transcription and translation as a means to perpetuate cycling, and in doing so, to dictate the timing of downstream events (Reppert and Weaver, 2001; for review, see Schibler, 2006, among others). Clock-related proteins rise and fall in concentration as a consequence of nuclear and cytosolic feedback loops, adjusted daily to maintain reproducible cycling timed at roughly 24-h intervals. Light is the primary zeitgeber, or "time-giver", in this process, and its actions on loop dynamics are thought to comprise the most important external influence on pacemaker function.In its simplest conception, the oscillations of the clock can run using four genes, Period (Per1, Per2) and Cryptochrome (Cry1, Cry2), that are activated in early circadian day by heterodimeric complexes of CLOCK and BMAL1 (Griffin et al., 1999;Kume et al., 1999;Cermakian and Boivin, 2003). Translated PER partners in the cytoplasm with CRY, the resultant heterodimer, readily gaining nuclear entry and opposing activation by Clock and BMAL1. A feedback loop is...

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Phosphodiesterases (PDEs) comprise a family of enzymes that regulate the levels of cyclic nucleotides, key second messengers that mediate a diverse array of functions. PDE2A is an evolutionarily conserved cGMP-stimulated cAMP and cGMP PDE. In the present study, the regional and cellular distribution of PDE2A in tissues of rats, mice, cynomolgus monkeys, dogs, and humans was evaluated by immunohistochemistry. A polyclonal antibody directed to the C-terminal portion of PDE2A specifically detected PDE2A by Western blotting and by immunohistochemistry. The pattern of PDE2A immunoreactivity (ir) was consistent across all species. Western blot analysis demonstrated that PDE2A was most abundant in the brain relative to peripheral tissues. PDE2A ir was heterogeneously distributed within brain and was selectively expressed in particular peripheral tissues. In the brain, prominent immunoreactivity was apparent in components of the limbic system, including the isocortex, hippocampus, amygdala, habenula, basal ganglia, and interpeduncular nucleus. Cytoplasmic PDE2A staining was prominent in several peripheral tissues, including the adrenal zona glomerulosa, neurons of enteric ganglia, endothelial cells in all organs, lymphocytes of spleen and lymph nodes, and pituitary. These studies suggest that PDE2A is evolutionarily conserved across mammalian species and support the hypothesis that the enzyme plays a fundamental role in signal transduction.

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Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a highaffinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.