George S. Baillie

DISC1 and PDE4B Are Interacting Genetic Factors in Schizophrenia That Regulate cAMP Signaling

The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3′,5′-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood. We show that DISC1 interacts with the UCR2 domain of PDE4B and that elevation of cellular cAMP leads to dissociation of PDE4B from DISC1 and an increase in PDE4B activity. We propose a mechanistic model whereby DISC1 sequesters PDE4B in resting cells and releases it in an activated state in response to elevated cAMP.

β-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-adrenoceptor switching from Gs to Gi

Phosphorylation of the β2 adrenoreceptor (β2AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (Gs) to inhibitory guanine nucleotide regulatory protein (Gi). β-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the β2AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the β2AR. In human embryonic kidney 293 cells overexpressing a recombinant β2AR, stimulation with isoprenaline recruits β-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the β2AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the β2AR. Rolipram and PDE4D5-D556A also enhance β2AR-mediated activation of extracellular signal-regulated kinases ERK1/2. This is consistent with a switch in coupling of the receptor from Gs to Gi, because the ERK1/2 activation is sensitive to both inhibitors of PKA (H89) and Gi (pertussis toxin). In cardiac myocytes, the β2AR also switches from Gs to Gi coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK1/2. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK1/2 activation. Thus, receptor-stimulated β-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the β2AR in a physiological system, the cardiac myocyte.

cAMP-Specific Phosphodiesterase-4 Enzymes in the Cardiovascular System A Molecular Toolbox for Generating Compartmentalized cAMP Signaling

Cyclic AMP regulates a vast number of distinct events in all cells. Early studies established that its hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) controlled both the magnitude and the duration of its influence. Recent evidence shows that PDEs also act as coincident detectors linking cyclic-nucleotide- and non-cyclic-nucleotide-based cellular signaling processes and are tethered with great selectively to defined intracellular structures, thereby integrating and spatially restricting their cellular effects in time and space. Although 11 distinct families of PDEs have been defined, and cells invariably express numerous individual PDE enzymes, a large measure of our increased appreciation of the roles of these enzymes in regulating cyclic nucleotide signaling has come from studies on the PDE4 family. Four PDE4 genes encode more than 20 isoforms. Alternative mRNA splicing and the use of different promoters allows cells the possibility of expressing numerous PDE4 enzymes, each with unique amino-terminal-targeting and/or regulatory sequences. Dominant negative and small interfering RNA-mediated knockdown strategies have proven that particular isoforms can uniquely control specific cellular functions. Thus the protein kinase A phosphorylation status of the beta(2) adrenoceptor and, thereby, its ability to switch its signaling to extracellular signal-regulated kinase activation, is uniquely regulated by PDE4D5 in cardiomyocytes. We describe how cardiomyocytes and vascular smooth muscle cells selectively vary both the expression and the catalytic activities of PDE4 isoforms to regulate their various functions and how altered regulation of these processes can influence the development, or resolution, of cardiovascular pathologies, such as heart failure, as well as various vasculopathies.

Role of dimorphism in the development of Candida albicans biofilms

Two model biofilm systems, involving growth on disks of catheter material or on cylindrical cellulose filters, were used to investigate the structure of Candida albicans biofilms. To assess the importance of dimorphism in biofilm development, biofilms produced by two wild-type strains were compared with those formed by two morphological mutants, incapable of yeast and hyphal growth, respectively. Scanning electron microscopy and thin sections of biofilms examined by light microscopy revealed that biofilms of the wild-type strains formed on catheter disks consisted of two distinct layers: a thin, basal yeast layer and a thicker, but more open, hyphal layer. The hypha- mutant produced only the basal layer, whereas the yeast- mutant formed a thicker, hyphal biofilm equivalent to the outer zone of the wild-type structures. Biofilms of the yeast- mutant were more easily detached from the catheter surface than the others, suggesting that the basal yeast layer has an important role in anchoring the biofilm to the surface. Biofilms formed on cylindrical cellulose filters were quite different in appearance. The hypha- mutant and both wild types produced exclusively yeast-form biofilms whereas the yeast- mutant generated a dense hyphal mat on the top of the filter. All these biofilms, irrespective of morphological form, were resistant to the antifungal agent, amphotericin B. Overall, these results indicate that the structure of a C. albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems.

Sleep deprivation impairs cAMP signalling in the hippocampus

Millions of people regularly obtain insufficient sleep. Given the effect of sleep deprivation on our lives, understanding the cellular and molecular pathways affected by sleep deprivation is clearly of social and clinical importance. One of the major effects of sleep deprivation on the brain is to produce memory deficits in learning models that are dependent on the hippocampus. Here we have identified a molecular mechanism by which brief sleep deprivation alters hippocampal function. Sleep deprivation selectively impaired 3′, 5′-cyclic AMP (cAMP)- and protein kinase A (PKA)-dependent forms of synaptic plasticity in the mouse hippocampus, reduced cAMP signalling, and increased activity and protein levels of phosphodiesterase 4 (PDE4), an enzyme that degrades cAMP. Treatment of mice with phosphodiesterase inhibitors rescued the sleep-deprivation-induced deficits in cAMP signalling, synaptic plasticity and hippocampus-dependent memory. These findings demonstrate that brief sleep deprivation disrupts hippocampal function by interfering with cAMP signalling through increased PDE4 activity. Thus, drugs that enhance cAMP signalling may provide a new therapeutic approach to counteract the cognitive effects of sleep deprivation.

The MAP kinase ERK2 inhibits the cyclic AMP‐specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579

The extracellular receptor stimulated kinase ERK2 (p42MAPK)‐phosphorylated human cAMP‐specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced (∼75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579→Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579→Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059‐ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579→Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF‐induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2‐induced inhibition of PDE4D3. We identify a novel means of cross‐talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.

Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract.

Understanding the molecular organisation of intracellular signalling pathways is a topic of considerable research interest. Since many signalling enzymes are widely distributed and have several substrates, a critical component in signal transduction is the control of specificity. This is achieved, in part by the assembly of multiprotein complexes where clusters of signalling enzymes create focal points to disseminate the intracellular action of many hormones. This is particularly true for the cAMP dependent protein kinase (PKA) that is localised throughout the cell via its association with A‐kinase anchoring proteins (AKAPs). Recent data suggest that some AKAPs also interact with phosphodiesterases (PDEs). Compartmentalisation of PDEs not only provides an elegant means to control PKA activation by monitoring the local cAMP flux, but also serves to concentrate and segregate the action of these important regulatory enzymes.

Mixed species biofilms of Candida albicans and Staphylococcus epidermidis

A simple catheter disk model system was used to study the development in vitro of mixed species biofilms of Candida albicans and Staphylococcus epidermidis, two organisms commonly found in catheter-associated infections. Two strains of S. epidermidis were used: a slime-producing wild type (strain RP62A) and a slime-negative mutant (strain M7). In mixed fungal-bacterial biofilms, both staphylococcal strains showed extensive interactions with C. albicans. The susceptibility of 48-h biofilms to fluconazole, vancomycin and mixtures of the drugs was determined colorimetrically. The results indicated that the extracellular polymer produced by S. epidermidis RP62A could inhibit fluconazole penetration in mixed fungal-bacterial biofilms. Conversely, the presence of C. albicans in a biofilm appeared to protect the slime-negative staphylococcus against vancomycin. Overall, the findings suggest that fungal cells can modulate the action of antibiotics, and that bacteria can affect antifungal activity in mixed fungal-bacterial biofilms.

Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis

Background and purpose:  Apremilast is an orally administered phosphodiesterase‐4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro‐inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis.

Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in Upstream Conserved Region 1 (UCR1)

Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP‐specific phosphodiesterase long isoforms. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. The PKA inhibitor, H89, ablated forskolin activation of wild‐type long PDE4 isoforms. Activated PKA caused the in vitro phosphorylation of recombinant wild‐type long PDE4 isoforms, but not those where the STR was mutated to alanine. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin‐challenged cells treated with H89. Neither was it evident in forskolin‐challenged cells expressing long isoforms where the STR had been mutated to alanine. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform‐specific N‐terminal region. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. Long PDE4 isoforms from all four sub‐families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed.