Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

The sex-specific metabolic signature of C57BL/6NRj mice during aging

Due to intact reactive oxygen species homeostasis and glucose metabolism, C57BL/6NRj mice are especially suitable to study cellular alterations in metabolism. We applied Nuclear Magnetic resonance spectroscopy to analyze five different tissues of this mouse strain during aging and included female and male mice aged 3, 6, 12, and 24 months. Metabolite signatures allowed separation between the age groups in all tissues, and we identified the most prominently changing metabolites in female and male tissues. A refined analysis of individual metabolite levels during aging revealed an early onset of age-related changes at 6 months, sex-specific differences in the liver, and a biphasic pattern for various metabolites in the brain, heart, liver, and lung. In contrast, a linear decrease of amino acids was apparent in muscle tissues. Based on these results, we assume that age-related metabolic alterations happen at a comparably early aging state and are potentially associated with a metabolic switch. Moreover, identified differences between female and male tissues stress the importance of distinguishing between sexes when studying age-related changes and developing new treatment approaches. Besides, metabolomic features seem to be highly dependent on the genetic background of mouse strains.ISSN:2045-232

FOXO transcription factors differ in their dynamics and intra/intermolecular interactions

Transcription factors play key roles in orchestrating a plethora of cellular mechanisms and controlling cellular homeostasis. Transcription factors share distinct DNA binding domains, which allows to group them into protein families. Among them, the Forkhead box O (FOXO) family contains transcription factors crucial for cellular homeostasis, longevity and response to stress. The dysregulation of FOXO signaling is linked to drug resistance in cancer therapy or cellular senescence, however, selective drugs targeting FOXOs are limited, thus knowledge about structure and dynamics of FOXO proteins is essential. Here, we provide an extensive study of structure and dynamics of all FOXO family members. We identify residues accounting for different dynamic and structural features. Furthermore, we show that the auto-inhibition of FOXO proteins by their C-terminal trans-activation domain is conserved throughout the family and that these interactions are not only possible intra-, but also inter-molecularly. This indicates a model in which FOXO transcription factors would modulate their activities by interacting mutually

Associations between Endothelial Lipase, High-Density Lipoprotein, and Endothelial Function Differ in Healthy Volunteers and Metabolic Syndrome Patients

Metabolic syndrome (MS) is characterized by endothelial- and high-density lipoprotein (HDL) dysfunction and increased endothelial lipase (EL) serum levels. We examined the associations between EL serum levels, HDL (serum levels, lipid content, and function), and endothelial function in healthy volunteers (HV) and MS patients. Flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NMD), serum levels of HDL subclasses (measured by nuclear magnetic resonance (NMR) spectroscopy), and EL serum levels differed significantly between HV and MS patients. The serum levels of triglycerides in large HDL particles were significantly positively correlated with FMD and NMD in HV, but not in MS patients. Cholesterol (C) and phospholipid (PL) contents of large HDL particles, calculated as HDL1-C/HDL1-apoA-I and HDL1-PL/HDL1-apoA-I, respectively, were significantly negatively correlated with FMD in HV, but not in MS patients. Cholesterol efflux capacity and arylesterase activity of HDL, as well as EL, were correlated with neither FMD nor NMD. EL was significantly negatively correlated with HDL-PL/HDL-apoA-I in HV, but not in MS patients, and with serum levels of small dense HDL containing apolipoprotein A-II in MS patients, but not in HV. We conclude that MS modulates the association between HDL and endothelial function, as well as between EL and HDL. HDL cholesterol efflux capacity and arylesterase activity, as well as EL serum levels, are not associated with endothelial function in HV or MS patients

Enhanced inter-compartmental Ca2+ flux modulates mitochondrial metabolism and apoptotic threshold during aging

Background: Senescence is characterized by a gradual decline in cellular functions, including changes in energy homeostasis and decreased proliferation activity. As cellular power plants, contributors to signal transduction, sources of reactive oxygen species (ROS) and executors of programmed cell death, mitochondria are in a unique position to affect aging-associated processes of cellular decline. Notably, metabolic activation of mitochondria is tightly linked to Ca2+ due to the Ca2+ -dependency of several enzymes in the Krebs cycle, however, overload of mitochondria with Ca2+ triggers cell death pathways. Consequently, a machinery of proteins tightly controls mitochondrial Ca2+ homeostasis as well as the exchange of Ca2+ between the different cellular compartments, including Ca2+ flux between mitochondria and the endoplasmic reticulum (ER). Methods: In this study, we investigated age-related changes in mitochondrial Ca2+ homeostasis, mitochondrial-ER linkage and the activity of the main ROS production site, the mitochondrial respiration chain, in an in vitro aging model based on porcine aortic endothelial cells (PAECs), using high-resolution live cell imaging, proteomics and various molecular biological methods. Results: We describe that in aged endothelial cells, increased ER-mitochondrial Ca2+ crosstalk occurs due to enhanced ER-mitochondrial tethering. The close functional inter-organelle linkage increases mitochondrial Ca2+ uptake and thereby the activity of the mitochondrial respiration, but also makes senescent cells more vulnerable to mitochondrial Ca2+-overload-induced cell death. Moreover, we identified the senolytic properties of the polyphenol resveratrol, triggering cell death via mitochondrial Ca2+ overload exclusively in senescent cells. Conclusion: By unveiling aging-related changes in the inter-organelle tethering and Ca2+ communications we have advanced the understanding of endothelial aging and highlighted a potential basis to develop drugs specifically targeting senescent cells. Keywords: Mitochondria, Endoplasmic reticulum, Calcium signaling, ER-mitochondrial coupling, Vascular agin

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose

BACKGROUND/AIMS: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored