Inactivation of Pde8b enhances memory,motor performance,and protects against age‐induced motor coordination decay

Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor‐coordination, and have an attenuation of age‐induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value .     

Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae

The efficiency of translation termination in yeast can vary several 100-fold, depending on the context around the stop codon. We performed a computer analysis designed to identify yeast open reading frames (ORFs) containing a readthrough motif surrounding the termination codon. Eight ORFs were found to display inefficient stop codon recognition, one of which, PDE2, encodes the high-affinity cAMP phosphodiesterase. We demonstrate that Pde2p stability is very impaired by the readthrough-dependent extension of the protein. A 20-fold increase in readthrough of PDE2 was observed in a [PSI+] as compared with a [psi-] strain. Consistent with this observation, an important increase in cAMP concentration was observed in suppressor backgrounds. These results provide a molecular explanation for at least some of the secondary phenotypes associated with suppressor backgrounds.     

The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses

The Ras-cyclic AMP (cAMP) pathway is a major determinant of intrinsic stress resistance of the yeast Saccharomyces cerevisiae. Here, we isolated IRA2, encoding the Ras GTPase activator, as a global stress response gene. Subsequently, we studied the other negative regulators on the separate branch of the Ras-cAMP pathway, the low- or high-affinity cAMP phosphodiesterase encoded by PDE1 or PDE2, respectively. Deletion of PDE2, similar to ira2 deletion, rendered cells sensitive to freeze-thawing, peroxides, paraquat, cycloheximide, heavy metals, NaCl, heat, or cold shock. However, deletion of PDE1 did not affect stress tolerance, although it exacerbated stress sensitivity caused by the pde2 deletion, indicating that PDE1 can partly compensate for PDE2. Deletion of IRA2 uniquely led to high sensitivity to cumene hydroperoxide, suggesting that IRA2 may have a distinct role for the response to this stress. Stress sensitivity of yeast cells in general correlated with the basal level of cAMP. Interestingly, yeast cells lacking PDE2 maintained higher cAMP levels in stationary phase than exponential growth phase, suggesting that Pde2p is the major regulator of cAMP levels in stationary phase. Depletion of Ras activity could not effectively suppress stress sensitivity caused by lack of cAMP phosphodiesterases although it could suppress stress sensitivity caused by lack of IRA2, indicating that cAMP accumulation in stationary phase can be mediated by other signaling proteins in addition to Ras. Our study shows that control of cAMP basal levels is important for determining intrinsic stress tolerance of yeast, and that the cAMP level during stationary phase is a result of a dynamic balance between its rates of synthesis and degradation.     

Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans

Frequent interconversion between yeasts, pseudohyphae and true hyphae is a hallmark of Candida albicans growth in mammalian tissues. The requirement for transient CAP1-dependent pulses of cAMP for generating true hyphae, Hwp1 and virulence raises questions about the role of yeast and pseudohyphal forms in the pathogenesis of candidiasis. In this study, hyperfilamentous mutants, limited in their capacity to produce buds, were generated by disrupting the high-affinity phosphodiesterase gene PDE2. Degradation of cAMP by the PDE2 gene product was confirmed by higher basal cAMP levels in the pde2/pde2 mutant and by accumulation of cAMP to levels permitting germ tube formation upon disrupting PDE2 in the cap1/cap1 mutant. Similar phenotypes of the C. albicans and Saccharomyces cerevisiae pde2/pde2 mutants were found, including sensitivity to nutritional starvation and exogenous cAMP and defective entry into stationary phase. Importantly, the hyperfilamentous mutants were as avirulent as hypofilamentous mutants in a systemic model of candidiasis. Growth in a multiplicity of forms appears to be a virulence attribute that is controlled by tight coupling of cAMP synthesis and degradation. Delayed increases in PDE2 mRNA in cAMP-deficient cap1/cap1 mutants during germ tube-inducing conditions suggested a mechanism of control involving cAMP-dependent induction of PDE2 mRNA.     

Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells. The glucose-induced artefactual increase occasionally observed in repressed cells is probably caused by the fact that their low intracellular pH is only restored after the ATP level has increased to such an extent that it is no longer limiting for cAMP synthesis. It is unclear why the artefactual increases are not always observed. Measurement of glucose- and DNP-induced activation of trehalase confirmed the physiological validity of the changes observed in the cAMP level. Our results are consistent with the idea that the glucose-induced signaling pathway contains a glucose-repressible protein and that the protein is located before the point where intracellular acidification triggers activation of the pathway.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - Mes 4-morpholineethanesulfonic acid     

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.     

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.     

Molecular Cloning and Expression of a Human Phosphodiesterase 4C

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5′ end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5′ splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the K_m for cAMP of the enzyme produced in COS cells was 0.6 μM compared to 2.6 μM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC₅₀ of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [³H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.     

Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene

The essential interactions between cAMP and the yeast low Km cAMP-phosphodiesterase have been analyzed using cAMP analogues and phosphodiesterase inhibitors. cAMP specificity is conferred by hydrogen bonding at the N-6 and N-7 positions. In contrast to the other yeast phosphodiesterase, (Rp)-adenosine 3',5'-monophosphorothioate is not hydrolyzed. Eleven standard phosphodiesterase inhibitors were not highly effective. In Chinese hamster ovary (CHO) cells that express the yeast cAMP-phosphodiesterase (PDE2) gene, cAMP levels cannot be raised by cholera toxin. cAMP analogues that are efficiently hydrolyzed by the yeast cAMP-phosphodiesterase had no effect on the growth of CHO cells that express the PDE2 gene, even though they block the growth and alter the morphology of control cells. cAMP analogues that are not hydrolyzed by the yeast enzyme inhibited the growth and changed the morphology of both control and PDE2 expressing CHO cells. We have developed a method for creating cell lines in which cAMP levels can be reduced by expression of an exogenous cAMP-phosphodiesterase gene. By employing cAMP analogues that are not hydrolyzed by this phosphodiesterase, the inhibitory effects of the enzyme can be bypassed.