Development of a chemogenetic approach to manipulate intracellular pH

Chemogenetic Operation of iNTRacellular prOton Levels(pH-Control)is a novel substrate-based enzymatic method that enables precise spatiotemporalcontrol of ultralocal acidification in cultured cell lines and primaryneurons. The genetically encoded biosensor SypHer3s showed that pH-Controleffectively acidifies cytosolic, mitochondrial, and nuclear pH exclusivelyin the presence of beta-chloro-d-alanine in living cellsin a concentration-dependent manner. The pH-Control approach is promisingfor investigating the ultralocal pH imbalance associated with manydiseases.CE254SWXHI ; NN254SWPZX ; CP254SWT2

Chemogenetic approaches to dissect the role of H2O2 in redox-dependent pathways using genetically encoded biosensors

Chemogenetic tools are recombinant enzymes that can be targeted to specific organelles and tissues. The provision or removal of the enzyme substrate permits control of its biochemical activities. Yeast-derived enzyme D-amino acid oxidase (DAAO) represents the first of its kind for a substrate-based chemogenetic approach to modulate H2O2 concentrations within cells. Combining these powerful enzymes with multiparametric imaging methods exploiting genetically encoded biosensors has opened new lines of investigations in life sciences. In recent years, the chemogenetic DAAO approach has proven beneficial to establish a new role for (patho)physiological oxidative stress on redoxdependent signaling and metabolic pathways in cultured cells and animal model systems. This mini-review covers established or emerging methods and assesses newer approaches exploiting chemogenetic tools combined with genetically encoded biosensors

Genetically encoded biosensors unveil neuronal injury dynamics via multichromatic ATP and calcium imaging

When a cell sustains damage, it liberates cytosolic ATP, which can serve as an injury signal, affecting neighboring cells. This study presents a methodological approach that employs in vitro axotomy and in vivo laser ablation to simulate cellular injury. Specially tailored biosensors are employed to monitor ATP dynamics and calcium transients in injured cells and their surroundings. To simultaneously visualize extracellular and cytosolic ATP, we developed bicistronic constructs featuring GRABATP1.0 and MaLionR biosensors alongside the calcium sensor RCaMP, enabling multiparametric imaging. In addition to transducing primary neuron cultures, we developed another method where we cocultured dorsal root ganglion neurons together with specialized “sniffer” cell lines expressing the bicistronic biosensors. Exploiting these approaches, we successfully demonstrated the release of ATP from the injured neurons and its extracellular diffusion in response to cellular injury in vitro and in vivo. Axotomy triggered intracellular calcium mobilization not only in the injured neuron but also in the intact neighboring cells, providing new insights into ATP’s role as an injury signal. The tools developed in this study have demonstrated remarkable efficiency in unraveling the intricacies of ATP-mediated injury signaling

Development of a chemogenetic approach to manipulate intracellular pH

Chemogenetic Operation of iNTRacellular prOton Levels (pH-Control) is a novel substrate-based enzymatic method that enables precise spatio-temporal control of ultra-local acidification in cultured cell lines and primary neurons. The genetically encoded biosensor SypHer3s showed that pH-Control effectively acidifies cytosolic, mitochondrial, and nuclear pH exclusively in the presence of beta-Chloro-D-alanine in living cells in a concentra-tion-dependent manner. The pH-Control approach is promising for investigating the ultra-local pH imbalance associated with many diseases

A co-culture-based multiparametric imaging technique to dissect local H2O2 signals with targeted HyPer7

Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method's potential as a practicable and informative multiparametric live-cell imaging technique.BAGEP award of the Science Academy Turkey ; Integration Projects of Sabanci University ; Austrian Science Fund (FWF) ; Medical University of Gra

Probing intracellular potassium dynamics in neurons with the genetically encoded sensor lc-LysM GEPII 1.0 in vitro and in vivo

Abstract Neuronal activity is accompanied by a net outflow of potassium ions (K+) from the intra- to the extracellular space. While extracellular [K+] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K+ biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K+] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K+] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K+] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K+]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K+] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials

Environmental exposures associated with honey bee health

Bee health is declining on a global scale, yet the exact causes and their interactions responsible for the decline remain unknown. To more objectively study bee health, recently biomarkers have been proposed as an essential tool, because they can be rapidly quantified and standardized, serving as a comparable measure across bee species and varying environments. Here, we used a systems biology approach to draw associations between endogenous and exogenous chemical profiles, with pesticide exposure, or the abundance of the 21 most common honey bee diseases. From the analysis we identified chemical biomarkers for both pesticide exposure and bee diseases along with the mechanistic biological pathways that may influence disease onset and progression. We found a total of 2352 chemical features, from 30 different hives, sampled from seven different locations. Of these, a total of 1088 significant associations were found that could serve as chemical biomarker profiles for predicting both pesticide exposure and the presence of diseases in a bee colony. In almost all cases we found novel external environmental exposures within the top seven associations with bee diseases and pesticide exposures, with the majority having previously unknown connections to bee health. We highlight the exposure-outcome paradigm and its ability to identify previously uncategorized interactions from different environmental exposures associated with bee diseases, pesticides, mechanisms, and potential synergistic interactions of these that are responsible for honey bee health decline

Complexities of the chemogenetic toolkit: Differential mDAAO activation by D-amino substrates and subcellular targeting

A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H2O2 to living cells or tissues. However, the addition of H2O2 to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. D-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of H2O2 on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate D-amino acids. Much has been learned about the intracellular transport of H2O2 through studies using DAAO, yet these valuable tools remain incompletely characterized in many cultured cells. In this study, we describe and characterize in detail the features of a new modified variant of DAAO (termed mDAAO) with improved catalytic activities. We tested mDAAO functionality in several cultured cell lines employing live-cell imaging techniques. Our imaging experiments show that mDAAO is suitable for the generation of H2O2 under hypoxic conditions imaged with the novel ultrasensitive H2O2 sensor (HyPer7). Moreover, this approach was suitable for generating H2O2 in a reversible and concentration-dependent manner in subcellular locales. Furthermore, we show that the choice of D-amino acids differentially affects mDAAO-dependent intracellular H2O2 generation. When paired with the hydrogen sulfide (H2S) sensor hsGFP, administration of the sulfur-containing amino acid D-cysteine to cells expressing mDAAO generates robust H2S signals. We also show that chemogenetic H2O2 generation in different cell types yields distinct HyPer7 profiles. These studies fully characterize the new mDAAO as a novel chemogenetic tool and provide multiparametric approaches for cell manipulation that may open new lines of investigations for redox biochemists to dissect the role of ROS signaling pathways with high spatial and temporal precision