The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing.

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

p53 is required for etoposide-induced apoptosis of human embryonic stem cells.

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.

Overexpression of the chromosome 21 transcription factor Ets2 induces neuronal apoptosis.

Down syndrome (trisomy 21) neurons display an increased rate of apoptosis in vitro. The genes on chromosome 21 that mediate this increased cell death remain to be elucidated. Here we show that the chromosome 21 transcription factor Ets2, a gene that is overexpressed in Down syndrome, is expressed in neurons, and that moderate overexpression of Ets2 leads to increased apoptosis of primary neuronal cultures from Ets2 tg mice that involves activation of caspase-3. Our data therefore suggest that overexpression of ETS2 may contribute to the increased rate of apoptosis of neurons in Down syndrome.

ETS2 overexpression in transgenic models and in Down syndrome predisposes to apoptosis via the p53 pathway.

ETS2 is a transcription factor encoded by a gene on human chromosome 21 and alterations in its expression have been implicated in the pathophysiological features of Down syndrome (DS). This study demonstrates that overexpression of ETS2 results in apoptosis. This is shown in a number of circumstances, including ETS2-overexpressing transgenic mice and cell lines and in cells from subjects with DS. Indeed we report for the first time that the ETS2 overexpression transgenic mouse develops a smaller thymus and lymphocyte abnormalities similar to that observed in DS. In all circumstances of ETS2 overexpression, the increased apoptosis correlated with increased p53 and alterations in downstream factors in the p53 pathway. In the human HeLa cancer cell line, transfection with functional p53 enables ETS2 overexpression to induce apoptosis. Furthermore, crossing the ETS2 transgenic mice with p53(-/-) mice genetically rescued the thymic apoptosis phenotype. Therefore, we conclude that overexpression of human chromosome 21-encoded ETS2 induces apoptosis that is dependent on p53. These results have important consequences for understanding DS and oncogenesis and may provide new insights into therapeutic interventions.

Ets-2 is induced by oxidative stress and sensitizes cells to H(2)O(2)-induced apoptosis: implications for Down's syndrome.

An elevated production of hydrogen peroxide mediates the increased rate of apoptosis of cells derived from individuals with Down's syndrome. The mechanism via which this occurs is unknown. Here we show that Ets-2, a transcription factor located on human chromosome 21 and already overexpressed in multiple tissues in Down syndrome (DS, trisomy 21), is induced by low concentrations of hydrogen peroxide. Moreover, cells with an imbalance in the antioxidant enzymes SOD-1/GPX-1, such as occurs in DS through the overexpression of the chromosome 21 gene SOD-1, also results in increased Ets-2 expression. The increase in Ets-2 expression is dependent on mRNA transcription. Importantly, we further demonstrate that 3T3 fibroblasts that overexpress Ets-2 are sensitized to hydrogen peroxide-induced apoptosis. These data implicate Ets-2 in the regulation of oxidant-induced apoptosis and provide a possible rationale for both the (5- to 7-) fold increase in Ets-2 protein level in DS tissues, above the expected gene dosage of 1.5-fold, and the elevated rate of apoptosis in DS cells.

Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis?

Hydrogen peroxide has been shown to induce either apoptosis or features of senescence in different cultured cell lines. We now show that both processes can be induced in the same culture of primary human diploid fibroblasts and that the outcome of apoptosis or the senescence-like phenotype is determined by the H2O2 concentrations. At 50 and 100 microM, H2O2 predominantly induced the senescence-like state, characterized by a reduced rate of proliferation, an increased number of cells in G0-G1, typically enlarged and flattened morphology, and increased CIP1 and fibronectin expression. At 300 and 400 microM, H2O2 mainly triggered apoptosis. At the intermediate 200 microM H2O2, features of both senescence and apoptosis were observed in the same culture. Thus, the higher the H2O2 concentration, the higher the proportion of cells undergoing apoptosis, suggesting a key role of the level of damage in the choice of a cell population to enter apoptosis and/or the senescence-like state. Before the induction of one or the other process, cells entered a transient "shock state" characterized by a typical morphological change, cell cycle arrest in G0-G1, and the induction of CIP1 and BCL-2.

Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators.

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specific in vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADH:ferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADH:ferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group of groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.

Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin.

Activation of the plasma membrane NADH-oxidoreductase (PMOR) system by addition of growth factors or extracellular electron acceptors stimulates cellular proliferation. We now show that the vanilloids capsaicin, dihydrocapsaicin, and resiniferatoxin are inhibitors of the NADH-oxidase activity of the PMOR system and that both these and two previously identified PMOR inhibitors (chloroquine and retinoic acid) induce apoptosis in human B-cell and mouse myeloid cell lines. At the optimal concentration, PMOR inhibitors can induce between 50 and 70% of apoptosis in mouse myeloid and human B-cell lines within 8-12 h, provided these cell lines do not express Bcl-2. The immunosuppressants cyclosporin A and fujimycin (tacrolimus) inhibit PMOR inhibitor-induced apoptosis. By using combinations of these immunosuppressants and excess amounts of their nonimmunosuppressive analogues, we demonstrate that in human B-cell lines the Bcl-2-sensitive apoptotic pathway triggered by PMOR inhibitors involves signaling through the protein phosphatase calcineurin. We suggest that the PMOR system is a redox sensor that can, depending on the ambient redox environment and the availability of growth factors, regulate plasma membrane calcium fluxes and signal for apoptosis through calcineurin. Bcl-2, a protein that is thought to inhibit apoptosis by regulating reactive oxygen species and calcium fluxes in the cell, inhibits this apoptotic pathway.

Didemnin B induces cell death by apoptosis: the fastest induction of apoptosis ever described.

Didemnin B, a cyclic N-methylated peptolide induces apoptosis in human HL-60 cells. When incubated with 1 microM didemnin B, unsynchronized HL-60 cultures undergo apoptosis to 100% within 140 minutes. Apoptosis has been assessed by the typical apoptotic morphology, the presence of double-stranded DNA fragments within the cytosol and the generation of DNA ladders. None of these characteristics of apoptosis are seen when HL-60 cells are pretreated with 1mM Zn2+ immediately before treatment with didemnin B.

Mitochondrial respiratory chain inhibitors induce apoptosis.

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

Factors influencing the latency of the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in permeabilized human skin fibroblasts.

In selectively permeabilized fibroblasts suspended in a medium mimicking the composition of the cytosol the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) was found to exhibit about 80% latency (Wolvetang, E.J., Tager, J.M. and Wanders, R.J.A. (1990) Biochem. Biophys. Res. Commun. 1035, 6-11). We investigated which components of the cytosol mimicking medium are important for latency of DHAP-AT and unmasking of latent DHAP-AT activity by ATP. We show that the latency of DHAP-AT is critically dependent upon the presence of reduced glutathione in the medium and that the in vivo prevailing GSH/GSSG ratio is sufficient to maintain DHAP-AT latency. Although thiol-groups in the peroxisomal membrane seem to be essential for the integrity of peroxisomes in selectively permeabilized fibroblasts no latency of DHAP-AT is observed in buffered sucrose media or in cell homogenates, irrespective of the presence of GSH in the medium used. We suggest that during homogenization irreversible damage is inflicted upon the peroxisomal membrane and/or that more factors than at present investigated are involved in maintaining peroxisomal integrity. Furthermore, we demonstrate that cations play a role in the stimulatory effect of ATP on latent DHAP-AT activity while a proton gradient is not directly involved in the stimulatory effect of ATP on latent DHAP-AT activity.

Latency of the peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase in digitonin-permeabilized fibroblasts: the effect of ATP and ATPase inhibitors.

We have studied the activity of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAP-AT) in fibroblasts treated with low concentrations of digitonin so that the cytoplasmic compartment was freely accessible to the substrates of DHAP-AT while intracellular membranes remained intact. DHAP-AT activity exhibited 70% latency under these conditions. This latency could be overcome by addition of ATP, resulting in a four-fold stimulation of DHAP-AT activity. Virtually no stimulatory effect of ATP on DHAP-AT activity was observed in sonicated fibroblasts or when a non-hydrolyzable ATP analogue was used. Furthermore the stimulatory effect of ATP was prevented in part by DCCD. N-ethylmaleimide and high concentrations of oligomycin; bafilomycin had no effect. This pattern of inhibitor sensitivity is similar to that of the ATPase activity in peroxisomal fractions from rat liver. We conclude that peroxisomes in situ exhibit structure linked latency and that ATP is required for the transport of at least one of the substrates of DHAP-AT.

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase.

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the beta-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F1F0-ATPase and the lysosomal V-type ATPase.

3-Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism.

3-Methyladenine is generally used as an inhibitor of autophagy [P. O. Seglen & P. B. Gordon (1982) Proc. Natl Acad. Sci. USA 79, 1889-1892]. Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well. 1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by cAMP, which slightly increased in the presence of 3-methyladenine. 2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.

Energy dependence of autophagic protein degradation in isolated rat hepatocytes.

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.