Brian J. Hawkins

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1–2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial–temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.

The Common Inhalational Anesthetic Isoflurane Induces Apoptosis via Activation of Inositol 1,4,5-Trisphosphate Receptors

Background:Isoflurane induces cell apoptosis by an unknown mechanism. The authors hypothesized that isoflurane activates inositol 1,4,5-trisphosphate (IP3) receptors on the endoplasmic reticulum (ER) membrane, causing excessive calcium release, triggering apoptosis. Methods:The authors determined isoflurane-induced cytotoxicity by measuring caspase-3 activity, lactate dehydrogenase release, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduction, and imaging analysis of cell damage markers (annexin V and propidium iodide staining) in different cell types. The authors used the chicken B lymphocyte with a total knock-out of IP3 receptors, PC12 cells with elevated IP3 receptor activity (transfected with L286V presenilin 1), striatal cells with a knock-in of Q111 Huntingtin, and each cell line’s corresponding wild-type controls. The authors also measured the isoflurane-evoked changes of calcium concentration in cytosol and/or mitochondria in these cells. Results:Isoflurane induced apoptosis concentration- and time-dependently, and sequentially elevated cytosolic and then mitochondrial calcium in the chicken B-lymphocyte wild-type but not the IP3 receptor total knock-out cells. Thapsigargin, a calcium adenosine triphosphatase inhibitor on ER membranes, induced apoptosis and elevations of calcium in cytosol and mitochondria in both chicken B-lymphocyte wild-type and IP3 receptor total knock-out cells. Isoflurane induced significantly more neurotoxicity and greater calcium release from the ER in L286V PC12 and Q111 Huntingtin striatal cells than in their corresponding wild-type controls, both of which were significantly inhibited by the IP3 receptor antagonist xestospongin C. Conclusion:These findings suggest that isoflurane activates the ER membrane IP3 receptor, producing excessive calcium release and triggering apoptosis. Neurons with enhanced IP3 receptor activity, as in certain cases of familial Alzheimer or Huntington disease, may be especially vulnerable to isoflurane cytotoxicity.

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress induces constitutive calcium entry by tacking glutathiones onto the Orai CRAC channel activator STIM1.

Inhalational Anesthetics Induce Cell Damage by Disruption of Intracellular Calcium Homeostasis with Different Potencies

Background:The authors hypothesized that inhalational anesthetics induced cell damage by causing abnormal calcium release from the endoplasmic reticulum via excessive activation of inositol 1,4,5-trisphosphate (IP3) receptors, with isoflurane having greater potency than sevoflurane or desflurane. Methods:The authors treated DT40 chicken B lymphocytes with total IP3 receptor knockout or their corresponding wild-type control cells with equipotent exposure to isoflurane, sevoflurane, and desflurane. The authors then determined the degree of cell damage by counting the percentage of annexin V– or propidium iodide–positively stained cells or measuring caspase-3 activity. They also studied the changes of calcium concentrations in the endoplasmic reticulum, cytosol, and mitochondria evoked by equipotent concentrations of isoflurane, sevoflurane, and desflurane in both types of DT40 cells. Results:Prolonged use of 2 minimal alveolar concentration sevoflurane or desflurane (24 h) induced significant cell damage only in DT40 wild-type and not in IP3 receptor total knockout cells, but with significantly less potency than isoflurane. In accord, all three inhalational anesthetics induced significant decrease of calcium concentrations in the endoplasmic reticulum, accompanied by a subsequent significant increase in the cytosol and mitochondrial calcium concentrations only in DT40 wild-type and not in IP3 receptor total knockout cells. Isoflurane treatment showed significantly greater potency of effect than sevoflurane or desflurane. Conclusion:Inhalational anesthetics may induce cell damage by causing abnormal calcium release from the endoplasmic reticulum via excessive activation of IP3 receptors. Isoflurane has greater potency than sevoflurane or desflurane to cause calcium release from the endoplasmic reticulum and to induce cell damage.

Selective role for superoxide in InsP3 receptor–mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2 .− that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2 .−-triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2 .− and could be prevented by [Ca2+]i buffering. This study provides evidence that O2 .− facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.

Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis.

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.

Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.

Atrial fibrillation is the most common clinical cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium, but the source and mechanism of these beats remains unclear. The melanin synthesis enzyme dopachrome tautomerase (DCT) is involved in intracellular calcium and reactive species regulation in melanocytes. Given that dysregulation of intracellular calcium and reactive species has been described in patients with atrial fibrillation, we investigated the role of DCT in this process. Here, we characterize a unique DCT-expressing cell population within murine and human hearts that populated the pulmonary veins, atria, and atrioventricular canal. Expression profiling demonstrated that this population expressed adrenergic and muscarinic receptors and displayed transcriptional profiles distinct from dermal melanocytes. Adult mice lacking DCT displayed normal cardiac development but an increased susceptibility to atrial arrhythmias. Cultured primary cardiac melanocyte-like cells were excitable, and those lacking DCT displayed prolonged repolarization with early afterdepolarizations. Furthermore, mice with mutations in the tyrosine kinase receptor Kit lacked cardiac melanocyte-like cells and did not develop atrial arrhythmias in the absence of DCT. These data suggest that dysfunction of melanocyte-like cells in the atrium and pulmonary veins may contribute to atrial arrhythmias.

PECAM-targeted delivery of SOD inhibits endothelial inflammatory response

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH‐oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule‐1 (VCAM‐1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM‐1) bind specifically to endothelium and inhibit effects of corresponding ROS, H2O2, and superoxide anion. In this study, anti‐PECAM/SOD, but not anti‐PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)‐SOD, inhibited 2‐ to 3‐fold VCAM expression caused by tumor necrosis factor (TNF), interleukin‐1β, and lipopolysaccharide. Anti‐PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide‐caused VCAM‐1 expression in mice. Anti‐PECAM/SOD colocalized with EEA‐1‐positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF‐induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti‐PECAM/SOD abolished VCAM expression caused by poly(I:C)‐induced activation of toll‐like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site‐specific interception of this signal attained by targeted delivery of anti‐PECAM/SOD into endothelial endo‐somes may have anti‐inflammatory effects.—Shuvaev, V. V., Han, J., Yu, K. J., Huang, S., Hawkins, B. J., Madesh, M., Nakada, M., and Muzykantov, V. R. PECAM‐targeted delivery of SOD inhibits endothelial inflammatory response. FASEB J. 25, 348–357 (2011). www.fasebj.org

Methamphetamine Causes Mitrochondrial Oxidative Damage in Human T Lymphocytes Leading to Functional Impairment

Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.

HIV-1 Tat protein promotes neuronal dysfunction through disruption of microRNAs.

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.