Altered Mitochondrial Dynamics Contributes to Propofol-induced Cell Death in Human Stem Cell-derived Neurons.

BACKGROUND: Studies in developing animals have shown that anesthetic agents can lead to neuronal cell death and learning disabilities when administered early in life. Development of human embryonic stem cell-derived neurons has provided a valuable tool for understanding the effects of anesthetics on developing human neurons. Unbalanced mitochondrial fusion and fission lead to various pathological conditions including neurodegeneration. The aim of this study was to dissect the role of mitochondrial dynamics in propofol-induced neurotoxicity. METHODS: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick-end labeling staining was used to assess cell death in human embryonic stem cell-derived neurons. Mitochondrial fission was assessed using TOM20 staining and electron microscopy. Expression of mitochondrial fission-related proteins was assessed by Western blot, and confocal microscopy was used to assess opening time of the mitochondrial permeability transition pore (mPTP). RESULTS: Exposure to 6 h of 20 µg/ml propofol increased cell death from 3.18 ± 0.17% in the control-treated group to 9.6 ± 0.95% and led to detrimental increases in mitochondrial fission (n = 5 coverslips per group) accompanied by increased expression of activated dynamin-related protein 1 and cyclin-dependent kinase 1, key proteins responsible for mitochondrial fission. Propofol exposure also induced earlier opening of the mPTP from 118.9 ± 3.1 s in the control-treated group to 73.3 ± 1.6 s. Pretreatment of the cells with mdivi-1, a mitochondrial fission blocker rescued the propofol-induced toxicity, mitochondrial fission, and mPTP opening time (n = 75 cells per group). Inhibiting cyclin-dependent kinase 1 attenuated the increase in cell death and fission and the increase in expression of activated dynamin-related protein 1. CONCLUSION: These data demonstrate for the first time that propofol-induced neurotoxicity occurs through a mitochondrial fission/mPTP-mediated pathway.

Up-regulation of microRNA-21 mediates isoflurane-induced protection of cardiomyocytes.

BACKGROUND: Anesthetic cardioprotection reduces myocardial infarct size after ischemia-reperfusion injury. Currently, the role of microRNA in this process remains unknown. MicroRNAs are short, noncoding nucleotide sequences that negatively regulate gene expression through degradation or suppression of messenger RNA. In this study, the authors uncovered the functional role of microRNA-21 (miR-21) up-regulation after anesthetic exposure. METHODS: MicroRNA and messenger RNA expression changes were analyzed by quantitative real-time polymerase chain reaction in cardiomyocytes after exposure to isoflurane. Lactate dehydrogenase release assay and propidium iodide staining were conducted after inhibition of miR-21. miR-21 target expression was analyzed by Western blot. The functional role of miR-21 was confirmed in vivo in both wild-type and miR-21 knockout mice. RESULTS: Isoflurane induces an acute up-regulation of miR-21 in both in vivo and in vitro rat models (n = 6, 247.8 ± 27.5% and 258.5 ± 9.0%), which mediates protection to cardiomyocytes through down-regulation of programmed cell death protein 4 messenger RNA (n = 3, 82.0 ± 4.9% of control group). This protective effect was confirmed by knockdown of miR-21 and programmed cell death protein 4 in vitro. In addition, the protective effect of isoflurane was abolished in miR-21 knockout mice in vivo, with no significant decrease in infarct size compared with nonexposed controls (n = 8, 62.3 ± 4.6% and 56.2 ± 3.2%). CONCLUSIONS: The authors demonstrate for the first time that isoflurane mediates protection of cardiomyocytes against oxidative stress via an miR-21/programmed cell death protein 4 pathway. These results reveal a novel mechanism by which the damage done by ischemia/reperfusion injury may be decreased.

Down-regulation of microRNA-21 is involved in the propofol-induced neurotoxicity observed in human stem cell-derived neurons.

BACKGROUND: Recent studies in various animal models have suggested that anesthetics such as propofol, when administered early in life, can lead to neurotoxicity. These studies have raised significant safety concerns regarding the use of anesthetics in the pediatric population and highlight the need for a better model to study anesthetic-induced neurotoxicity in humans. Human embryonic stem cells are capable of differentiating into any cell type and represent a promising model to study mechanisms governing anesthetic-induced neurotoxicity. METHODS: Cell death in human embryonic stem cell-derived neurons was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling staining, and microRNA expression was assessed using quantitative reverse transcription polymerase chain reaction. miR-21 was overexpressed and knocked down using an miR-21 mimic and antagomir, respectively. Sprouty 2 was knocked down using a small interfering RNA, and the expression of the miR-21 targets of interest was assessed by Western blot. RESULTS: Propofol dose and exposure time dependently induced significant cell death (n = 3) in the neurons and down-regulated several microRNAs, including miR-21. Overexpression of miR-21 and knockdown of Sprouty 2 attenuated the increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells following propofol exposure. In addition, miR-21 knockdown increased the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells by 30% (n = 5). Finally, activated signal transducer and activator of transcription 3 and protein kinase B (Akt) were down-regulated, and Sprouty 2 was up-regulated following propofol exposure (n = 3). CONCLUSIONS: These data suggest that (1) human embryonic stem cell-derived neurons represent a promising in vitro human model for studying anesthetic-induced neurotoxicity, (2) propofol induces cell death in human embryonic stem cell-derived neurons, and (3) the propofol-induced cell death may occur via a signal transducer and activator of transcription 3/miR-21/Sprouty 2-dependent mechanism.