Phosphodiesterase‐1b deletion confers depression‐like behavioral resistance separate from stress‐related effects in mice

Phosphodiesterase‐1b (Pde1b) is highly expressed in striatum, dentate gyrus, CA3 and substantia nigra. In a new Floxed Pde1b × Cre^CMV global knockout (KO) mouse model, we show an immobility‐resistance phenotype that recapitulates that found in constitutive Pde1b KO mice. We use this new mouse model to show that the resistance to acute stress‐induced depression‐like phenotype is not the product of changes in locomotor activity or reactivity to other stressors (learned helplessness, novelty suppressed feeding or dexamethasone suppression), and is not associated with anhedonia using the sucrose preference test. Using tamoxifen inducible Cre, we show that the immobility‐resistant phenotype depends on the age of induction. The effect is present when Pde1b is Reduced from conception, P0 or P32, but not if reduced as adults (P60). We also mapped regional brain expression of PDE1B protein and of the Cre driver. These data add to the suggestion that PDE1B may be a target for drug development with therapeutic potential in depression alone or in combination with existing antidepressants.     

shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa

In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca²⁺) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP‐gated calcium sodium channels (CNG) to increase Na⁺ and Ca²⁺ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na⁺ and Ca²⁺ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca²⁺ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca²⁺ to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b^H620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b^H620Q mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.     

The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin

Inhibition of adipocyte lipolysis by insulin is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance and type 2 diabetes mellitus. The main target of the antilipolytic action of insulin is believed to be phosphodiesterase 3B (PDE3B), whose phosphorylation by Akt leads to accelerated degradation of the prolipolytic second messenger cyclic AMP (cAMP). To test this hypothesis genetically, brown adipocytes lacking PDE3B were examined for their regulation of lipolysis. In Pde3b knockout (KO) adipocytes, insulin was unable to suppress β-adrenergic receptor-stimulated glycerol release. Reexpressing wild-type PDE3B in KO adipocytes fully rescued the action of insulin against lipolysis. Surprisingly, a mutant form of PDE3B that ablates the major Akt phosphorylation site, murine S273, also restored the ability of insulin to suppress lipolysis. Taken together, these data suggest that phosphorylation of PDE3B by Akt is not required for insulin to suppress adipocyte lipolysis.     

Infertility and Male Mating Behavior Deficits Associated With Pde1c in Drosophila melanogaster

Pde1c is a calcium/calmodulin-regulated, dual-specificity cyclic nucleotide phosphodiesterase. We have used a transposon insertion line to investigate the physiological function of Pde1c in Drosophila melanogaster and to show that the insertion leads to male sterility and male mating behavior defects that include reduced copulation rates. Sterility appears to be primarily due to elimination of sperm from the female reproductive system. The male mating behavior defects were fully rescued by expression of exogenous Pde1c under the control of either a Pde1c or a pan-neuronal promoter, whereas the sterility could be only partially rescued by expression of exogenous Pde1c under the control of these promoters. We also show that Pde1c has a male-specific expression pattern in the CNS with an increased number of Pde1c-expressing neurons in the abdominal ganglion in males.THE cyclic nucleotides, cyclic AMP (cAMP) and cyclic GMP (cGMP), have been known for many years to regulate a wide variety of physiological processes in all animals (e.g., Siegel et al. 1994). Similarly, there is a large body of research focused on an understanding of the structure and regulation of the enzymes that synthesize cAMP and cGMP, the adenylyl and guanylyl cyclases, respectively. Although the concentrations of cyclic nucleotides within a cell are regulated by both their synthesis and their degradation, less attention has been devoted to the function and regulation of the enzymes that break down cyclic nucleotides, the phosphodiesterases (PDEs).Mammals have >20 genes that code for cyclic nucleotide PDEs, which have been subdivided into 11 families on the basis of their sequences, substrate specificities, and regulatory properties (Conti and Beavo 2007). Insects also have a wide variety of PDEs with Drosophila melanogaster containing 6 genes that code for cyclic nucleotide PDEs (Morton and Hudson 2002; Day et al. 2005). On the basis of their sequence similarity to the mammalian PDEs, these 6 genes have been classified into 6 of the 11 families: Pde1c, Pde4, Pde6, Pde8, Pde9, and Pde11 (Day et al. 2005). When their biochemical properties have been investigated, they match well with other members of the same family (Day et al. 2005).Despite the importance of cyclic nucleotides in insect physiology, only one of the Drosophila PDEs has been associated with a mutant phenotype. This gene, Pde4, also known as dunce, was one of the first learning and memory mutants discovered (Byers et al. 1981; Davis et al. 1995). In this study, we have investigated the phenotypes associated with reduced expression of Pde1c, a PDE that has dual specificity for both cAMP and cGMP and that is stimulated in the presence of calcium and calmodulin (Day et al. 2005). Here we show that Pde1c is required for male fertility and male mating behavior. Male sterility appears to be primarily due to females rejecting sperm or failing to store the sperm from mutant males.     

Select 3′,5′-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain

3′,5′-cyclic nucleotide phosphodiesterases (PDEs) are the only known enzymes to compartmentalize cAMP and cGMP, yet little is known about how PDEs are dynamically regulated across the lifespan. We mapped mRNA expression of all 21 PDE isoforms in the adult rat and mouse central nervous system (CNS) using quantitative polymerase chain reaction (qPCR) and in situ hybridization to assess conservation across species. We also compared PDE mRNA and protein in the brains of old (26 months) versus young (5 months) Sprague–Dawley rats, with select experiments replicated in old (9 months) versus young (2 months) BALB/cJ mice. We show that each PDE isoform exhibits a unique expression pattern across the brain that is highly conserved between rats, mice, and humans. PDE1B, PDE1C, PDE2A, PDE4A, PDE4D, PDE5A, PDE7A, PDE8A, PDE8B, PDE10A, and PDE11A showed an age-related increase or decrease in mRNA expression in at least 1 of the 4 brain regions examined (hippocampus, cortex, striatum, and cerebellum). In contrast, mRNA expression of PDE1A, PDE3A, PDE3B, PDE4B, PDE7A, PDE7B, and PDE9A did not change with age. Age-related increases in PDE11A4, PDE8A3, PDE8A4/5, and PDE1C1 protein expression were confirmed in hippocampus of old versus young rodents, as were age-related increases in PDE8A3 protein expression in the striatum. Age-related changes in PDE expression appear to have functional consequences as, relative to young rats, the hippocampi of old rats demonstrated strikingly decreased phosphorylation of GluR1, CaMKIIα, and CaMKIIβ, decreased expression of the transmembrane AMPA regulatory proteins γ2 (a.k.a. stargazin) and γ8, and increased trimethylation of H3K27. Interestingly, expression of PDE11A4, PDE8A4/5, PDE8A3, and PDE1C1 correlate with these functional endpoints in young but not old rats, suggesting that aging is not only associated with a change in PDE expression but also a change in PDE compartmentalization.     

A new model of Pde4d deficiency: genetic knock-down of PDE4D enzyme in rats produces an antidepressant phenotype without spatial cognitive effects

Phosphodiesterases (PDEs) are a superfamily of intracellular second messenger cyclic nucleotide hydrolyzing enzymes composed of 12 families. The Pde4 family has been implicated in depression and cognition, and PDE4 inhibitors have been evaluated as antidepressants and possible cognitive enhancers. Pde4d(-/-) mice show an antidepressant phenotype and learning enhancement on some tests, but not others as do mice treated with PDE4 inhibitors. Here, we report for the first time the behavioral phenotype of a new Pde4d knock-down (KD) rat model of PDE4D deficiency. Consistent with other data on PDE4D deficiency, Pde4d KD rats showed depression resistance in the Porsolt forced swim test and hyperreactivity of the acoustic startle response with no differential response on prepulse inhibition, suggesting no sensorimotor gating defect. Pde4d KD rats also exhibited a small exploratory activity reduction but no difference following habituation, and no enhanced spatial learning or reference memory in the Morris water maze. A selective improvement in route-based learning in the Cincinnati water maze was seen as well as enhanced contextual and cued fear conditioning and a more rapid rate of cued extinction from their higher freezing level that declined to wild-type (WT) levels only after ～20 extinction trials. The rat model confirms Pde4d's role in depression but not in spatial learning or memory enhancement and shows for the first time higher fear conditioning and altered extinction compared with controls. The new model provides a tool by which to better understand the role of PDE4D in neuropsychiatric disorders and for the development of alternate treatment approaches.     

Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation

The regulation of the secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is crucial in the hormonal regulation of bone metabolism. Both cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 families (PDE1-11). We compared the PDEs of cultured human osteoblasts (NHOst) and SaOS-2 osteosarcoma cells. The PDE activity of NHOst cells consisted of PDE1, PDE3 and PDE7, whereas PDE1, PDE7 and PDE4, but no PDE3 activity was detected in SaOS-2 cells. In line with the difference in the PDE profiles, rolipram, a PDE4 inhibitor, increased the accumulation of cAMP in SaOS-2, but not in NHOst cells. Expression of PDE subtypes PDE1C, PDE3A, PDE4A, PDE4B, PDE7A and PDE7B was detected in both cell types. NHOst cells additionally expressed PDE1A.     

Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format.     

Function of the asparagine 74 residue of the inhibitory γ-subunit of retinal rod cGMP-phophodiesterase (PDE) in vivo

The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αβ. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g^tm1 mice. This transgenic line was crossed with Pde6g^tm1/Pde6g^tm1 mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αβ may be more dynamic and context-dependent than was replicated in vitro.     

The effects of inhibitors and magnesium ions on the activity of the thermostable extracellular cAMP-Specific phosphodiesterase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> plasmodium

Plasmodium of myxomycete Physarum polycephalum produces cyclic nucleotide phosphodiesterase (PDE). The extracellular PDE is cAMP-specific and highly thermostable. This study demonstrates that the extracellular PDE of Ph. polycephalum is weakly inhibited by caffeine, isobutylmethylxantine and theophiline (type I mammalian PDE nonspecific inhibitors), dipyridamole (mammalian PDE5, PDE6, PDE8 and PDE10 inhibitors), and erythro-9-[3-(2-hydroxynonyl)]-adenine (mammalian PDE2 inhibitor). The enzyme does not require Mg²⁺ for the activity. The results show that the Ph. polycephalum extracellular PDE differs from class I PDEs, represented by mammalian PDE1-PDE11, and, most likely, belongs to a poorly investigated class II PDEs.     

OPC-13013, a cyclic nucleotide phosphodiesterase type III, inhibitor, inhibits cell proliferation and transdifferentiation of cultured rat hepatic stellate cells.

Activated hepatic stellate cells (HSC; lipocytes; Ito cells) proliferate and are responsible for extracellular matrix synthesis during hepatic fibrogenesis. During activation, HSC undergo transdifferentiation into myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA). Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is an ubiquitous intracellular signaling molecule, and is upregulated by the activation of adenylate cyclase and downregulated via hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). Recently, increased intracellular cyclic AMP has been shown to inhibit HSC activation. The aim of the current study was to determine the effects of inhibition of PDEs on cell proliferation and transdifferentiation in cultured rat HSC. Cell proliferation was determined by [3H]thymidine incorporation, and Western blot analysis was performed for detection of alpha-SMA, a phenotypic marker of transdifferentiation into myofibroblast. When the cells were exposed to 3-isobutyl-1-methylxanthine (IBMX; 50-1000 microM), a nonselective PDE inhibitor, serum-stimulated [3H]thymidine incorporation was suppressed in a dose-dependent manner with a maximum inhibition of 66% at a concentration of 500 microM OPC-13013 (1-60 microM), a selective PDE III isoenzyme inhibitor, induced a dose-dependent inhibitory effect on serum-stimulated DNA synthesis that reached a maximum inhibition of 95% at a concentration of 60 microM, while neither 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX), a PDE I isoenzyme inhibitor, nor Ro-20-1724, a PDE IV isoenzyme inhibitor, had an inhibitory effect. Western blot analysis revealed that IBMX or OPC-13013 decreased alpha-SMA expression, while other selective PDE isoenzyme inhibitors did not have a suppressive effect. IBMX, OPC-13013 or Ro-20-1724, but not 8-MMX augmented forskolin-induced increase in intracellular cyclic AMP levels although cyclic AMP levels were not affected by treatment with any of these PDE inhibitors alone. These data indicate that inhibition of PDEs, especially PDE III isoenzyme, can produce an inhibitory effect on HSC activation. The PDE III isoenzyme may contribute to the regulation of HSC activation during fibrogenesis. In addition, OPC-13013 may have the potential to inhibit initiation and progression of hepatic fibrosis by interfering with HSC activation.     

The Phosphodiesterase 9 Inhibitor PF‐04449613 Promotes Dendritic Spine Formation and Performance Improvement after Motor Learning

The cyclic nucleotide cGMP is an intracellular second messenger with important roles in neuronal functions and animals' behaviors. The phosphodiesterases (PDEs) are a family of enzymes that hydrolyze the second messengers cGMP and cAMP. Inhibition of phosphodiesterase 9 (PDE9), a main isoform of PDEs hydrolyzing cGMP, has been shown to improve learning and memory as well as cognitive function in rodents. However, the role of PDE9 in regulating neuronal structure and function in vivo remains unclear. Here we used in vivo two‐photon microscopy to investigate the effect of a selective PDE9 inhibitor PF‐04449613 on the activity and plasticity of dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex. We found that administration of PF‐04449613 increased calcium activity of dendrites and dendritic spines of layer V pyramidal neurons in mice under resting and running conditions. Chronic treatment of PF‐04449613 over weeks increased dendritic spine formation and elimination under basal conditions. Furthermore, PF‐04449613 treatment over 1–7 days increased the formation and survival of new spines as well as performance improvement after rotarod motor training. Taken together, our studies suggest that elevating the level of cGMP with the PDE9 inhibitor PF‐04449613 increases synaptic calcium activity and learning‐dependent synaptic plasticity, thereby contributing to performance improvement after learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2018     

Effect of the ILE86TER mutation in the γ subunit of cGMP phosphodiesterase (PDE6) on rod photoreceptor signaling

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g^tm1/Pde6g^tm1 mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile⁸⁶ and Ile⁸⁷ residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100 μM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile⁸⁶ and Ile⁸⁷ residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.     

Role of Wdr45b in maintaining neural autophagy and cognitive function

ABSTRACT

Macroautophagy/autophagy functions as a quality control mechanism by degrading misfolded proteins and damaged organelles and plays an essential role in maintaining neural homeostasis. The phosphoinositide phosphatidylinositol-3-phosphate (PtdIns3P) effector Atg18 is essential for autophagosome formation in yeast. Mammalian cells contain four Atg18 homologs, belonging to two subclasses, WIPI1 (WD repeat domain, phosphoinositide interacting 1), WIPI2 and WDR45B/WIPI3 (WD repeat domain 45B), WDR45/WIPI4. The role of Wdr45b in autophagy and in neural homeostasis, however, remains unknown. Recent human genetic studies have revealed a potential causative role of WDR45B in intellectual disability. Here we demonstrated that mice deficient in Wdr45b exhibit motor deficits and learning and memory defects. Histological analysis reveals that wdr45b knockout (KO) mice exhibit a large number of swollen axons and show cerebellar atrophy. SQSTM1- and ubiquitin-positive aggregates, which are autophagy substrates, accumulate in various brain regions in wdr45b KO mice. Double KO mice, wdr45b and wdr45, die within one day after birth and exhibit more severe autophagy defects than either of the single KO mice, suggesting that these two genes act cooperatively in autophagy. Our studies demonstrated that WDR45B is critical for neural homeostasis in mice. The wdr45b KO mice provide a model to study the pathogenesis of intellectual disability.

Abbreviations: ACSF: artificial cerebrospinal fluid; AMC: aminomethylcoumarin; BPAN: beta-propeller protein-associated neurodegeneration; CALB1: calbindin 1; CNS: central nervous system; DCN: deep cerebellar nuclei; fEPSP: field excitatory postsynaptic potential; IC: internal capsule; ID: intellectual disability; ISH: in situ hybridization; KO: knockout; LTP: long-term potentiation; MBP: myelin basic protein; MGP: medial globus pallidus; PtdIns3P: phosphoinositide phosphatidylinositol-3-phosphate; WDR45B: WD repeat domain 45B; WIPI1: WD repeat domain, phosphoinositide interacting 1; WT: wild type.     

Differential phosphodiesterase expression and cytosolic Ca2+ in human CNS tumour cells and in non-malignant and malignant cells of rat origin

A promising attempt in the field of tumour therapy is the modulation of intracellular, proliferation-associated signalling pathways. The role of cyclic nucleotide phosphodiesterases (PDEs), key enzymes in cAMP/cGMP signal transduction, was investigated in two human CNS tumour cell lines as well as in the rat glioblastoma cell line C6 in comparison with rat cerebellar astrocytes with the emphasis on target evaluation. We found differential PDE expression patterns in human CNS tumour cell lines as well as in CNS cells of rat origin. In human glioblastoma cells, intracellular cAMP and Ca(2+) levels correlated well with the PDE expression pattern. There were, however, marked differences in PDE expression and Ca(2+) kinetics between the human glioblastoma cell lines. In contrast to human epithelial tumour cells, shown earlier by us to express significantly enhanced cAMP-specific PDE activity, this was not the case in rat glioblastoma cells compared with non-malignant rat astrocytes. Despite different levels of PDE1 and PDE4 expression and activity, cyclic nucleotide and Ca(2+) levels in non-malignant and malignant rat CNS cells were similar. These in vitro data do not support the concept of PDE1C representing a target exploitable for drug treatment of malignant CNS tumours.     

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.     

Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h^−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h^−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h^−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.     

PDE2 inhibition: Potential for the treatment of cognitive disorders

Phosphodiesterase inhibition has received much attention in the past 20 years for the potential treatment of CNS disorders. A primary focus of this work is the enhancement of memory and/or cognitive functioning. The role of PDEs in the augmentation of cyclic nucleotide signaling makes these enzymes attractive targets for enhancing the effects of neuronal communication. This review focuses on recent findings with respect to the role of PDE2 inhibition in cognitive functioning. Special attention is paid to recently disclosed, selective tool compounds and the use of these tool compounds to support the role of PDE2 inhibition in cognition. Recently reported SAR and modeling work will be presented along with discussion of the entry of new PDE2 inhibitors into the clinic.