Formation and evolution of carbonaceous asteroid Ryugu: Direct evidence from returned samples.

Samples of the carbonaceous asteroid Ryugu were brought to Earth by the Hayabusa2 spacecraft. We analyzed 17 Ryugu samples measuring 1 to 8 millimeters. Carbon dioxide-bearing water inclusions are present within a pyrrhotite crystal, indicating that Ryugu's parent asteroid formed in the outer Solar System. The samples contain low abundances of materials that formed at high temperatures, such as chondrules and calcium- and aluminum-rich inclusions. The samples are rich in phyllosilicates and carbonates, which formed through aqueous alteration reactions at low temperature, high pH, and water/rock ratios of <1 (by mass). Less altered fragments contain olivine, pyroxene, amorphous silicates, calcite, and phosphide. Numerical simulations, based on the mineralogical and physical properties of the samples, indicate that Ryugu's parent body formed ~2 million years after the beginning of Solar System formation.

Cytokine-induced nuclear factor kappa B activation promotes the survival of developing neurons.

Ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and interleukin 6 (IL-6) comprise a group of structurally related cytokines that promote the survival of subsets of neurons in the developing peripheral nervous system, but the signaling pathways activated by these cytokines that prevent neuronal apoptosis are unclear. Here, we show that these cytokines activate NF-kappaB in cytokine-dependent developing sensory neurons. Preventing NF-kappaB activation with a super-repressor IkappaB-alpha protein markedly reduces the number of neurons that survive in the presence of cytokines, but has no effect on the survival response of the same neurons to brain-derived neurotrophic factors (BDNF), an unrelated neurotrophic factor that binds to a different class of receptors. Cytokine-dependent sensory neurons cultured from embryos that lack p65, a transcriptionally active subunit of NF-kappaB, have a markedly impaired ability to survive in response to cytokines, but respond normally to BDNF. There is increased apoptosis of cytokine- dependent neurons in p65(-/)- embryos in vivo, resulting in a reduction in the total number of these neurons compared with their numbers in wild-type embryos. These results demonstrate that NF-kappaB plays a key role in mediating the survival response of developing neurons to cytokines.

GFR alpha-4 and the tyrosine kinase Ret form a functional receptor complex for persephin.

Glial-cell-line-derived neurotrophic factor (GDNF), neurturin and persephin are structurally related, secreted proteins that are widely expressed in the nervous system and other tissues and promote the survival of a variety of neurons during development. GDNF and neurturin signal through multicomponent receptors that consist of the Ret receptor tyrosine kinase and one of two structurally related glycosyl-phosphatidylinositol (GPI)-linked ligand-binding subunits: GFR alpha-1 is the preferred ligand-binding subunit for GDNF, and GFR alpha-2 is the preferred ligand-binding subunit for neurturin. Two additional members of the GFR alpha family of GPI-linked proteins have recently been cloned: GFR alpha-3 and GFR alpha-4. We have shown that persephin binds efficiently only to GFR alpha-4, and labelled persephin is effectively displaced from cells expressing GFR alpha-4 by persephin but not by GDNF or neurturin. Using microinjection to introduce expression plasmids into cultured neurons, we have also shown that coexpression of Ret with GFR alpha-4, confers a marked survival response to persephin but not to GDNF or neurturin. These results demonstrate that GFR alpha-4 is the ligand-binding subunit for persephin and that persephin, like GDNF and neurturin, also requires Ret for signalling.

Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells.

The compound 13-L-hydroperoxylinoleic acid (LOOH) triggered the death of clonal rat pheochromocytoma PC12h cells (LD50 = about 8 microM). LOOH induced nuclear condensation and DNA fragmentation, which was prevented by cycloheximide (a protein synthesis inhibitor) and NGF, indicating that LOOH triggered apoptosis in PC12h cells. LOOH produced reactive oxygen species (ROS) in PC12h cells in a time- and dose-dependent manner, as measured by flow cytometry using the ROS-specific fluorescent indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Antioxidants such as N,N'-diphenyl-p-phenylenediamine (DPPD), vitamin E and N-acetylcysteine, and a ferric iron chelator, deferoxamine, inhibited the LOOH-triggered apoptosis and simultaneously decreased the generation of ROS, whereas an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO), enhanced the apoptosis and increased the generation of ROS. These results indicate that LOOH triggers the apoptosis of PC12h cells by increasing the production of ROS. A confocal analysis with the Ca(2+)-specific fluorescent indicator, fluo-3, demonstrated that LOOH at concentrations up to 200 microM, did not increase the intracellular Ca2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhanced production of ROS, not through increasing the permeability of Ca2+.

In vitro model of hypoxia: basic fibroblast growth factor can rescue cultured CNS neurons from oxygen-deprived cell death.

We established an in vitro hypoxia model and investigated the protective effect of basic fibroblast growth factor (bFGF) against neuronal cell death caused by hypoxia. Hippocampal neurons obtained from rats on embryonic day (E) 17 and 20 and on postnatal day (P) 4 were cultured for 6-24 h in an oxygen-deprived state. This in vitro hypoxia study showed that the cultured neurons were sensitive to the oxygen deprivation. The cultured P4 rat hippocampal neurons seemed to be weaker in the hypoxia condition than those of E17 and E20 rats, suggesting that the cultured postnatal cells might be sensitive to hypoxia. bFGF, but not nerve growth factor, prevented the neuronal cell death caused by hypoxia in a dose-dependent manner.

Apoptotic cell death occurs in hippocampal neurons cultured in a high oxygen atmosphere.

When embryonic rat hippocampal neurons were cultured in a 50% oxygen atmosphere, neurons gradually died after 20 h in culture. This death pattern was found to be mediated by an intracellular active death program, so called apoptosis, as follows: (1) Cycloheximide and actinomycin-D, protein and RNA synthesis inhibitors, respectively, prevented cell death, indicating that cell death required new protein biosynthesis. (2) DNA fragmentation (called a "DNA ladder"), a specific biochemical marker of apoptosis, was detected during the course of cell death. (3) Depolarization with high K+ medium (26-50 mM) prevented cell death. This effect was suppressed by some dihydropyridine derivatives, L-type Ca channel blockers, such as nifedipine and nicardipine. These results indicate that increased levels of oxygen activate an apoptotic mechanism in the cultured hippocampal neurons, and suggest that neuronal activity may protect the neurons from oxygen-induced apoptosis.

Free radical-independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide-triggered apoptosis.

To investigate the mechanism of oxidative stress induced death of PC12 cells, we performed confocal and flow cytometric analysis with a reactive oxygen species (ROS)-specific fluorogen, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Hydrogen peroxide significantly decreased the number of viable PC12 cells after 24 h. Hydrogen peroxide caused membrane blebbing, nuclear condensation and DNA fragmentation, indicating that the PC12 cells died due to apoptosis. The hydrogen peroxide-triggered apoptosis of PC12 cells was associated with enhanced ROS production in a dose-dependent manner by measuring with C-DCDHF-DA. Nerve growth factor (NGF) and Bcl-2 inhibited the hydrogen peroxide-induced apoptosis of PC12 cells. Neither of them, however, reduced the ROS production in PC12 cells. These data suggest that NGF or Bcl-2 protects PC12 cells from hydrogen peroxide-triggered apoptosis independently from ROS production.

High oxygen atmosphere for neuronal cell culture with nerve growth factor. II. Survival and growth of clonal rat pheochromocytoma PC12h cells.

When clonal rat pheochromocytoma PC12h cells were cultured in a 50% O2 atmosphere, cells gradually died during the cultivation. On the other hand, the addition of NGF at the final concentration of 50 ng/ml could rescue the cells from death. The culture in a 40% O2 atmosphere had little effect on the growth of PC12h cells, as compared with the culture in a normal 20% O2 condition. A very high O2 concentration, as 60%, caused severe damage to PC12h cell growth, and the restoration of cell growth by NGF seemed to be insufficient. PC12h cells were fully differentiated and extended dense long neurites by NGF even in a 50% O2 atmosphere. However, the neurite extension in the culture in a 60% O2 atmosphere was suppressed. The cell-saving effect of NGF on cell death in culture under a 50% O2 atmosphere was dose-dependent, and the ED50 value of NGF was 5 ng/ml. Basic fibroblast growth factor and epidermal growth factor also had a potent effect to rescue the cell death in the high O2 culture, but insulin had no effect. Since the differentiation effects of NGF on PC12h cells are thought to offer a model system to investigate the effect of NGF on neurons, the present observations suggest that a protection machinery for high O2 toxicity to neurons may exist in the neuronal differentiated PC12h cells by NGF, but not in the undifferentiated cells.

Involvement of c-Jun N-terminal kinase and caspase 3-like protease in DNA damage-induced, p53-mediated apoptosis of cultured mouse cerebellar granule neurons.

In the previous studies, we have demonstrated that the tumor suppressor gene p53 is required for DNA strand break-induced neuronal apoptosis in organotypic slice cultures of cerebellum as well as in dissociated cerebellar neuron cultures. In this study, we further investigated the role of p53 in neuronal apoptosis, by examining whether caspases and c-Jun N-terminal kinase (JNK) are involved in the DNA strand break-induced apoptosis. The protein level of phospho-JNK increased in p53 wild-type mouse cerebellar granule neurons after exposure to bleomycin. On the other hand, the response was not observed in cerebellar granule neurons of p53-deficient mice. Caspase-3-like protease was activated and poly(ADP-ribose) polymerase (PARP) was cleaved in the bleomycin-induced apoptosis. Caspase-3-like protease inhibitor decreased the number of TUNEL-positive but not p53- or c-Jun-positive neurons in bleomycin-induced death. These results suggest that JNK and caspase-3-like protease are involved in the signaling cascade of DNA strand break-induced, p53-dependent apoptosis.

Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells.

To investigate the involvement of reactive oxygen species (ROS) in neuronal apoptosis, we performed confocal and flow cytometric analysis with a ROS-specific fluorogen, 6-carboxy-2', 7'-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Serum deprivation significantly increased the level of ROS in PC12 cells and rat cortical neurons. N,N'-diphenyl-p-phenylenediamine (DPPD), an antioxidant, reduced ROS production induced by serum deprivation and recovered cell survival. However, some survival factors such as nerve growth factor and Bcl-2, which prevented the apoptosis of PC12 cells, did not affect the up-regulation of ROS induced by serum deprivation. Epidermal growth factor which prevented the apoptosis of cortical neurons, did not affect the increase of ROS. These data suggest that survival factors rescue the serum deprivation induced apoptosis independently of ROS production.

Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture.

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats.

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.

Generation of reactive oxygen species, release of L-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture.

Oxygen-induced cell death in embryonic neurons is a useful in vitro model of neuronal apoptosis to study the molecular mechanisms underlying the cell death induced by oxidative stress. In the present study, we examined the involvement of reactive oxygen species and glutamate in the high (50%) oxygen-induced death of cultured hippocampal neurons. During the course of cell death, increases in O2- and hydrogen peroxide (H2O2) levels were observed. On the other hand, superoxide dismutase (SOD), catalase and deferoxamine (DFX), which have inhibitory effects on the generation of O2-, H2O2 and hydroxyl radicals, respectively, protected the neurons. These results suggested that both O2- and H2O2 play important roles in this apoptosis. Antagonists of NMDA and AMPA/kinate (AMPA/KA) receptors and an inhibitor of glutamate release partially prevented the apoptosis, suggesting that exposure to high oxygen enhances glutamate release, which results in activation of NMDA receptor and AMPA/KA receptor. In addition, specific nitric oxide (NO) scavenger and NO synthetase inhibitors blocked the apoptosis, indicating that NO and/or peroxynitrite are involved in this mechanism of cell death. Caspase inhibitors also blocked the neuronal apoptosis. These results suggested that multiple effectors including generation of reactive oxygen species, release of L-glutamate and activation of caspases are activated during the death induced by high oxygen.

Oxygen-induced apoptosis in PC12 cells with special reference to the role of Bcl-2.

We previously reported that PC12h cells are killed by a high oxygen atmosphere. In this study, we further characterized this oxygen-induced cell death and found apoptotic features, as follows. Firstly, chromatin condensation was observed in cells cultured in a 50% O2 atmosphere. Secondly, cycloheximide and cordycepin, protein and RNA synthesis inhibitors, respectively, prevented the oxygen-induced cell death in PC12h cells, suggesting that it is mediated by an intracellular death program. Thirdly, NGF, CPT-cAMP and depolarization by high potassium medium also effectively inhibited this apoptotic cell death in PC12h cells. The effect of high K+ is thought to be mediated by the influx of Ca2+ into cells through voltage-dependent Ca2+ channels, because nifedipine, an L-type Ca2+ channel blocker, inhibited the effect of high K+. In addition, since the oxygen-induced apoptosis was blocked by the antioxidant vitamin E, this oxygen toxicity is suggested to be mediated by reactive oxygen species. To further characterize this oxygen-induced apoptosis at the molecular level, we used PC12 cells overexpressing the proto-oncogene bcl-2. Although a large number of PC12 cells transfected with the control vector died in a 50% O2 atmosphere within 6 days, bcl-2-transfected PC12 cells survived and proliferated. These findings suggested that our system using PC12 cells will be a useful model with which to analyze the molecular mechanisms of apoptosis induced by oxidative stress in neuronal cells.

Changes in c-Jun but not Bcl-2 family proteins in p53-dependent apoptosis of mouse cerebellar granule neurons induced by DNA damaging agent bleomycin.

Tumor suppressor gene p53 is a critical regulator of the cellular response to DNA damage. To examine the function of p53 in postmitotic CNS neurons, we cultured cerebellar granule cells from 15-day-old wild type and p53-deficient mice, and analyzed changes of protein expression in apoptosis elicited by DNA damage. When cerebellar granule cells from wild type mice were treated with bleomycin, a DNA strand-break inducing agent, neuronal death occurred. In contrast, cells from p53-deficient mice were resistant to bleomycin-induced neuronal death. Furthermore, cells from p53 heterozygous mice showed an intermediate resistance between wild type and p53-deficient mice. These results show that p53 is required for the bleomycin-induced cerebellar granule cell death. To examine which proteins are involved in this apoptosis, we examined changes in protein levels of the Bcl-2 family, including Bcl-2, Bcl-X and Bax. The relative amounts of these proteins did not change after bleomycin treatment, suggesting that the changes in the levels of these Bcl-2 family proteins are not necessary for apoptosis in this system. In contrast, the levels of c-Jun protein significantly increased 6 h after treatment with bleomycin in wild type but not in p53-deficient cerebellar granule cells. These results raise the possibility that c-Jun is required for p53-dependent neuronal apoptosis induced by bleomycin.

Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture.

Cerebellar granule neurons obtained from 9-day-old rats were grown in vitro for 4 days in high K+ (26 mM) medium. The culture medium was then replaced with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determine whether the granule neurons respond directly to BDNF, we analyzed the induction of the Fos protein in these neurons. Individual cells that synthesize Fos protein after exposure to neurotrophin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF, but few of them were immunoreactive in the absence of BDNF or in the presence of NGF. Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+.

Expression of cyclin A decreases during neuronal apoptosis in cultured rat cerebellar granule neurons.

Cultured cerebellar granule neurons died in an apoptotic manner when the K+ concentration in culture medium was lowered to the normal level (5 mM) after maturation of cells with a high concentration of K+ (26 mM). The changes in expression of 14 cell cycle-related genes in this CNS apoptosis model were analyzed by quantitative RT-PCR. Most of the genes analyzed were stable during apoptosis. The expression of cyclin A mRNA, however, transiently decreased 1 h after the induction of apoptosis, and recovered within 3 h to above the basal level. In this system, the level of cyclin D1, which has been reported to be up-regulated in apoptosis of NGF-deprived cultured sympathetic neurons, did not change. These results suggest that the molecular mechanisms in these two apoptosis models are different. To determine cyclin A protein level, we used an immunostaining method. The number of cyclin A-positive neurons decreased during apoptosis. Moreover, the numbers of MAP2- and cdk2-positive neurons also decreased in a similar manner. Taken together, these results suggest that there is a relationship between apoptosis and cell cycle, and that morphological changes during apoptosis result from cytoskeletal structure degradation.

Signaling pathways and survival effects of BDNF and NT-3 on cultured cerebellar granule cells.

We investigated the signaling pathways exerted by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in relation to their survival-promoting effects on dissociated cultures of cerebellar granule cells prepared from postnatal 9-day-old rats. Granule neuron survival in culture was supported by BDNF, but not significantly by either nerve growth factor (NGF) or NT-3. BDNF and NT-3 resulted in not only the respective autophosphorylation of the Trk receptors, TrkB or TrkC, but also tyrosine phosphorylation of SHC, a protein involved in controlling p21ras activity, and phosphatidylinositol-3' (PI-3') kinase. NGF does not result in TrkA phosphorylation. In parallel, c-fos was induced within 30 min, in response to BDNF and NT-3. NT-3 induced the phosphorylation of these proteins to a lesser extent than BDNF. BDNF also induced the tyrosine phosphorylation of phospholipase C gamma (PLC gamma), but the NT-3-induced one was not detected. We postulate that no survival promotion by NT-3 is due to lesser level of trkC expression and of the NT-3-induced signaling in the cultured cerebellar granule neurons. Wortmannin, a specific inhibitor of PI-3' inhibited the BDNF effect on neuronal survival. PI-3' kinase-dependent pathways might be involved in the promotion of cerebellar granule cell survival by BDNF.

Involvement of phosphatidylinositol-3 kinase in prevention of low K(+)-induced apoptosis of cerebellar granule neurons.

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.

The tumor suppressor p53 gene plays a key role in controlling the cell cycle checkpoint and in apoptosis following the exposure of normal cells to DNA damage. To investigate the role of p53 in cytosine arabinoside (Ara C)-induced cell death of CNS neurons, we examined the effect of Ara C on the survival of cultured cerebellar granule neurons from normal wild-type and p53 null mutant mice. When the neurons from wild-type mice were cultured with Ara C, they gradually died after 24 h in culture. In contrast, the neurons from p53 null mutant mice showed a resistance to the Ara C neurotoxicity. These results indicate that p53 involves Ara C-induced apoptosis in cultured cerebellar granule neurons, in which DNA damage may initiate the apoptotic death program of the neurons.