Synaptic and mitochondrial mechanisms behind alcohol-induced imbalance of excitatory/inhibitory synaptic activity and associated cognitive and behavioral abnormalities.

Alcohol consumption during pregnancy can significantly impact the brain development of the fetus, leading to long-term cognitive and behavioral problems. However, the underlying mechanisms are not well understood. In this study, we investigated the acute and chronic effects of binge-like alcohol exposure during the third trimester equivalent in postnatal day 7 (P7) mice on brain cell viability, synapse activity, cognitive and behavioral performance, and gene expression profiles at P60. Our results showed that alcohol exposure caused neuroapoptosis in P7 mouse brains immediately after a 6-hour exposure. In addition, P60 mice exposed to alcohol during P7 displayed impaired learning and memory abilities and anxiety-like behaviors. Electrophysiological analysis of hippocampal neurons revealed an excitatory/inhibitory imbalance in alcohol-treated P60 mice compared to controls, with decreased excitation and increased inhibition. Furthermore, our bioinformatic analysis of 376 dysregulated genes in P60 mouse brains following alcohol exposure identified 50 synapse-related and 23 mitochondria-related genes. These genes encoded proteins located in various parts of the synapse, synaptic cleft, extra-synaptic space, synaptic membranes, or mitochondria, and were associated with different biological processes and functions, including the regulation of synaptic transmission, transport, synaptic vesicle cycle, metabolism, synaptogenesis, mitochondrial activity, cognition, and behavior. The dysregulated synapse and mitochondrial genes were predicted to interact in overlapping networks. Our findings suggest that altered synaptic activities and signaling networks may contribute to alcohol-induced long-term cognitive and behavioral impairments in mice, providing new insights into the underlying synaptic and mitochondrial molecular mechanisms and potential neuroprotective strategies.

Integrated Excitatory/Inhibitory Imbalance and Transcriptomic Analysis Reveals the Association between Dysregulated Synaptic Genes and Anesthetic-Induced Cognitive Dysfunction.

Emerging evidence from human epidemiologic and animal studies has demonstrated that developmental anesthesia neurotoxicity could cause long-term cognitive deficits and behavioral problems. However, the underlying mechanisms remain largely unknown. We conducted an electrophysiological analysis of synapse activity and a transcriptomic assay of 24,881 mRNA expression on hippocampal tissues from postnatal day 60 (P60) mice receiving propofol exposure at postnatal day 7 (P7). We found that developmentally propofol-exposed P60 mouse hippocampal neurons displayed an E/I imbalance, compared with control mice as evidenced by the decreased excitation and increased inhibition. We found that propofol exposure at P7 led to the abnormal expression of 317 mRNAs in the hippocampus of P60 mice, including 23 synapse-related genes. Various bioinformatic analyses revealed that these abnormally expressed synaptic genes were associated with the function and development of synapse activity and plasticity, E/I balance, behavior, and cognitive impairment. Our findings suggest that the altered E/I balance may constitute a mechanism for propofol-induced long-term impaired learning and memory in mice. The transcriptomic and bioinformatic analysis of these dysregulated genes related to synaptic function paves the way for development of therapeutic strategies against anesthetic neurodegeneration through the restoration of E/I balance and the modification of synaptic gene expression.

Identification of a dihydropyridine scaffold that blocks ryanodine receptors.

Ryanodine receptors (RyRs) are large, intracellular ion channels that control Ca2+ release from the sarco/endoplasmic reticulum. Dysregulation of RyRs in skeletal muscle, heart, and brain has been implicated in various muscle pathologies, arrhythmia, heart failure, and Alzheimer's disease. Therefore, there is considerable interest in therapeutically targeting RyRs to normalize Ca2+ homeostasis in scenarios involving RyR dysfunction. Here, a simple invertebrate screening platform was used to discover new chemotypes targeting RyRs. The approach measured Ca2+ signals evoked by cyclic adenosine 5'-diphosphate ribose, a second messenger that sensitizes RyRs. From a 1,534-compound screen, FLI-06 (currently described as a Notch "inhibitor") was identified as a potent blocker of RyR activity. Two closely related tyrosine kinase inhibitors that stimulate and inhibit Ca2+ release through RyRs were also resolved. Therefore, this simple screen yielded RyR scaffolds tractable for development and revealed an unexpected linkage between RyRs and trafficking events in the early secretory pathway.

Isolation and Culture of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoid Cells.

The advent of human-induced pluripotent stem cell (iPSC)-derived three-dimensional (3D) cerebral organoids provides unprecedented opportunities of modeling human brains in states of health and disorder. Emerging data supports that cerebral organoids allow for more relevant in vitro systems for studying the human brain system and diseases than the current widely used 2D monolayer cell culture. Thus, the ability to isolate, culture, and maintain human brain cells from cerebral organoids is highly needed, particularly for studies on organoid-derived cell-type-specific signaling and their electrophysiological properties. Here we present a protocol to isolate and culture brain cells from 2-month human iPSC-derived cerebral organoids. The dissociation and plating of cells from organoids takes 3-4 h. The dissociated cells can be maintained in culture for up to at least 3 weeks. Some cells expressed the neuron-specific marker microtubule-associated protein 2 and exhibited spontaneous action potentials.

The importance of non-coding RNAs in environmental stress-related developmental brain disorders: A systematic review of evidence associated with exposure to alcohol, anesthetic drugs, nicotine, and viral infections.

Brain development is a dynamic and lengthy process that includes cell proliferation, migration, neurogenesis, gliogenesis, synaptogenesis, and pruning. Disruption of any of these developmental events can result in long-term outcomes ranging from brain structural changes, to cognitive and behavioral abnormality, with the mechanisms largely unknown. Emerging evidence suggests non-coding RNAs (ncRNAs) as pivotal molecules that participate in normal brain development and neurodevelopmental disorders. NcRNAs such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are transcribed from the genome but not translated into proteins. Many ncRNAs have been implicated as tuners of cell fate. In this review, we started with an introduction of the current knowledge of lncRNAs and miRNAs, and their potential roles in brain development in health and disorders. We then reviewed and discussed the evidence of ncRNA involvement in abnormal brain development resulted from alcohol, anesthetic drugs, nicotine, and viral infections. The complex connections among these ncRNAs were also discussed, along with potential overlapping ncRNA mechanisms, possible pharmacological targets for therapeutic/neuroprotective interventions, and potential biomarkers for brain developmental disorders.

Expression Signature of lncRNAs and mRNAs in Sevoflurane-Induced Mouse Brain Injury: Implication of Involvement of Wide Molecular Networks and Pathways.

Sevoflurane, one of the most commonly used pediatric anesthetics, was found to cause developmental neurotoxicity. To understand specific risk groups and develop countermeasures, a better understanding of its mechanisms is needed. We hypothesize that, as in many other brain degeneration pathways, long non-coding RNAs (lncRNAs) are involved in the sevoflurane-induced neurotoxicity. Postnatal day 7 (PD7) mice were exposed to 3% sevoflurane for 6 h. To quantify neurotoxicity in these mice, we (1) detected neural apoptosis through analysis of caspase 3 expression level and activity and (2) assessed long-term learning ability via the Morris water maze at PD60. To elucidate specific mechanisms, profiles of 27,427 lncRNAs and 18,855 messenger RNAs (mRNAs) in mouse hippocampi were analyzed using microarray assays. Sevoflurane-induced abnormal lncRNA and mRNA expression-associated function pathways were predicted by bioinformatic analysis. We found that sevoflurane induced significant neurotoxicity, causing acute neuroapoptosis and abnormal expression of 148 mRNAs and 301 lncRNAs on PD7 in mouse hippocampus. Additionally, exposed mice exhibited impaired memory on PD60. Bioinformatic analysis predicted that the dysregulated mRNAs, which are highly correlated with their co-expressed dysregulated lncRNAs, might be involved in 34 neurodegenerative signaling pathways (e.g., brain cell apoptosis and intellectual developmental disorder). Our study reveals for the first time that neonatal exposure to 3% sevoflurane induces abnormal lncRNA and mRNA expression profiles. These dysregulated lncRNAs/mRNAs form wide molecular networks that might contribute to various functional neurological disease pathways in the hippocampus, resulting in the observed acute apoptosis and impaired long-term memory.

Biphasic effect of metformin on human cardiac energetics.

Metformin is the first-line medication for treatment of type 2 diabetes and has been shown to reduce heart damage and death. However, mechanisms by which metformin protects human heart remain debated. The aim of the study was to evaluate the cardioprotective effect of metformin on cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) and mitochondria isolated from human cardiac tissue. At concentrations ≤2.5 mM, metformin significantly increased oxygen consumption rate (OCR) in the hiPSC-CMs by activating adenosine monophosphate activated protein kinase (AMPK)-dependent signaling and enhancing mitochondrial biogenesis. This effect was abrogated by compound C, an inhibitor of AMPK. At concentrations >5 mM, metformin inhibited the cellular OCR and triggered metabolic reprogramming by enhancing glycolysis and glutaminolysis in the cardiomyocytes. In isolated cardiac mitochondria, metformin did not increase the OCR at any concentrations but inhibited the OCR starting at 1 mM through direct inhibition of electron-transport chain complex I. This was associated with reduction of superoxide production and attenuation of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in the mitochondria. Thus, in human heart, metformin might improve cardioprotection due to its biphasic effect on mitochondria: at low concentrations, it activates mitochondrial biogenesis via AMPK signaling and increases the OCR; at high concentrations, it inhibits the respiration by directly affecting the activity of complex I, reduces oxidative stress and delays mPTP formation. Moreover, metformin at high concentrations causes metabolic reprogramming by enhancing glycolysis and glutaminolysis. These effects can be a beneficial adjunct to patients with impaired endogenous cardioprotective responses.

Modeling alcohol-induced neurotoxicity using human induced pluripotent stem cell-derived three-dimensional cerebral organoids.

Maternal alcohol exposure during pregnancy can substantially impact the development of the fetus, causing a range of symptoms, known as fetal alcohol spectrum disorders (FASDs), such as cognitive dysfunction and psychiatric disorders, with the pathophysiology and mechanisms largely unknown. Recently developed human cerebral organoids from induced pluripotent stem cells are similar to fetal brains in the aspects of development and structure. These models allow more relevant in vitro systems to be developed for studying FASDs than animal models. Modeling binge drinking using human cerebral organoids, we sought to quantify the downstream toxic effects of alcohol (ethanol) on neural pathology phenotypes and signaling pathways within the organoids. The results revealed that alcohol exposure resulted in unhealthy organoids at cellular, subcellular, bioenergetic metabolism, and gene expression levels. Alcohol induced apoptosis on organoids. The apoptotic effects of alcohol on the organoids depended on the alcohol concentration and varied between cell types. Specifically, neurons were more vulnerable to alcohol-induced apoptosis than astrocytes. The alcohol-treated organoids exhibit ultrastructural changes such as disruption of mitochondria cristae, decreased intensity of mitochondrial matrix, and disorganized cytoskeleton. Alcohol exposure also resulted in mitochondrial dysfunction and metabolic stress in the organoids as evidenced by (1) decreased mitochondrial oxygen consumption rates being linked to basal respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity, and (2) increase of non-mitochondrial respiration in alcohol-treated organoids compared with control groups. Furthermore, we found that alcohol treatment affected the expression of 199 genes out of 17,195 genes analyzed. Bioinformatic analyses showed the association of these dysregulated genes with 37 pathways related to clinically relevant pathologies such as psychiatric disorders, behavior, nervous system development and function, organismal injury and abnormalities, and cellular development. Notably, 187 of these genes are critically involved in neurodevelopment, and/or implicated in nervous system physiology and neurodegeneration. Furthermore, the identified genes are key regulators of multiple pathways linked in networks. This study extends for the first time animal models of binge drinking-related FASDs to a human model, allowing in-depth analyses of neurotoxicity at tissue, cellular, subcellular, metabolism, and gene levels. Hereby, we provide novel insights into alcohol-induced pathologic phenotypes, cell type-specific vulnerability, and affected signaling pathways and molecular networks, that can contribute to a better understanding of the developmental neurotoxic effects of binge drinking during pregnancy.

Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles.

BACKGROUND: The development of 3D cerebral organoid technology using human-induced pluripotent stem cells (iPSCs) provides a promising platform to study how brain diseases are appropriately modeled and treated. So far, understanding of the characteristics of organoids is still in its infancy. The current study profiled, for the first time, the electrophysiological properties of organoids at molecular and cellular levels and dissected the potential age equivalency of 2-month-old organoids to human ones by a comparison of gene expression profiles among cerebral organoids, human fetal and adult brains. RESULTS: Cerebral organoids exhibit heterogeneous gene and protein markers of various brain cells, such as neurons, astrocytes, and vascular cells (endothelial cells and smooth muscle cells) at 2 months, and increases in neural, glial, vascular, and channel-related gene expression over a 2-month differentiation course. Two-month organoids exhibited action potentials, multiple channel activities, and functional electrophysiological responses to the anesthetic agent propofol. A bioinformatics analysis of 20,723 gene expression profiles showed the similar distance of gene profiles in cerebral organoids to fetal and adult brain tissues. The subsequent Ingenuity Pathway Analysis (IPA) of select canonical pathways related to neural development, network formation, and electrophysiological signaling, revealed that only calcium signaling, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signaling in neurons, glutamate receptor signaling, and synaptogenesis signaling were predicted to be downregulated in cerebral organoids relative to fetal samples. Nearly all cerebral organoid and fetal pathway phenotypes were predicted to be downregulated compared with adult tissue. CONCLUSIONS: This novel study highlights dynamic development, cellular heterogeneity and electrophysiological activity. In particular, for the first time, electrophysiological drug response recapitulates what occurs in vivo, and neural characteristics are predicted to be highly similar to the human brain, further supporting the promising application of the cerebral organoid system for the modeling of the human brain in health and disease. Additionally, the studies from these characterizations of cerebral organoids in multiple levels and the findings from gene comparisons between cerebral organoids and humans (fetuses and adults) help us better understand this cerebral organoid-based cutting-edge platform and its wide uses in modeling human brain in terms of health and disease, development, and testing drug efficacy and toxicity.

Fatty Acid-Treated Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes Exhibit Adult Cardiomyocyte-Like Energy Metabolism Phenotypes.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. As an example, glycolysis is a major energy source for immature CMs. As CMs mature, the mitochondrial oxidative capacity increases, with fatty acid ß-oxidation becoming a key energy source to meet the heart's high energy demand. The immaturity of iPSC-CMs thereby limits their applications. The aim of this study was to investigate whether the energy substrate fatty acid-treated iPSC-CMs exhibit adult CM-like metabolic properties. After 20 days of differentiation from human iPSCs, iPSC-CMs were sequentially cultured with CM purification medium (lactate+/glucose-) for 7 days and maturation medium (fatty acids+/glucose-) for 3-7 days by mimicking the adult CM's preference of utilizing fatty acids as a major metabolic substrate. The purity and maturity of iPSC-CMs were characterized via the analysis of: (1) Expression of CM-specific markers (e.g., troponin T, and sodium and potassium channels) using RT-qPCR, Western blot or immunofluorescence staining and electron microscopy imaging; and (2) cell energy metabolic profiles using the XF96 Extracellular Flux Analyzer. iPSCs-CMs (98% purity) cultured in maturation medium exhibited enhanced elongation, increased mitochondrial numbers with more aligned Z-lines, and increased expression of matured CM-related genes, suggesting that fatty acid-contained medium promotes iPSC-CMs to undergo maturation. In addition, the oxygen consumption rate (OCR) linked to basal respiration, ATP production, and maximal respiration and spare respiratory capacity (representing mitochondrial function) was increased in matured iPSC-CMs. Mature iPSC-CMs also displayed a larger change in basal and maximum respirations due to the utilization of exogenous fatty acids (palmitate) compared with non-matured control iPSC-CMs. Etomoxir (a carnitine palmitoyltransferase 1 inhibitor) but not 2-deoxyglucose (an inhibitor of glycolysis) abolished the palmitate pretreatment-mediated OCR increases in mature iPSC-CMs. Collectively, our data demonstrate for the first time that fatty acid treatment promotes metabolic maturation of iPSC-CMs (as evidenced by enhanced mitochondrial oxidative function and strong capacity of utilizing fatty acids as energy source). These matured iPSC-CMs might be a promising human CM source for broad biomedical application.

Propofol Alters Long Non-Coding RNA Profiles in the Neonatal Mouse Hippocampus: Implication of Novel Mechanisms in Anesthetic-Induced Developmental Neurotoxicity.

BACKGROUND: Propofol induces acute neurotoxicity (e.g., neuroapoptosis) followed by impairment of long-term memory and learning in animals. However, underlying mechanisms remain largely unknown. Long non-coding RNAs (lncRNAs) are found to participate in various pathological processes. We hypothesized that lncRNA profile and the associated signaling pathways were altered, and these changes might be related to the neurotoxicity observed in the neonatal mouse hippocampus following propofol exposure. METHODS: In this laboratory experiment, 7-day-old mice were exposed to a subanesthetic dose of propofol for 3 hours, with 4 animals per group. Hippocampal tissues were harvested 3 hours after propofol administration. Neuroapoptosis was analyzed based on caspase 3 activity using a colorimetric assay. A microarray was performed to investigate the profiles of 35,923 lncRNAs and 24,881 messenger RNAs (mRNAs). Representative differentially expressed lncRNAs and mRNAs were validated using reverse transcription quantitative polymerase chain reaction. All mRNAs dysregulated by propofol and the 50 top-ranked, significantly dysregulated lncRNAs were subject to bioinformatics analysis for exploring the potential mechanisms and signaling network of propofol-induced neurotoxicity. RESULTS: Propofol induced neuroapoptosis in the hippocampus, with differential expression of 159 lncRNAs and 100 mRNAs (fold change ± 2.0, P< 0.05). Bioinformatics analysis demonstrated that these lncRNAs and their associated mRNAs might participate in neurodegenerative pathways (e.g., calcium handling, apoptosis, autophagy, and synaptogenesis). CONCLUSION: This novel report emphasizes that propofol alters profiles of lncRNAs, mRNAs, and their cooperative signaling network, which provides novel insights into molecular mechanisms of anesthetic-induced developmental neurodegeneration and preventive targets against the neurotoxicity.

Signaling network between the dysregulated expression of microRNAs and mRNAs in propofol-induced developmental neurotoxicity in mice.

Mounting evidence has demonstrated that general anesthetics could induce acute neuroapoptosis in developing animals followed by long-term cognitive dysfunction, with the mechanisms remaining largely unknown. The aim of this study was to investigate the effect of the intravenous anesthetic propofol on the profiles of microRNAs (miRNAs) and messenger RNAs (mRNAs), and their interactive signaling networks in the developing mouse hippocampus. Postnatal day 7 (P7) mice were exposed to propofol for 3 hours. Hippocampi were harvested from both P7 (3 hours after exposure) and P60 mice for the analysis of the expression of 726 miRNAs and 24,881 mRNAs, and apoptosis. Long-term memory ability of P60 mice was analyzed using the Morris Water Maze. Propofol induced acute apoptosis in the hippocampus, and impaired memory function of mice. There were 100 altered mRNAs and 18 dysregulated miRNAs in the propofol-treated hippocampi compared with the intralipid-treated control tissues on P7. Bioinformatics analysis of these abnormally expressed genes on P7 indicated that 34 dysregulated miRNA-mRNA target pairs were related to pathological neurological and developmental disorder processes such as cell viability, cell morphology and migration, neural stem cell proliferation and neurogenesis, oligodendrocyte myelination, reactive oxygen species, and calcium signaling. Neonatal propofol exposure also resulted in the abnormal expression of 49 mRNAs and 4 miRNAs in P60 mouse hippocampi. Specifically, bioinformatics analysis indicates that among these dysregulated mRNAs and miRNAs, there were 2 dysregulated miRNA-mRNA targets pairs (Fam46a/miR-363-3p and Rgs3/miR-363-3p) that might be related to the effect of propofol on long-term cognitive function. Collectively, our novel investigation indicates that acute and long-term dysregulated miRNA-mRNA signaling networks potentially participate in propofol-induced developmental neurotoxicity.

Propofol Induces Apoptosis of Neurons but Not Astrocytes, Oligodendrocytes, or Neural Stem Cells in the Neonatal Mouse Hippocampus.

It has been shown that propofol can induce widespread apoptosis in neonatal mouse brains followed by long-term cognitive dysfunction. However, selective brain area and cell vulnerability to propofol remains unknown. This study was aimed to dissect toxic effect of propofol on multiple brain cells, including neurons, astrocytes, oligodendrocytes, and neural stem cells (NSCs). Seven-day-old mice were intraperitoneally administrated propofol or intralipid as a vehicle control for 6 hours. To identify vulnerable cells undergoing apoptosis following propofol exposure, brain sagittal sections were co-stained with antibodies against an apoptosis marker along with neuron, astrocyte, oligodendrocyte, or NSC markers using immunofluorescence staining. The results showed widespread apoptosis in propofol-treated brains (apoptotic cells: 1.55 ± 0.04% and 0.06 ± 0.01% in propofol group and intralipid-treated control group, respectively). Apoptotic cell distribution exhibits region- and cell-specific patterns. Several brain regions (e.g., cerebral cortex and hippocampus) were more vulnerable to propofol than other brain regions. Most apoptotic cells in the hippocampus were located in the cornus ammonis 1 (CA1) subfield. These apoptotic cells were only detected in neurons and not astrocytes, oligodendrocytes, or NSCs. These data demonstrate that different brain regions, subfields, and different types of neuronal cells in mice exhibit various vulnerabilities to propofol. Understanding region- and cell-specific susceptibility to propofol will help to better understand cellular contribution to developmental neurotoxicity and further develop novel therapeutic targets.

Insufficient Astrocyte-Derived Brain-Derived Neurotrophic Factor Contributes to Propofol-Induced Neuron Death Through Akt/Glycogen Synthase Kinase 3ß/Mitochondrial Fission Pathway.

BACKGROUND: Growing animal evidence demonstrates that prolonged exposure to propofol during brain development induces widespread neuronal cell death, but there is little information on the role of astrocytes. Astrocytes can release neurotrophic growth factors such as brain-derived neurotrophic factor (BDNF), which can exert the protective effect on neurons in paracrine fashion. We hypothesize that during propofol anesthesia, BDNF released from developing astrocytes may not be sufficient to prevent propofol-induced neurotoxicity. METHODS: Hippocampal astrocytes and neurons isolated from neonatal Sprague Dawley rats were exposed to propofol at a clinically relevant dose of 30 µM or dimethyl sulfoxide as control for 6 hours. Propofol-induced cell death was determined by propidium iodide (PI) staining in astrocyte-alone cultures, neuron-alone cultures, or cocultures containing either low or high density of astrocytes (1:9 or 1:1 ratio of astrocytes to neurons ratio [ANR], respectively). The astrocyte-conditioned medium was collected 12 hours after propofol exposure and measured by protein array assay. BDNF concentration in astrocyte-conditioned medium was quantified using enzyme-linked immunosorbent assay. Neuron-alone cultures were treated with BDNF, tyrosine receptor kinase B inhibitor cyclotraxin-B, glycogen synthase kinase 3ß (GSK3ß) inhibitor CHIR99021, or mitochondrial fission inhibitor Mdivi-1 before propofol exposure. Western blot was performed for quantification of the level of protein kinase B and GSK3ß. Mitochondrial shape was visualized through translocase of the outer membrane 20 staining. RESULTS: Propofol increased cell death in neurons by 1.8-fold (% of PI-positive cells [PI%] = 18.6; 95% confidence interval [CI], 15.2-21.9, P < .05) but did not influence astrocyte viability. The neuronal death was attenuated by a high ANR (1:1 cocultures; fold change [FC] = 1.17, 95% CI, 0.96-1.38, P < .05), but not with a low ANR [1:9 cocultures; FC = 1.87, 95% CI, 1.48-2.26, P > .05]). Astrocytes secreted BDNF in a cell density-dependent way and propofol decreased BDNF secretion from astrocytes. Administration of BDNF, CHIR99021, or Mdivi-1 significantly attenuated the propofol-induced neuronal death and aberrant mitochondria in neuron-alone cultures (FC = 0.8, 95% CI, 0.62-0.98; FC = 1.22, 95% CI, 1.11-1.32; FC = 1.35, 95% CI, 1.16-1.54, respectively, P < .05) and the cocultures with a low ANR (1:9; FC = 0.85, 95% CI, 0.74-0.97; FC = 1.08, 95% CI, 0.84-1.32; FC = 1.25, 95% CI, 1.1-1.39, respectively, P < .05). Blocking BDNF receptor or protein kinase B activity abolished astrocyte-induced neuroprotection in the cocultures with a high ANR (1:1). CONCLUSIONS: Astrocytes attenuate propofol-induced neurotoxicity through BDNF-mediated cell survival pathway suggesting multiple neuroprotective strategies such as administration of BDNF, astrocyte-conditioned medium, decreasing mitochondrial fission, or inhibition of GSK3ß.

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.

MicroRNA-21 Mediates Isoflurane-induced Cardioprotection against Ischemia-Reperfusion Injury via Akt/Nitric Oxide Synthase/Mitochondrial Permeability Transition Pore Pathway.

BACKGROUND: The role of microRNA-21 in isoflurane-induced cardioprotection is unknown. The authors addressed this issue by using microRNA-21 knockout mice and explored the underlying mechanisms. METHODS: C57BL/6 and microRNA-21 knockout mice were echocardiographically examined. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in vivo or ex vivo in the presence or absence of 1.0 minimum alveolar concentration of isoflurane administered before ischemia. Cardiac Akt, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) proteins were determined by Western blot analysis. Opening of the mitochondrial permeability transition pore (mPTP) in cardiomyocytes was induced by photoexcitation-generated oxidative stress and detected by rapid dissipation of tetramethylrhodamine ethyl ester fluorescence using a confocal microscope. RESULTS: Genetic disruption of miR-21 gene did not alter phenotype of the left ventricle, baseline cardiac function, area at risk, and the ratios of phosphorylated-Akt/Akt, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS. Isoflurane decreased infarct size from 54 ± 10% in control to 36 ± 10% (P < 0.05, n = 8 mice per group), improved cardiac function after reperfusion, and increased the ratios of phosphorylated-Akt/AKT, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS in C57BL/6 mice subjected to ischemia-reperfusion injury. These beneficial effects of isoflurane were lost in microRNA-21 knockout mice. There were no significant differences in time of the mPTP opening induced by photoexcitation-generated oxidative stress in cardiomyocytes isolated between C57BL/6 and microRNA-21 knockout mice. Isoflurane significantly delayed mPTP opening in cardiomyocytes from C57BL/6 but not from microRNA-21 knockout mice. CONCLUSIONS: Isoflurane protects mouse hearts from ischemia-reperfusion injury by a microRNA-21-dependent mechanism. The Akt/NOS/mPTP pathway is involved in the microRNA-21-mediated protective effect of isoflurane.

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.

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death.

Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

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.