Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications.

Galactokinase catalyses the site- and stereospecific phosphorylation of α-d-galactose. As such it has attracted interest as a biocatalyst for the introduction of phosphate groups into monosaccharides. However, attempts to broaden the substrate range of human galactokinase have generally resulted in substantially reduced activity. The enzyme also has biotechnological potential in enzyme replacement therapy (ERT) for type II galactosaemia. The return-to-consensus approach can be used to identify residues that can be altered to increase protein stability and enzyme activity. This approach identified six residues of potential interest in human galactokinase. Some of the single consensus variants (M60V, D268E, A334S and G373S) increased the catalytic turnover of the enzyme, but none resulted in improved stability. When all six changes were introduced into the protein (M60V/M180V/D268E/A334S/R366Q/G373S), thermal stability was increased. Molecular dynamics simulations suggested that these changes altered the protein's conformation at key sites. The number of salt bridges and hydrogen bonds was also increased. Combining the six consensus variations with Y379W (a variant with greater substrate promiscuity) increased the stability of this variant and its turnover towards some substrates. Thus, the six consensus variants can be used to stabilise catalytically interesting variants of human galactokinase and might also be useful if the protein were to be used in ERT.

Unscheduled changes in pre-clinical stroke model housing contributes to variance in physiological and behavioural data outcomes: A post hoc analysis.

Ischaemic stroke presents a significant problem worldwide with no neuroprotective drugs available. Many of the failures in the search for neuroprotectants are attributed to failure to translate from pre-clinical models to humans, which has been combatted with rigorous pre-clinical stroke research guidelines. Here, we present post hoc analysis of a pre-clinical stroke trial, conducted using intraluminal filament transient middle cerebral artery occlusion in the stroke-prone spontaneously hypertensive rat, whereby unscheduled changes were implemented in the animal housing facility. These changes severely impacted body weight post-stroke resulting in a change from the typical body weight of 90.6% of pre-surgery weight post-stroke, to on average 80.5% of pre-surgery weight post-stroke. The changes also appeared to impact post-stroke blood pressure, with an increase from 215.4 to 240.3 mmHg between housing groups, and functional outcome post-stroke, with a 38% increased latency to contact in the sticky label test. These data highlight the importance of tightly controlled housing conditions when using physiological or behavioural measurements as a primary outcome.

The counter regulatory axis of the renin angiotensin system in the brain and ischaemic stroke: Insight from preclinical stroke studies and therapeutic potential.

Stroke is the 2nd leading cause of death worldwide and the leading cause of physical disability and cognitive issues. Although we have made progress in certain aspects of stroke treatment, the consequences remain substantial and new treatments are needed. Hypertension has long been recognised as a major risk factor for stroke, both haemorrhagic and ischaemic. The renin angiotensin system (RAS) plays a key role in blood pressure regulation and this, plus local expression and signalling of RAS in the brain, both support the potential for targeting this axis therapeutically in the setting of stroke. While historically, focus has been on suppressing classical RAS signalling through the angiotensin type 1 receptor (AT1R), the identification of a counter-regulatory axis of the RAS signalling via the angiotensin type 2 receptor (AT2R) and Mas receptor has renewed interest in targeting the RAS. This review describes RAS signalling in the brain and the potential of targeting the Mas receptor and AT2R in preclinical models of ischaemic stroke. The animal and experimental models, and the route and timing of intervention, are considered from a translational perspective.

UK consensus on pre-clinical vascular cognitive impairment functional outcomes assessment: Questionnaire and workshop proceedings.

Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.

Altered Extracellular Vesicle MicroRNA Expression in Ischemic Stroke and Small Vessel Disease.

Active transport of microRNAs (miRNA) in extracellular vesicles (EV) occurs in disease. Circulating EV-packaged miRNAs in the serum of stroke patients were compared to stroke mimics with matched cardio- and cerebrovascular risk factors, with corroboration of results in a pre-clinical model. An unbiased miRNA microarray was performed in stroke vs. stroke mimic patients (n = 39). Results were validated (n = 173 patients) by real-time quantitative polymerase chain reaction. miRNA expression was quantified in total serum/EV (n = 5-7) of naïve adult spontaneously hypertensive stroke-prone rats (SHRSP), their normotensive reference strain (Wistar Kyoto, WKY) and in circulating EV (n = 3), peri-infarct brain (n = 6), or EV derived from this region (n = 3) in SHRSP following transient middle cerebral artery occlusion (tMCAO). Circulating EV concentration did not differ between stroke and stroke mimic patients. The microarray identified many altered EV-packaged miRNAs: levels of miRNA-17-5p, -20b-5p and -93-5p (miRNA-17 family members) and miRNA-27b-3p were significantly (p ≤ 0.05) increased in stroke vs. stroke mimic patients. Patients with small vessel disease (SVD) consistently had the highest miRNA levels. Circulating EV concentration was unaltered between naïve SHRSP and WKY but levels of miRNA-17-5p and -93-5p were significantly increased in SHRSP. tMCAO in SHRSP did not further alter circulating EV miRNA-17 family member expression and nor did it change total miRNA-17 family levels in peri-infarct brain tissue or in EV isolated from this region at 24 h post-tMCAO. Changes in EV packaged miRNA expression was validated in patients with stroke, particularly those with SVD and corroborated pre-clinically. Together, altered circulating EV levels of miRNA-17 family members may reflect the chronic sequelae underlying cerebrovascular SVD rather than the acute ischemic stroke itself.