Moritz Lindner

Probing the regulation of TASK potassium channels by PI(4,5)P2 with switchable phosphoinositide phosphatases

Non‐technical summary  The electrical activity of nerve cells is produced by the flux of ions through specialized membrane proteins called ion channels. Some ion channels can be regulated by the signalling lipid PIP2, a component of the channels’ membrane environment. Here we examine the relevance of PIP2 for the regulation of one specific channel type, termed TASK. Many chemical transmitters in the brain change neural activity by shutting off TASK channels and it has been suggested that this results from reduction of PIP2. By using novel techniques to alter the concentration of PIP2 in living cells, we find that the activity of TASK is independent of PIP2. Besides demonstrating that another signalling mechanism must control the activity of nerve cells via TASK inhibition, we delineate a general approach for clarifying the relevance of PIP2 in many cell types and organs.

Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors.

The two-pore domain potassium (K2P) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) are important determinants of background K(+) conductance and membrane potential. TASK-1/3 activity is regulated by hormones and transmitters that act through G protein-coupled receptors (GPCR) signalling via G proteins of the Gαq/11 subclass. How the receptors inhibit channel activity has remained unclear. Here, we show that TASK-1 and -3 channels are gated by diacylglycerol (DAG). Receptor-initiated inhibition of TASK required the activity of phospholipase C, but neither depletion of the PLC substrate PI(4,5)P2 nor release of the downstream messengers IP3 and Ca(2+). Attenuation of cellular DAG transients by DAG kinase or lipase suppressed receptor-dependent inhibition, showing that the increase in cellular DAG-but not in downstream lipid metabolites-mediates channel inhibition. The findings identify DAG as the signal regulating TASK channels downstream of GPCRs and define a novel role for DAG that directly links cellular DAG dynamics to excitability.

The inhibition of the potassium channel TASK-1 in rat cardiac muscle by endothelin-1 is mediated by phospholipase C

AIMS The two-pore-domain potassium channel TASK-1 is robustly inhibited by the activation of receptors coupled to the Gα(q) subgroup of G-proteins, but the signal transduction pathway is still unclear. We have studied the mechanisms by which endothelin receptors inhibit the current carried by TASK-1 channels (I(TASK)) in cardiomyocytes. METHODS AND RESULTS Patch-clamp measurements were carried out in isolated rat cardiomyocytes. I(TASK) was identified by extracellular acidification to pH 6.0 and by the application of the TASK-1 blockers A293 and A1899. Endothelin-1 completely inhibited I(TASK) with an EC(50) of <10 nM; this effect was mainly mediated by endothelin-A receptors. Application of 20 nM endothelin-1 caused a significant increase in action potential duration under control conditions; this was significantly reduced after pre-incubation of the cardiomyocytes with 200 nM A1899. The inhibition of I(TASK) by endothelin-1 was not affected by inhibitors of protein kinase C or rho kinase, but was strongly reduced by U73122, an inhibitor of phospholipase C (PLC). The ability of endothelin-1 to activate PLC-mediated signalling pathways was examined in mammalian cells transfected with TASK-1 and the endothelin-A receptor using patch-clamp measurements and total internal reflection microscopy. U73122 prevented the inhibition of I(TASK) by endothelin-1 and blocked PLC-mediated signalling, as verified with a fluorescent probe for phosphatidylinositol-(4,5)-bisphosphate hydrolysis. CONCLUSION Our results show that I(TASK) in rat cardiomyocytes is controlled by endothelin-1 and suggest that the inhibition of TASK-1 via endothelin receptors is mediated by the activation of PLC. The prolongation of the action potential observed with 20 nM endothelin-1 was mainly due to the inhibition of I(TASK).

Imaging Protocols in Clinical Studies in Advanced Age-Related Macular Degeneration: Recommendations from Classification of Atrophy Consensus Meetings

PURPOSE To summarize the results of 2 consensus meetings (Classification of Atrophy Meeting [CAM]) on conventional and advanced imaging modalities used to detect and quantify atrophy due to late-stage non-neovascular and neovascular age-related macular degeneration (AMD) and to provide recommendations on the use of these modalities in natural history studies and interventional clinical trials. DESIGN Systematic debate on the relevance of distinct imaging modalities held in 2 consensus meetings. PARTICIPANTS A panel of retina specialists. METHODS During the CAM, a consortium of international experts evaluated the advantages and disadvantages of various imaging modalities on the basis of the collective analysis of a large series of clinical cases. A systematic discussion on the role of each modality in future studies in non-neovascular and neovascular AMD was held. MAIN OUTCOME MEASURES Advantages and disadvantages of current retinal imaging technologies and recommendations for their use in advanced AMD trials. RESULTS Imaging protocols to detect, quantify, and monitor progression of atrophy should include color fundus photography (CFP), confocal fundus autofluorescence (FAF), confocal near-infrared reflectance (NIR), and high-resolution optical coherence tomography volume scans. These images should be acquired at regular intervals throughout the study. In studies of non-neovascular AMD (without evident signs of active or regressed neovascularization [NV] at baseline), CFP may be sufficient at baseline and end-of-study visit. Fluorescein angiography (FA) may become necessary to evaluate for NV at any visit during the study. Indocyanine-green angiography (ICG-A) may be considered at baseline under certain conditions. For studies in patients with neovascular AMD, increased need for visualization of the vasculature must be taken into account. Accordingly, these studies should include FA (recommended at baseline and selected follow-up visits) and ICG-A under certain conditions. CONCLUSIONS A multimodal imaging approach is recommended in clinical studies for the optimal detection and measurement of atrophy and its associated features. Specific validation studies will be necessary to determine the best combination of imaging modalities, and these recommendations will need to be updated as new imaging technologies become available in the future.

Directional Kinetics of Geographic Atrophy Progression in Age-Related Macular Degeneration with Foveal Sparing

PURPOSE To describe the directional kinetics of the spread of geographic atrophy (GA) spread in eyes with age-related macular degeneration and foveal sparing. DESIGN Prospective, noninterventional natural history study: Fundus Autofluorescence Imaging in Age-Related Macular Degeneration (FAM; clinicaltrials.gov identifier, NCT00393692). SUBJECTS Participants of the FAM study exhibiting foveal sparing of GA. METHODS Eyes were examined longitudinally with fundus autofluorescence (FAF; excitation wavelength, 488 nm; emission wavelength, >500 nm) and near infrared (NIR) reflectance imaging (Spectralis HRA+OCT or HRA2; Heidelberg Engineering, Heidelberg, Germany). Areas of foveal sparing and GA were measured by 2 independent readers using a semiautomated software tool that allows for combined NIR reflectance and FAF image grading (RegionFinder; Heidelberg Engineering). A linear mixed effect model was used to model GA kinetics over time. MAIN OUTCOME MEASURE Change of GA lesion size over time (central vs. peripheral progression). RESULTS A total of 47 eyes of 36 patients (mean age, 73.8±7.5 years) met the inclusion criteria. Mean follow-up time was 25.2±16.9 months (range, 5.9-74.6 months). Interreader agreement for measurements of GA and foveal-sparing size were 0.995 and 0.946, respectively. Mean area progression of GA toward the periphery was 2.27±0.22 mm(2)/year and 0.25±0.03 mm(2)/year toward the center. Analysis of square root-transformed data revealed a 2.8-fold faster atrophy progression toward the periphery than toward the fovea. Faster atrophy progression toward the fovea correlated with faster progression toward the periphery in presence of marked interindividual differences. CONCLUSIONS The results demonstrate a significantly faster centrifugal than centripetal GA spread in eyes with GA and foveal sparing. Although the underlying pathomechanisms for differential GA progression remain unknown, local factors may be operative that protect the foveal retina-retinal pigment epithelial complex. Quantification of directional spread characteristics and modeling may be useful in the design of interventional clinical trials aiming to prolong foveal survival in eyes with GA.

A Circulating MicroRNA Profile Is Associated with Late-Stage Neovascular Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in Western populations over 55 years. A growing number of gene variants have been identified which are strongly associated with an altered risk to develop AMD. Nevertheless, gene-based biomarkers which could be dysregulated at defined stages of AMD may point toward key processes in disease mechanism and thus may support efforts to design novel treatment regimens for this blinding disorder. Circulating microRNAs (cmiRNAs) which are carried by nanosized exosomes or microvesicles in blood plasma or serum, have been recognized as valuable indicators for various age-related diseases. We therefore aimed to elucidate the role of cmiRNAs in AMD by genome-wide miRNA expression profiling and replication analyses in 147 controls and 129 neovascular AMD patients. We identified three microRNAs differentially secreted in neovascular (NV) AMD (hsa-mir-301-3p, pcorrected = 5.6*10−5, hsa-mir-361-5p, pcorrected = 8.0*10−4 and hsa-mir-424-5p, pcorrected = 9.6*10−3). A combined profile of the three miRNAs revealed an area under the curve (AUC) value of 0.727 and was highly associated with NV AMD (p = 1.2*10−8). To evaluate subtype-specificity, an additional 59 AMD cases with pure unilateral or bilateral geographic atrophy (GA) were analyzed for microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p. While we found no significant differences between GA AMD and controls neither individually nor for a combined microRNAs profile, hsa-mir-424-5p levels remained significantly higher in GA AMD when compared to NV (pcorrected<0.005). Pathway enrichment analysis on genes predicted to be regulated by microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p, suggests canonical TGFβ, mTOR and related pathways to be involved in NV AMD. In addition, knockdown of hsa-mir-361-5p resulted in increased neovascularization in an in vitro angiogenesis assay.

Direct modulation of TRPM4 and TRPM3 channels by the phospholipase C inhibitor U73122

Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122‐sensitive ion channels.

Test-Retest Reliability of Scotopic and Mesopic Fundus-Controlled Perimetry Using a Modified MAIA (Macular Integrity Assessment) in Normal Eyes

Purpose: To assess the intrasession test-retest reliability of scotopic cyan and scotopic red fundus-controlled perimetry (FCP) in normal subjects using a modified MAIA “microperimeter” (macular integrity assessment) device. Methods: Forty-seven normal eyes of 30 subjects (aged 33.8 years) underwent duplicate mesopic (achromatic stimuli, 400-800 nm), scotopic cyan (505 nm), and scotopic red (627 nm) FCP, using a grid of 49 stimuli over 14° of the central retina. Test-retest reliability for pointwise sensitivity (PWS), stability of fixation, reaction time and test duration were analyzed using mixed-effects models. Results: PWS test-retest reliability was good among all 3 types of retinal sensitivity assessments (coefficient of repeatability of 4.75 dB for mesopic, 5.26 dB for scotopic cyan, and 4.06 dB for scotopic red testing). While the mean sensitivity decreased with eccentricity for both mesopic and scotopic red testing, it was highest at 7° eccentricity for the scotopic cyan assessment (p < 0.001). Conclusions: The modified MAIA device allows for reliable scotopic FCP in normal subjects. Our findings suggest that testing of scotopic cyan sensitivity largely reflects rod function.

Technical principles of OCT angiography

ZusammenfassungHintergrundDie OCT-Angiographie (OCT-A) ist eine neue klinische Untersuchungsmethode, die eine nichtinvasive dreidimensionale Darstellung der vaskulären Strukturen der Netzhaut und Aderhaut erlaubt. Technisch handelt es sich bei der OCT-A um eine Weiterentwicklung der optischen Kohärenztomographie (OCT). Durch leistungsfähigere Soft- und Hardware ermöglicht die OCT-A neben morphologischen Analysen auch eine dreidimensionale retinale und choroidale Perfusionsanalyse. Wir erläutern die Grundlagen sowie die Anwendung der OCT-A im Vergleich mit anderen nichtinvasiven Untersuchungsverfahren der retinalen und choroidalen Blutzirkulation.MethodenDer Arbeit liegen eine selektive Literaturrecherche und die Auswertung eigener Daten zugrunde.ErgebnisseVorteile der OCT-A bestehen in der einfachen Anwendung, die keiner Mydriasis oder intravenösen Fluoreszenzfarbstoffverabreichung bedarf. Sie gestattet eine exakte tiefensensitive Lokalisation vaskulärer Veränderungen. Bei retinalen Pathologien können Diskrepanzen zwischen softwareassistierter automatischer Segmentierung und realen Netzhautschichten bestehen, die bei der klinischen Interpretation zu beachten sind.SchlussfolgerungDie OCT-A ist von allen nichtinvasiven Perfusionsanalysen die einzige, die bereits in den klinischen Alltag implementiert werden kann. Mit diesem neuen bildgebenden Untersuchungsverfahren können vaskuläre retinale und choroidale Veränderungen tiefenselektiv und ohne Maskierungseffekt durch Pooling- oder Stainingphänomene detektiert werden.AbstractBackgroundOptical coherence tomography angiography (OCT-A) is a new diagnostic non-invasive method by which the vascular structures of the retina and choroid can be visualized three-dimensionally without need for using fluorescence dyes. The technology of OCT-A is an advancement of the OCT. By means of more powerful software and hardware used for OCT-A not only morphological but also retinal and choroidal vascular perfusion analyses can be performed. In this article, the principles and applications of OCT-A are discussed and compared to other non-invasive diagnostic devices for visualization of the retinal and choroidal blood circulation.MethodsThis article is based on a selective literature review and analyses of own data.ResultsThe advantages of OCT-A include easy application without the need for mydriasis or intravenous injection of fluorescence dyes and also the exact three-dimensional localization of vascular changes. In the case of retinal pathologies there is a considerable difference between software-assisted automatic segmentation and the real architecture of the retina, which must be taken into consideration in the clinical interpretation.ConclusionOf all noninvasive devices for visualization of the retinal and choroidal circulation, OCT-A is the only one which can already be implemented into the clinical routine. With this novel imaging device retinal and choroidal alterations can be visualized in a depth- selective manner and without masking affects, such as pooling or staining phenomena.BACKGROUND Optical coherence tomography angiography (OCT-A) is a new diagnostic non-invasive method by which the vascular structures of the retina and choroid can be visualized three-dimensionally without need for using fluorescence dyes. The technology of OCT-A is an advancement of the OCT. By means of more powerful software and hardware used for OCT-A not only morphological but also retinal and choroidal vascular perfusion analyses can be performed. In this article, the principles and applications of OCT-A are discussed and compared to other non-invasive diagnostic devices for visualization of the retinal and choroidal blood circulation. METHODS This article is based on a selective literature review and analyses of own data. RESULTS The advantages of OCT-A include easy application without the need for mydriasis or intravenous injection of fluorescence dyes and also the exact three-dimensional localization of vascular changes. In the case of retinal pathologies there is a considerable difference between software-assisted automatic segmentation and the real architecture of the retina, which must be taken into consideration in the clinical interpretation. CONCLUSION Of all noninvasive devices for visualization of the retinal and choroidal circulation, OCT-A is the only one which can already be implemented into the clinical routine. With this novel imaging device retinal and choroidal alterations can be visualized in a depth- selective manner and without masking affects, such as pooling or staining phenomena.

Progression of Late-Onset Stargardt Disease.

Purpose Identification of sensitive biomarkers is essential to determine potential effects of emerging therapeutic trials for Stargardt disease. This study aimed to describe the natural history of late-onset Stargardt, and demonstrates the accuracy of retinal pigment epithelium (RPE) atrophy progression as an outcome measure. Methods We performed a retrospective cohort study collecting multicenter data from 47 patients (91 eyes) with late-onset Stargardt, defined by clinical phenotype, at least one ABCA4 mutation, and age at disease onset ≥ 45 years. We analyzed RPE atrophy progression on fundus autofluorescence and near-infrared reflectance imaging using semiautomated software and a linear mixed model. We performed sample size calculations to assess the power in a simulated 2-year interventional study and assessed visual endpoints using time-to-event analysis. Results Over time, progression of RPE atrophy was observed (mean: 0.22 mm/year, 95% confidence interval [CI]: 0.19-0.27). By including only patients with bilateral RPE atrophy in a future trial, 32 patients are needed to reach a power of 83.9% (95% CI: 83.1-84.6), assuming a fixed therapeutic effect size of 30%. We found a median interval between disease onset and visual acuity decline to 20/32, 20/80, and 20/200 of 2.74 (95% CI: 0.54-4.41), 10.15 (95% CI: 6.13-11.38), and 11.38 (95% CI: 6.13-13.34) years, respectively. Conclusions We show that RPE atrophy represents a robust biomarker to monitor disease progression in future therapeutic trials. In contrast, the variability in terms of the course of visual acuity was high.