Beta-amyloid interacts with and activates the long-form phosphodiesterase PDE4D5 in neuronal cells to reduce cAMP availability.

Inhibition of the cyclic-AMP degrading enzyme phosphodiesterase type 4 (PDE4) in the brains of animal models is protective in Alzheimer's disease (AD). We show for the first time that enzymes from the subfamily PDE4D not only colocalize with beta-amyloid (Aß) plaques in a mouse model of AD but that Aß directly associates with the catalytic machinery of the enzyme. Peptide mapping suggests that PDE4D is the preferential PDE4 subfamily for Aß as it possesses a unique binding site. Intriguingly, exogenous addition of Aß to cells overexpressing the PDE4D5 longform caused PDE4 activation and a decrease in cAMP. We suggest a novel mechanism where PDE4 longforms can be activated by Aß, resulting in the attenuation of cAMP signalling to promote loss of cognitive function in AD.

Nucleus accumbens dopamine release reflects the selective nature of pair bonds.

In monogamous species, prosocial behaviors directed toward partners are dramatically different from those directed toward unknown individuals and potential threats. Dopamine release in the nucleus accumbens has a well-established role in social reward and motivation, but how this mechanism may be engaged to drive the highly divergent social behaviors directed at a partner or unfamiliar conspecific remains unknown. Using monogamous prairie voles, we first employed receptor pharmacology in partner preference and social operant tasks to show that dopamine is critical for the appetitive drive for social interaction but not for low-effort, unconditioned consummatory behaviors. We then leveraged the subsecond temporal resolution of the fluorescent biosensor, GRABDA, to ask whether differential dopamine release might distinguish between partner and novel social access and interaction. We found that partner seeking, anticipation, and interaction resulted in more accumbal dopamine release than the same events directed toward a novel vole. Further, partner-associated dopamine release decreased after prolonged partner separation. Our results are consistent with a model in which dopamine signaling plays a prominent role in the appetitive aspects of social interactions. Within this framework, differences in partner- and novel-associated dopamine release reflect the selective nature of pair bonds and may drive the partner- and novel-directed social behaviors that reinforce and cement bonds over time. This provides a potential mechanism by which highly conserved reward systems can enable selective, species-appropriate social behaviors.

Emergent intra-pair sex differences and organized behavior in pair bonded prairie voles (Microtus ochrogaster).

In pair bonding animals, coordinated behavior between partners is required for the pair to accomplish shared goals such as raising young. Despite this, experimental designs rarely assess the behavior of both partners within a bonded pair. Thus, we lack an understanding of the interdependent behavioral dynamics between partners that likely facilitate relationship success. To identify intra-pair behavioral correlates of pair bonding, we used socially monogamous prairie voles (Microtus ochrogaster) and tested both partners using social choice and non-choice tests at short- and long-term pairing timepoints. Females developed a preference for their partner more rapidly than males, with preference driven by different behaviors in each sex. Further, as bonds matured, intra-pair behavioral sex differences and organized behavior emerged-females consistently huddled more with their partner than males did regardless of overall intra-pair affiliation levels. When animals were allowed to freely interact with a partner or a novel vole in sequential free interaction tests, pairs spent more time interacting together than either animal did with a novel vole, consistent with partner preference in the more commonly employed choice test. Total pair interaction in freely moving voles was correlated with female, but not male, behavior. Via a social operant paradigm, we found that pair-bonded females, but not males, are more motivated to access and huddle with their partner than a novel vole. Together, our data indicate that as pair bonds mature, sex differences and organized behavior emerge within pairs, and that these intra-pair behavioral changes are likely organized and driven by the female animal.

A neuronal signature for monogamous reunion.

Pair-bond formation depends vitally on neuromodulatory signaling within the nucleus accumbens, but the neuronal dynamics underlying this behavior remain unclear. Using 1-photon in vivo Ca2+ imaging in monogamous prairie voles, we found that pair bonding does not elicit differences in overall nucleus accumbens Ca2+ activity. Instead, we identified distinct ensembles of neurons in this region that are recruited during approach to either a partner or a novel vole. The partner-approach neuronal ensemble increased in size following bond formation, and differences in the size of approach ensembles for partner and novel voles predict bond strength. In contrast, neurons comprising departure ensembles do not change over time and are not correlated with bond strength, indicating that ensemble plasticity is specific to partner approach. Furthermore, the neurons comprising partner and novel-approach ensembles are nonoverlapping while departure ensembles are more overlapping than chance, which may reflect another key feature of approach ensembles. We posit that the features of the partner-approach ensemble and its expansion upon bond formation potentially make it a key neuronal substrate associated with bond formation and maturation.

Increase in Ca2+ current by sustained cAMP levels enhances proliferation rate in GH3 cells.

AIMS: Ca2+ and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca2+ currents and proliferation in pituitary tumor GH3 cells. MAIN METHODS: Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca2+ current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. KEY FINDINGS: Sustained forskolin treatment (24 and 48h) induced a significant increase in total Ca2+ current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca2+ current density. However, the maximum effect of dbcAMP occurred only after 72h incubation, whereas forskolin showed maximal effect at 48h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca2+ channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. SIGNIFICANCE: We conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca2+ current density and this phenomenon impacts proliferation rate in GH3 cells.

Selective inhibition of phosphodiesterases 4, 5 and 9 induces HSP20 phosphorylation and attenuates amyloid beta 1-42-mediated cytotoxicity.

Phosphodiesterase (PDE) inhibitors are currently under evaluation as agents that may facilitate the improvement of cognitive impairment associated with Alzheimer's disease. Our aim was to determine whether inhibitors of PDEs 4, 5 and 9 could alleviate the cytotoxic effects of amyloid beta 1-42 (Aß1-42) via a mechanism involving the small heatshock protein HSP20. We show that inhibition of PDEs 4, 5 and 9 but not 3 induces the phosphorylation of HSP20 which, in turn, increases the colocalisation between the chaperone and Aß1-42 to significantly decrease the toxic effect of the peptide. We conclude that inhibition of PDE9 is most effective to combat Aß1-42 cytotoxicity in our cell model.

Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling.

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin-collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets.

The Impact of Acculturation Level on Weight Status and Weight Outcomes in Hispanic Children.

BACKGROUND: Previous studies revealed that higher levels of acculturation are related to obesity in Hispanic adults. Conflicting findings exist regarding this relationship in children, and little is known about the impact of acculturation on children's success in pediatric weight management programs. The purposes of the study were to (1) examine the relationship between acculturation and overweight/obese weight status and (2) determine the impact of acculturation on the changes in weight status among overweight/obese children 12 and 24 months after having participated in a weight management intervention. METHODS: This is a secondary analysis of aggregated data from three randomized control trials that occurred between 2005 and 2009. Height, weight, and level of acculturation using the Child Short Scale for Hispanics (C-SASH) were measured in a sample of Hispanic children (n = 559). Logistic regression models were used to study phase 1 (n = 559) and phase 2 (n = 142), controlling for child and family characteristics. RESULTS: Children reporting high levels of acculturation had a 52 % lower odds of being overweight or obese. Among overweight/obese children who participated in the intervention, high levels of acculturation demonstrated greater reductions in standardized body mass index (zBMI) at 24 months. CONCLUSIONS: The results of this study indicate a need to tailor weight management programs for Hispanic children who have lower levels of acculturation.

Specific Inhibition of Phosphodiesterase-4B Results in Anxiolysis and Facilitates Memory Acquisition.

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and ß-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells.

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T-EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2'-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T-EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP.

The role and therapeutic targeting of α-, ß- and γ-secretase in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia in the elderly and its prevalence is set to increase rapidly in coming decades. However, there are as yet no available drugs that can halt or even stabilize disease progression. One of the main pathological features of AD is the presence in the brain of senile plaques mainly composed of aggregated ß amyloid (Aß), a derivative of the longer amyloid precursor protein (APP). The amyloid hypothesis proposes that the accumulation of Aß within neural tissue is the initial event that triggers the disease. Here we review research efforts that have attempted to inhibit the generation of the Aß peptide through modulation of the activity of the proteolytic secretases that act on APP and discuss whether this is a viable therapeutic strategy for treating AD.

The phosphorylation of Hsp20 enhances its association with amyloid-ß to increase protection against neuronal cell death.

Up-regulation of Hsp20 protein levels in response to amyloid fibril formation is considered a key protective response against the onset of Alzheimer's disease (AD). Indeed, the physical interaction between Hsp20 and Aß is known to prevent Aß oligomerisation and protects neuronal cells from Aß mediated toxicity, however, details of the molecular mechanism and regulatory cell signalling events behind this process have remained elusive. Using both conventional MTT end-point assays and novel real time measurement of cell impedance, we show that Hsp20 protects human neuroblastoma SH-SY5Y cells from the neurotoxic effects of Aß. In an attempt to provide a mechanism for the neuroprotection afforded by Hsp20, we used peptide array, co-immunoprecipitation analysis and NMR techniques to map the interaction between Hsp20 and Aß and report a binding mode where Hsp20 binds adjacent to the oligomerisation domain of Aß, preventing aggregation. The Hsp20/Aß interaction is enhanced by Hsp20 phosphorylation, which serves to increase association with low molecular weight Aß species and decrease the effective concentration of Hsp20 required to disrupt the formation of amyloid oligomers. Finally, using a novel fluorescent assay for the real time evaluation of morphology-specific Aß aggregation, we show that phospho-dependency of this effect is more pronounced for fibrils than for globular Aß forms and that 25mers corresponding to the Hsp20 N-terminal can be used as Aß aggregate inhibitors. Our report is the first to provide a molecular model for the Hsp20/Aß complex and the first to suggest that modulation of the cAMP/cGMP pathways could be a novel route to enhance Hsp20-mediated attenuation of Aß fibril neurotoxicity.

Real-time probing of ß-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes.

The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting ß-amyloid aggregates (Aß) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aß peptides and demonstrate that Aß self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of ß-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aß1-42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in Förster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.

Arrestin regulation of small GTPases.

The regulation of small GTPases by arrestins is a relatively new way by which arrestin can exert influence over cell signalling cascades, hence, molecular interactions and specific binding partners are still being discovered. A pathway showcasing the regulation of GTPase activity by ß-arrestin was first elucidated in 2001. Since this original study, growing evidence has emerged for arrestin modulation of GTPase activity through direct interactions and also via the scaffolding of GTPase regulatory proteins. Given the importance of small GTPases in a variety of essential cellular functions, pharmacological manipulation of this pathway may represent an area with therapeutic potential, particularly with respect to cancer pathology and cardiac hypertrophy.The regulation of small GTPases by arrestins is a relatively new way by which arrestin can exert influence over cell signalling cascades, hence, molecular interactions and specific binding partners are still being discovered. A pathway showcasing the regulation of GTPase activity by ß-arrestin was first elucidated in 2001. Since this original study, growing evidence has emerged for arrestin modulation of GTPase activity through direct interactions and also via the scaffolding of GTPase regulatory proteins. Given the importance of small GTPases in a variety of essential cellular functions, pharmacological manipulation of this pathway may represent an area with therapeutic potential, particularly with respect to cancer pathology and cardiac hypertrophy.

Chemical informatics uncovers a new role for moexipril as a novel inhibitor of cAMP phosphodiesterase-4 (PDE4).

PDE4 is one of eleven known cyclic nucleotide phosphodiesterase families and plays a pivotal role in mediating hydrolytic degradation of the important cyclic nucleotide second messenger, cyclic 3'5' adenosine monophosphate (cAMP). PDE4 inhibitors are known to have anti-inflammatory properties, but their use in the clinic has been hampered by mechanism-associated side effects that limit maximally tolerated doses. In an attempt to initiate the development of better-tolerated PDE4 inhibitors we have surveyed existing approved drugs for PDE4-inhibitory activity. With this objective, we utilised a high-throughput computational approach that identified moexipril, a well tolerated and safe angiotensin-converting enzyme (ACE) inhibitor, as a PDE4 inhibitor. Experimentally we showed that moexipril and two structurally related analogues acted in the micro molar range to inhibit PDE4 activity. Employing a FRET-based biosensor constructed from the nucleotide binding domain of the type 1 exchange protein activated by cAMP, EPAC1, we demonstrated that moexipril markedly potentiated the ability of forskolin to increase intracellular cAMP levels. Finally, we demonstrated that the PDE4 inhibitory effect of moexipril is functionally able to induce phosphorylation of the small heat shock protein, Hsp20, by cAMP dependent protein kinase A. Our data suggest that moexipril is a bona fide PDE4 inhibitor that may provide the starting point for development of novel PDE4 inhibitors with an improved therapeutic window.

The technology behind Colgate Simply White Toothpaste.

Colgate Simply White Toothpaste is a new advanced tooth whitening dentifrice that can be used every day. The synergy of abrasive stain removal with activated hydrogen peroxide delivers excellent performance in the removal of extrinsic and intrinsic tooth stain. Colgate Simply White Toothpaste provides other oral health benefits that have become the cost-of-entry into the toothpaste market: caries protection, tartar control, fresh breath, and a preferred flavor.