Different types of cell death are involved in brain damage of methylmalonic aciduria and glutaric aciduria type I

We previously showed that exposure of 3D organotypic rat brain cell cultures to 1mM 2-methylcitrate (2-MCA) or 3-hydroxyglutarate (3- OHGA) every 12h over three days (DIV11-DIV14) results in ammonium accumulation and cell death. The aim of this study was to define the time course (every 24h) of the observed effects. Ammonium in culture medium already increased at DIV12 staying stable on the following days under 3-OHGA exposure, while it increased consecutively up to much higher levels under 2-MCA exposure. Lactate increase and glucose decrease were observed from DIV13 and DIV14, respectively. We conclude that ammonium accumulation precedes alterations of energy metabolism. As observed by immunohistochemistry glial cells were the predominant dying cells. Immunoblotting and immunohistochemistry with cell death specific markers (caspase-3, alpha-fodrin, LC3) showed that 2-MCA exposure significantly increased apoptosis on DIV14, but did not alter autophagy or necrosis. In contrast, 3-OHGA exposure substantially increased necrosis already from DIV13, while no change was observed for apoptosis and autophagy. In conclusion, ammonium accumulation, secondary disturbance of energy metabolism and glial cell death are involved in the neuropathogenesis ofmethylmalonic aciduria and glutaric aciduria type I. Interestingly, brain cells are dying by necrosis under 3-OHGA exposure and by apoptosis under 2-MCA exposure.

La programmmation foetale de la dysfonction vasculaire pulmonaire chez la souris : rôle des mécanismes épigénétiques = Fetal programming of pulmonary vascular dysfunction in mice : role of epigenetic mechanisms

Rapport de synthèseDes événements pathologiques survenant pendant la période foetale prédisposent la descendance aux maladies cardiovasculaires systémiques. Il existe peu de connaissances au sujet de la circulation pulmonaire et encore moins quant aux mécanismes sous-jacents. La sous-alimentation maternelle pendant la grossesse peut représenter un modèle d'investigation de ces mécanismes, parce que chez l'animal et l'homme elle est associée à une dysfonction vasculaire systémique chez la progéniture. Chez le rat, la diète restrictive pendant la grossesse induit une augmentation du stress oxydatif dans le placenta. Les dérivés de l'oxygène sont connus pour induire des altérations épigénétiques et peuvent traverser la barrière placentaire. Nous avons dès lors spéculé que chez la souris la diète restrictive pendant la grossesse induit une dysfonction vasculaire pulmonaire chez sa progéniture qui serait liée à un mécanisme épigénétique.Pour tester cette hypothèse, nous avons examiné la fonction vasculaire pulmonaire et la méthylation de l'ADN pulmonaire à la fin de 2 semaines d'exposition à l'hypoxie chez la progéniture de souris soumises à une diète restrictive pendant la grossesse et des souris contrôles. Nous avons trouvé que la vasodilatation endothélium-dépendante de l'artère pulmonaire in vitro était défectueuse, et que l'hypertension pulmonaire et l'hypertrophie ventriculaire droite induites par l'hypoxie in vivo étaient exagérées chez la progéniture de souris soumises à une diète restrictive pendant la grossesse. Cette dysfonction vasculaire pulmonaire était associée avec une altération de la méthylation de l'ADN pulmonaire. L'administration d'inhibiteurs de la déacétylase des histones, le Butyrate et la Trichostatine-A à la progéniture de souris soumises à une diète restrictive pendant la grossesse a normalisé la méthylation de l'ADN et la fonction vasculaire pulmonaire. Finalement, l'administration du nitroxyde Tempol aux mères durant la diète restrictive pendant la grossesse a prévenu la dysfonction vasculaire et la dysméthylation chez la progéniture.Ces découvertes démontrent que chez la souris la sous-alimentation pendant la gestation induit une dysfonction vasculaire chez la progéniture qui est causée par un mécanisme épigénétique. Il est possible qu'un mécanisme similaire soit impliqué dans la programmation foetale de la dysfonction vasculaire chez les humains.

Attitudes towards hastened death in ALS: A prospective study of patients and family caregivers.

Abstract Amyotrophic lateral sclerosis (ALS) may be associated with the wish to hasten death (WTHD). We aimed to determine the prevalence and stability of WTHD and end-of-life attitudes in ALS patients, identify predictive factors, and explore communication about WTHD. We conducted a prospective questionnaire study among patients and their primary caregivers attending ALS clinics in Germany and Switzerland. We enrolled 66 patients and 62 caregivers. Half of the patients could imagine asking for assisted suicide or euthanasia; 14% expressed a current WTHD at the baseline survey. While 75% were in favour of non-invasive ventilation, only 55% and 27% were in favour of percutaneous endoscopic gastrostomy and invasive ventilation, respectively. These attitudes were stable over 13 months. The WTHD was predicted by depression, anxiety, loneliness, perceiving to be a burden to others, and a low quality of life (all p < 0.05). Lower religiosity predicted whether patients could imagine assisted suicide or euthanasia. Two-thirds of patients had communicated their WTHD to relatives; no-one talked to the physician about it, yet half of them would like to do so. In conclusion, physicians should consider proactively asking for WTHD, and be sensitive towards neglected psychosocial problems and psychiatric comorbidity.

Plasticity of fetal cartilaginous cells.

Tissue-specific stem cells found in adult tissues can participate in the repair process following injury. However, adult tissues, such as articular cartilage and intervertebral disc, have low regeneration capacity, whereas fetal tissues, such as articular cartilage, show high regeneration ability. The presence of fetal stem cells in fetal cartilaginous tissues and their involvement in the regeneration of fetal cartilage is unknown. The aim of the study was to assess the chondrogenic differentiation and the plasticity of fetal cartilaginous cells. We compared the TGF-β3-induced chondrogenic differentiation of human fetal cells isolated from spine and cartilage tissues to that of human bone marrow stromal cells (BMSC). Stem cell surface markers and adipogenic and osteogenic plasticity of the two fetal cell types were also assessed. TGF-β3 stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens, and variable levels of type X collagen. Although fetal cells showed the same pattern of surface stem cell markers as BMSCs, both type of fetal cells had lower adipogenic and osteogenic differentiation capacity than BMSCs. Fetal cells from femoral head showed higher adipogenic differentiation than fetal cells from spine. These results show that fetal cells are already differentiated cells and may be a good compromise between stem cells and adult tissue cells for a cell-based therapy.

79-OR: Measurement of β-tryptase in postmortem serum in cardiac death

Mast cells are well known for their role in hypersensitivity reactions. However, there is increasing evidence that they might also participate in both developing and weakening atherosclerotic plaques, potentially causing plaque instability. Some clinical studies have therefore postulated the existence of relationships between blood β-tryptase levels and acute coronary syndromes. In this study, we investigated postmortem serum β-tryptase levels in a series of 90 autopsy cases with various degrees of coronary atherosclerosisthat had undergone medico-legal investigations. β-tryptase concentrations in these cases were compared to levels observed in 6 fatal anaphylaxis cases following contrast material administration. Postmortem serum β-tryptase concentrations in the anaphylactic deaths ranged from 146 to 979 ng/ml. In 9 out of 90 cases of cardiac deaths, β-tryptase levels were higher than clinical reference values of 11.4 ng/ml and ranged from 21 to 65 ng/ml. These results indicate that increased postmortem serum β-tryptase levels can be observed, though not systematically, in cardiac deaths with varying degrees of coronary atherosclerosis disease, thereby suggesting that mast cell activation in this disease cannot be ascertained by postmortem serum β-tryptase measurements.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.

Autophagic cell death exists.

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.

T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Expressed isolated integrin beta1 subunit cytodomain induces endothelial cell death secondary to detachment.

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

Hyperactivation of retina by light in mice leads to photoreceptor cell death mediated by VEGF and retinal pigment epithelium permeability.

Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood-retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side - that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.

TWEAK can induce cell death via endogenous TNF and TNF receptor 1.

TWEAK is a recently cloned novel member of the TNF ligand family. Here we show that soluble TWEAK is sufficient to induce apoptosis in Kym-1 cells within 18 h. TWEAK-induced apoptosis is indirect and is mediated by the interaction of endogenous TNF and TNF receptor (TNFR)1, as each TNFR1-Fc, neutralizing TNF-specific antibodies and TNFR1-specific Fab fragments efficiently antagonize cell death induction. In addition to this indirect mode of action, co-stimulation of Kym-1 cells with TWEAK enhances TNFR1-mediated cell death induction. In contrast to TNF, TWEAK does only modestly activate NF-kappaB or c-jun N-terminal kinase (JNK) in Kym-1 cells. Although TWEAK binding to Kym-1 cells is easily detectable by flow cytometric analysis, we found neither evidence for expression of the recently identified TWEAK receptor Apo3/TRAMP/wsl/DR3/LARD, nor indications for direct interactions of TWEAK with TNFR. Together, these characteristics of TWEAK-induced signaling in Kym-1 cells argue for the existence of an additional, still undefined non-death domain-containing TWEAK receptor in Kym-1 cells.