High-content analysis and Kinetic Image Cytometry identify toxicity and epigenetic effects of HIV antiretrovirals on human iPSC-neurons and primary neural precursor cells.

INTRODUCTION: Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain antiretrovirals (ARVs) may contribute to HAND. METHODS: We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis. RESULTS: Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after 7 days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults. CONCLUSION: This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.

Assay of Calcium Transients and Synapses in Rat Hippocampal Neurons by Kinetic Image Cytometry and High-Content Analysis: An In Vitro Model System for Postchemotherapy Cognitive Impairment.

Postchemotherapy cognitive impairment (PCCI) is commonly exhibited by cancer patients treated with a variety of chemotherapeutic agents, including the endocrine disruptor tamoxifen (TAM). The etiology of PCCI is poorly understood. Our goal was to develop high-throughput assay methods to test the effects of chemicals on neuronal function applicable to PCCI. Rat hippocampal neurons (RHNs) were plated in 96- or 384-well dishes and exposed to test compounds (forskolin [FSK], 17ß-estradiol [ES]), TAM or fulvestrant [FUL], aka ICI 182,780) for 6-14 days. Kinetic Image Cytometry™ (KIC™) methods were developed to quantify spontaneously occurring intracellular calcium transients representing the activity of the neurons, and high-content analysis (HCA) methods were developed to quantify the expression, colocalization, and puncta formed by synaptic proteins (postsynaptic density protein-95 [PSD-95] and presynaptic protein Synapsin-1 [Syn-1]). As quantified by KIC, FSK increased the occurrence and synchronization of the calcium transients indicating stimulatory effects on RHN activity, whereas TAM had inhibitory effects. As quantified by HCA, FSK also increased PSD-95 puncta and PSD-95:Syn-1 colocalization, whereas ES increased the puncta of both PSD-95 and Syn-1 with little effect on colocalization. The estrogen receptor antagonist FUL also increased PSD-95 puncta. In contrast, TAM reduced Syn-1 and PSD-95:Syn-1 colocalization, consistent with its inhibitory effects on the calcium transients. Thus TAM reduced activity and synapse formation by the RHNs, which may relate to the ability of this agent to cause PCCI. The results illustrate that KIC and HCA can be used to quantify neurotoxic and neuroprotective effects of chemicals in RHNs to investigate mechanisms and potential therapeutics for PCCI.

Specific prediction of clinical QT prolongation by kinetic image cytometry in human stem cell derived cardiomyocytes.

INTRODUCTION: A priority in the development and approval of new drugs is assessment of cardiovascular risk. Current methodologies for screening compounds (e.g. HERG testing) for proarrhythmic risk lead to many false positive and false negative results, resulting in the attrition of potentially therapeutic compounds in early development, and the advancement of other candidates that cause adverse effects. With improvements in the technologies of high content imaging and human stem cell differentiation, it is now possible to directly screen compounds for arrhythmogenic tendencies in human stem cell derived cardiomyocytes (hSC-CMs). METHODS: A training panel of 90 compounds consisting of roughly equal numbers of QT-prolonging and negative control (non-QT-prolonging) compounds, and a follow-up blinded study of 35 compounds including 16 from the 90 compound panel and 2 duplicates, were evaluated for prolongation of the calcium transient in hSC-CMs using kinetic image cytometry (KIC), a specialized form of high content analysis. RESULTS: The KIC-hSC-CM assay identified training compounds that prolong the calcium transient with 98% specificity, 97% precision, 80% sensitivity, and 89% accuracy in predicting clinical QT prolongation by these compounds. The follow-up study of 35 blinded compounds confirmed the reproducibility and strong diagnostic accuracy of the assay. DISCUSSION: The correlation of the KIC-hSC-CM results to clinical observations met or surpassed traditional preclinical assessment of cardiac risk utilizing animal models. Thus, the KIC-hSC-CM assay, which can be accomplished in high throughput and at relatively low cost, is an effective new model system for testing chemicals for cardiovascular risk.

Automated methods for the analysis of skeletal muscle fiber size and metabolic type.

It is of interest to quantify the size, shape, and metabolic subtype of skeletal muscle fibers in many areas of biomedical research. To do so, skeletal muscle samples are sectioned transversely to the length of the muscle and labeled for extracellular or membrane proteins to delineate the fiber boundaries and additionally for biomarkers related to function or metabolism. The samples are digitally photographed and the fibers "outlined" for quantification of fiber cross-sectional area (CSA) using pointing devices interfaced to a computer, which is tedious, prone to error, and can be nonobjective. Here, we review methods for characterizing skeletal muscle fibers and describe new automated techniques, which rapidly quantify CSA and biomarkers. We discuss the applications of these methods to the characterization of mitochondrial dysfunctions, which underlie a variety of human afflictions, and we present a novel approach, utilizing images from the online Human Protein Atlas to predict relationships between fiber-specific protein expression, function, and metabolism.

Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

Sorcin modulates mitochondrial Ca(2+) handling and reduces apoptosis in neonatal rat cardiac myocytes.

Sorcin localizes in cellular membranes and has been demonstrated to modulate cytosolic Ca(2+) handling in cardiac myocytes. Sorcin also localizes in mitochondria; however, the effect of sorcin on mitochondrial Ca(2+) handling is unknown. Using mitochondrial pericam, we measured mitochondrial Ca(2+) concentration and fluxes in intact neonatal cardiac myocytes overexpressing sorcin. Our results showed that sorcin increases basal and caffeine-stimulated mitochondrial Ca(2+) concentration. This effect was associated with faster Ca(2+) uptake and release. The effect of sorcin was specific for mitochondria, since similar results were obtained with digitonin-permeabilized cells, where cytosolic Ca(2+) flux was disrupted. Furthermore, mitochondria of cardiac myocytes in which sorcin was overexpressed were more Ca(2+)-tolerant. Experiments analyzing apoptotic signaling demonstrated that sorcin prevented 2-deoxyglucose-induced cytochrome c release. Furthermore, sorcin prevented hyperglycemia-induced cytochrome c release and caspase activation. In contrast, antisense sorcin induced caspase-3 activation. Thus, sorcin antiapoptotic properties may be due to modulation of mitochondrial Ca(2+) handling in cardiac myocytes.

High throughput measurement of Ca²âº dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry.

Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca²âº indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca²âº](i) at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca²âº dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background.

Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.

Identification of MicroRNAs that control lipid droplet formation and growth in hepatocytes via high-content screening.

Hepatic lipid droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis C, and both alcoholic and nonalcoholic fatty liver disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the level of translation. Approximately 1000 different miRNA species are encoded within the human genome, and many are differentially expressed by healthy and diseased liver. However, few studies have investigated the role of miRNAs in regulating LD expression. Accordingly, a high-content assay (HCA) was performed in which human hepatocytes (Huh-7 cells) were transiently transfected with 327 unique human miRNAs; the cells were then fixed, labeled for nuclei and lipid droplets, and imaged with an automated digital microscopy workstation. LD expression was analyzed on a cell-by-cell basis, using automated image analysis. Eleven miRNAs were identified that altered LDs. MiR-181d was the most efficacious inhibitor, decreasing LDs by about 60%. miRNA-181d was also confirmed to reduce cellular triglycerides and cholesterol ester via biochemical assays. Furthermore, a series of proteins was identified via miRNA target analysis, and siRNAs directed against many of these proteins also modified LDs. Thus, HCA-based screening identified novel miRNA and protein regulators of LDs and cholesterol metabolism that may be relevant to hepatic diseases arising from obesity and alcohol abuse.

Quantification of lipid droplets and associated proteins in cellular models of obesity via high-content/high-throughput microscopy and automated image analysis.

Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-(c) agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc's CyteSeer((R)) image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z' values of 0.54-0.71). Regarding colocalization with lipid droplets, Pearson's correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and -0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, HuH-7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin C on lipid droplets (Z's > 0.50) and ADFP expression/colocalization. Oleic acid-induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.