Ethanol-induced ceramide production causes neuronal apoptosis by increasing MCL-1S-mediated ER-mitochondria contacts.

Heavy alcohol consumption causes neuronal cell death and cognitive impairment. Neuronal cell death induced by ethanol may result from increased production of the sphingolipid metabolite ceramide. However, the molecular mechanisms of neuronal cell death caused by ethanol-induced ceramide production have not been elucidated. Therefore, we investigated the mechanism through which ethanol-induced ceramide production causes neuronal cell apoptosis using human induced-pluripotent stem cell-derived neurons and SH-SY5Y cells and identified the effects of ceramide on memory deficits in C57BL/6 mice. First, we found that ethanol-induced ceramide production was decreased by inhibition of the de novo synthesis pathway, mediated by serine palmitoyltransferase (SPT). The associated alterations of the molecules related to the ceramide pathway suggest that the elevated level of ceramide activated protein phosphatase 1 (PP1), which inhibited the nuclear translocation of serine/arginine-rich splicing factor 1 (SRSF1). This led to aberrant splicing of myeloid cell leukemia 1 (MCL-1) pre-mRNA, which upregulated MCL-1S expression. Our results demonstrated that the interaction of MCL-1S with the inositol 1, 4, 5-trisphosphate receptor (IP3R) increases calcium release from the endoplasmic reticulum (ER) and then activated ER-bound inverted formin 2 (INF2). In addition, we discovered that F-actin polymerization through INF2 activation promoted ER-mitochondria contacts, which induced mitochondrial calcium influx and mitochondrial reactive oxygen species (mtROS) production. Markedly, MCL-1S silencing decreased mitochondria-associated ER membrane (MAM) formation and prevented mitochondrial calcium influx and mtROS accumulation, by inhibiting INF2-dependent actin polymerization interacting with mitochondria. Furthermore, the inhibition of ceramide production in ethanol-fed mice reduced MCL-1S expression, neuronal cell death, and cognitive impairment. In conclusion, we suggest that ethanol-induced ceramide production may lead to mitochondrial calcium overload through MCL-1S-mediated INF2 activation-dependent MAM formation, which promotes neuronal apoptosis.

Prenatal glucocorticoid exposure selectively impairs neuroligin 1-dependent neurogenesis by suppressing astrocytic FGF2-neuronal FGFR1 axis.

Exposure to maternal stress irreversibly impairs neurogenesis of offspring by inducing life-long effects on interaction between neurons and glia under raging differentiation process, culminating in cognitive and neuropsychiatric abnormalities in adulthood. We identified that prenatal exposure to stress-responsive hormone glucocorticoid impaired neurogenesis and induced abnormal behaviors in ICR mice. Then, we used human induced pluripotent stem cell (iPSC)-derived neural stem cell (NSC) to investigate how neurogenesis deficits occur. Following glucocorticoid treatment, NSC-derived astrocytes were found to be A1-like neurotoxic astrocytes. Moreover, cortisol-treated astrocytic conditioned media (ACM) then specifically downregulated AMPA receptor-mediated glutamatergic synaptic formation and transmission in differentiating neurons, by inhibiting localization of ionotropic glutamate receptor (GluR)1/2 into synapses. We then revealed that downregulated astrocytic fibroblast growth factor 2 (FGF2) and nuclear fibroblast growth factor receptor 1 (FGFR1) of neurons are key pathogenic factors for reducing glutamatergic synaptogenesis. We further confirmed that cortisol-treated ACM specifically decreased the binding of neuronal FGFR1 to the synaptogenic NLGN1 promoter, but this was reversed by FGFR1 restoration. Upregulation of neuroligin 1, which is important in scaffolding GluR1/2 into the postsynaptic compartment, eventually normalized glutamatergic synaptogenesis and subsequent neurogenesis. Moreover, pretreatment of FGF2 elevated neuroligin 1 expression and trafficking of GluR1/2 into the postsynaptic compartment of mice exposed to prenatal corticosterone, improving spatial memory and depression/anxiety-like behaviors. In conclusion, we identified neuroligin 1 restoration by astrocytic FGF2 and its downstream neuronal nuclear FGFR1 as a critical target for preventing prenatal stress-induced dysfunction in glutamatergic synaptogenesis, which recovered both neurogenesis and hippocampal-related behaviors.

Human embryonic stem cell-specific role of YAP in maintenance of self-renewal and survival.

Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs.

Cyanidin 3-O-arabinoside suppresses DHT-induced dermal papilla cell senescence by modulating p38-dependent ER-mitochondria contacts.

BACKGROUND: Androgenetic alopecia (AGA) is a genetic disorder caused by dihydrotestosterone (DHT), accompanied by the senescence of androgen-sensitive dermal papilla cells (DPCs) located in the base of hair follicles. DHT causes DPC senescence in AGA through mitochondrial dysfunction. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the protective role of cyanidins on DHT-induced mitochondrial dysfunction and DPC senescence and the regulatory mechanism involved. METHODS: DPCs were used to investigate the effect of DHT on mitochondrial dysfunction with MitoSOX and Rhod-2 staining. Senescence-associated ß-galactosidase activity assay was performed to examine the involvement of membrane AR-mediated signaling in DHT-induced DPC senescence. AGA mice model was used to study the cyanidins on DHT-induced hair growth deceleration. RESULTS: Cyanidin 3-O-arabinoside (C3A) effectively decreased DHT-induced mtROS accumulation in DPCs, and C3A reversed the DHT-induced DPC senescence. Excessive mitochondrial calcium accumulation was blocked by C3A. C3A inhibited p38-mediated voltage-dependent anion channel 1 (VDAC1) expression that contributes to mitochondria-associated ER membrane (MAM) formation and transfer of calcium via VDAC1-IP3R1 interactions. DHT-induced MAM formation resulted in increase of DPC senescence. In AGA mice models, C3A restored DHT-induced hair growth deceleration, which activated hair follicle stem cell proliferation. CONCLUSIONS: C3A is a promising natural compound for AGA treatments against DHT-induced DPC senescence through reduction of MAM formation and mitochondrial dysfunction.

Glucocorticoid impairs mitochondrial quality control in neurons.

Neurons are particularly vulnerable to mitochondrial dysfunction due to high energy demand and an inability to proliferate. Therefore, dysfunctional mitochondria cause various neuropathologies. Mitochondrial damage induces maintenance pathways to repair or eliminate damaged organelles. This mitochondrial quality control (MQC) system maintains appropriate morphology, localization, and removal/replacement of mitochondria to sustain brain homeostasis and counter progression of neurological disorders. Glucocorticoid release is an essential response to stressors for adaptation; however, it often culminates in maladaptation if neurons are exposed to chronic and severe stress. Long-term exposure to high levels of glucocorticoids induces mitochondrial dysfunction via genomic and nongenomic mechanisms. Glucocorticoids induce abnormal mitochondrial morphology and dysregulate fusion and fission. Moreover, mitochondrial trafficking is arrested by glucocorticoids and dysfunctional mitochondria are subsequently accumulated around the soma. These alterations lead to energy deficiency, particularly for synaptic transmission that requires large amounts of energy. Glucocorticoids also impair mitochondrial clearance by preventing mitophagy of damaged organelle and suppress mitochondrial biogenesis, resulting in the reduced number of healthy mitochondria. Failure to maintain MQC degrades brain function and contributes to neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, mechanisms of glucocorticoid action on the regulation of MQC during chronic stress conditions are not well understood. The present review discusses pathways involved in the impairment of MQC and the clinical significance of high glucocorticoid blood levels for neurodegenerative diseases.

A simple metastatic brain cancer model using human embryonic stem cell-derived cerebral organoids.

Every year, hundreds of thousands of people die because of metastatic brain cancer. Most metastatic cancer research uses 2D cell culture or animal models, but they have a few limitations, such as difficulty reproducing human tissue structures. This study developed a simple 3D in vitro model to better replicate brain metastasis using human cancer cells and human embryonic stem cell-derived cerebral organoids (metastatic brain cancer cerebral organoid [MBCCO]). The MBCCO model successfully reproduced metastatic cancer processes, including cell adhesion, proliferation, and migration, in addition to cell-cell interactions. Using the MBCCO model, we demonstrated that lung-specific X protein (LUNX) plays an important role in cell proliferation and migration or invasion. We also observed astrocyte accumulation around and their interaction with cancer cells through connexin 43 in the MBCCO model. We analyzed whether the MBCCO model can be used to screen drugs by measuring the effects of gefitinib, a well-known anticancer agent. We also examined the toxicity of gefitinib using normal cerebral organoids (COs). Therefore, the MBCCO model is a powerful tool for modeling human metastatic brain cancer in vitro and can also be used to screen drugs.

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with Gαq.

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.

Ethanol-activated CaMKII signaling induces neuronal apoptosis through Drp1-mediated excessive mitochondrial fission and JNK1-dependent NLRP3 inflammasome activation.

BACKGROUND: Neurodegeneration is a representative phenotype of patients with chronic alcoholism. Ethanol-induced calcium overload causes NOD-like receptor protein 3 (NLRP3) inflammasome formation and an imbalance in mitochondrial dynamics, closely associated with the pathogenesis of neurodegeneration. However, how calcium regulates this process in neuronal cells is poorly understood. Therefore, the present study investigated the detailed mechanism of calcium-regulated mitochondrial dynamics and NLRP3 inflammasome formation in neuronal cells by ethanol. METHODS: In this study, we used the SK-N-MC human neuroblastoma cell line. To confirm the expression level of the mRNA and protein, real time quantitative PCR and western blot were performed. Co-immunoprecipitation and Immunofluorescence staining were conducted to confirm the complex formation or interaction of the proteins. Flow cytometry was used to analyze intracellular calcium, mitochondrial dysfunction and neuronal apoptosis. RESULTS: Ethanol increased cleaved caspase-3 levels and mitochondrial reactive oxygen species (ROS) generation associated with neuronal apoptosis. In addition, ethanol increased protein kinase A (PKA) activation and cAMP-response-element-binding protein (CREB) phosphorylation, which increased N-methyl-D-aspartate receptor (NMDAR) expression. Ethanol-increased NMDAR induced intracellular calcium overload and calmodulin-dependent protein kinase II (CaMKII) activation leading to phosphorylation of dynamin-related protein 1 (Drp1) and c-Jun N-terminal protein kinase 1 (JNK1). Drp1 phosphorylation promoted Drp1 translocation to the mitochondria, resulting in excessive mitochondrial fission, mitochondrial ROS accumulation, and loss of mitochondrial membrane potential, which was recovered by Drp1 inhibitor pretreatment. Ethanol-induced JNK1 phosphorylation activated the NLRP3 inflammasome that induced caspase-1 dependent mitophagy inhibition, thereby exacerbating ROS accumulation and causing cell death. Suppressing caspase-1 induced mitophagy and reversed the ethanol-induced apoptosis in neuronal cells. CONCLUSIONS: Our results demonstrated that ethanol upregulated NMDAR-dependent CaMKII phosphorylation which is essential for Drp1-mediated excessive mitochondrial fission and the JNK1-induced NLRP3 inflammasome activation resulting in neuronal apoptosis. Video abstract.

Role of Microtubule-Associated Factors in HIF1α Nuclear Translocation.

Adaptation to hypoxia is essential for regulating the survival and functions of hypoxic cells; it is mainly mediated by the hypoxia-inducible factor 1 (HIF1). The alpha subunit of HIF1 (HIF1α) is a well-known regulatory component of HIF1, which is tightly controlled by various types of HIF1α-regulating processes. Previous research has shown that microtubule-regulated HIF1α nuclear translocation is a key factor for HIF1 activation under hypoxia. In this review, we summarize experimental reports on the role of microtubule-associated factors, such as microtubule, dynein, and dynein adaptor protein, in nuclear translocation of HIF1α. Based upon scientific evidence, we propose a bicaudal D homolog (BICD) as a novel HIF1α translocation regulating factor. A deeper understanding of the mechanism of the action of regulatory factors in controlling HIF1α nuclear translocation will provide novel insights into cell biology under hypoxia.

Astrocyte D-serine modulates the activation of neuronal NOS leading to the development of mechanical allodynia in peripheral neuropathy.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0-3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

Trolox-induced cardiac differentiation is mediated by the inhibition of Wnt/ß-catenin signaling in human embryonic stem cells.

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/ß-catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/ß-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/ß-catenin regulator.

Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aß Generation: Involvement of Lipid Raft-Mediated CREB Activation.

Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid ß (Aß) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aß production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aß showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment ß of amyloid precursor protein (C99) and Aß production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aß production induced by corticosterone treatment or intracerebroventricular injection of Aß more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aß generation.SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid due to intracellular glucocorticoid receptor deficiency. In this respect, the genomic mechanism of glucocorticoid faces difficulties in explaining the consistent amyloid ß (Aß) production. Therefore, it is necessary to investigate the novel pathway of glucocorticoid on Aß generation to find a more selective therapeutic approach to AD patients. In this study, we revealed the importance of nongenomic pathway induced by glucocorticoid where membrane glucocorticoid receptor plays an important role in Aß formation.

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach.

The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.

High Glucose-Induced Reactive Oxygen Species Stimulates Human Mesenchymal Stem Cell Migration Through Snail and EZH2-Dependent E-Cadherin Repression.

BACKGROUND/AIMS: Glucose plays an important role in stem cell fate determination and behaviors. However, it is still not known how glucose contributes to the precise molecular mechanisms responsible for stem cell migration. Thus, we investigate the effect of glucose on the regulation of the human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) migration, and analyze the mechanism accompanied by this effect. METHODS: Western blot analysis, wound healing migration assays, immunoprecipitation, and chromatin immunoprecipitation assay were performed to investigate the effect of high glucose on hUCB-MSC migration. Additionally, hUCB-MSC transplantation was performed in the mouse excisional wound splinting model. RESULTS: High concentration glucose (25 mM) elicits hUCB-MSC migration compared to normal glucose and high glucose-pretreated hUCB-MSC transplantation into the wound sites in mice also accelerates skin wound repair. We therefore elucidated the detailed mechanisms how high glucose induces hUCB-MSC migration. We showed that high glucose regulates E-cadherin repression through increased Snail and EZH2 expressions. And, we found high glucose-induced reactive oxygen species (ROS) promotes two signaling; JNK which regulates γ-secretase leading to the cleavage of Notch proteins and PI3K/Akt signaling which enhances GSK-3ß phosphorylation. High glucose-mediated JNK/Notch pathway regulates the expression of EZH2, and PI3K/Akt/GSK-3ß pathway stimulates Snail stabilization, respectively. High glucose enhances the formation of EZH2/Snail/HDAC1 complex in the nucleus, which in turn causes E-cadherin repression. CONCLUSION: This study reveals that high glucose-induced ROS stimulates the migration of hUCB-MSC through E-cadherin repression via Snail and EZH2 signaling pathways.

Avian blastoderm dormancy arrests cells in G2 and suppresses apoptosis.

In most avian species, the early embryo suspends development when the ambient temperature is too low; the resultant dormant state is called cold torpor. However, very little is known about dormant avian embryos at the cellular level. To investigate the molecular processes that occur in the chicken blastoderm during cold torpor, we performed transcriptome analysis and investigated cellular responses in dormant embryos. In embryos stored at low temperature, we observed up-regulation of genes and proteins related to endoplasmic reticulum stress and stress-activated protein kinase signaling. In addition, the proportion of early apoptotic cells rose dramatically during torpor, whereas the proportion of late apoptotic cells was unchanged. Cell cycle analysis revealed that mitotic arrest occurred at the G2 phase in a DNA damage-independent manner and that the arrest was alleviated after incubation at 37°C. Our data suggest that the dormant avian embryo tolerates cold stress by inducing stress-tolerance pathways, inhibiting late apoptosis, and triggering cell cycle arrest at the G2 phase.-Ko, M. H., Hwang, Y. S., Rim, J. S., Han, H. J., Han, J. Y. Avian blastoderm dormancy arrests cells in G2 and suppresses apoptosis.

Aß-Induced Drp1 phosphorylation through Akt activation promotes excessive mitochondrial fission leading to neuronal apoptosis.

Mitochondrial dysfunction is known as one of causative factors in Alzheimer's disease (AD), inducing neuronal cell death. Mitochondria regulate their functions through changing their morphology. The present work was undertaken to investigate whether Amyloid ß (Aß) affects mitochondrial morphology in neuronal cells to induce apoptosis. Aß treatment induced not only the fragmentation of mitochondria but also neuronal apoptosis in association with an increase in caspase-9 and -3 activity. Calcium influx induced by Aß up-regulated the activation of Akt through CaMKII resulting in changes to the phosphorylation level of Drp1 in a time-dependent manner. Translocation of Drp1 from the cytosol to mitochondria was blocked by CB-124005 (an Akt inhibitor). Recruitment of Drp1 to mitochondria led to ROS generation and mitochondrial fission, accompanied by dysfunction of mitochondria such as loss of membrane potential and ATP production. ROS generation and mitochondrial dysfunction by Aß were attenuated when treated with Mdivi-1, a selective Drp1 inhibitor. Furthermore, the sustained Akt activation induced not only the fragmentation of mitochondria but also the activation of mTOR, eventually suppressing autophagy. Inhibition of autophagic clearance of Aß led to increased ROS levels and aggravating mitochondrial defects, which were blocked by Rapamycin (an mTOR inhibitor). In conclusion, sustained phosphorylation of Akt by Aß directly activates Drp1 and inhibits autophagy through the mTOR pathway. Together, these changes elicit abundant mitochondrial fragmentation resulting in ROS-mediated neuronal apoptosis.

Purification of small molecule-induced cardiomyocytes from human induced pluripotent stem cells using a reporter system.

In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/ß-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research.

Ethanol-induced PGE2 up-regulates Aß production through PKA/CREB signaling pathway.

Ethanol abuse aggravates dementia-associated cognitive defects through the progression of Alzheimer's disease (AD) pathophysiology. Beta-site APP-cleaving enzyme 1 (BACE1) has been considered as a key regulator of AD pathogenesis by controlling amyloid beta peptide (Aß) accumulation. In addition, previous studies reported that endoplasmic reticulum (ER) stress and neuroinflammation have been proposed in ethanol-induced neurodegeneration. Thus, we investigated the role of ER stress and PGE2, a neuroinflammation mediator, in the ethanol-stimulated BACE1 expression and Aß production. Using the human-derived neuroblastoma cell line SK-N-MC, the results show that ethanol up-regulated BACE1 expression in a dose-dependent manner. Ethanol stimulated reactive oxygen species (ROS) production, which induced CHOP expression and eIF2α phosphorylation. PBA (an ER stress inhibitor) attenuated the ethanol-increased cyclooxygenase-2 (COX-2) expression and PGE2 production. By using salubrinal (an eIF2α dephosphorylation inhibitor) or EIF2A siRNA, we found that eIF2α phosphorylation mediated the ethanol-induced COX-2 expression. In addition, COX-2-induced BACE1 up-regulation was abolished by NS-398 (a selective COX-2 inhibitor). And, PF-04418948 (an EP-2 receptor inhibitor) pretreatment reduced ethanol-induced PKA activation and CREB phosphorylation as well as ethanol-stimulated Aß production. Furthermore, 14-22 amide (a PKA inhibitor) pretreatment or CREB1 siRNA transfection suppressed the ethanol-induced BACE1 expression. In conclusion, ethanol-induced eIF2α phosphorylation stimulates COX-2 expression and PGE2 production which induces the BACE1 expression and Aß production via EP-2 receptor-dependent PKA/CREB pathway.