Publikationsserver der Universitätsbibliothek Marburg

Titel:Autophagy and apoptosis contribute to neuronal survival in a model system of oxytosis in vitro
Autor:Neunteibl, Stefanie
Weitere Beteiligte: Culmsee, Carsten (Prof. Dr.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0463
DOI: https://doi.org/10.17192/z2014.0463
URN: urn:nbn:de:hebis:04-z2014-04630
DDC: Naturwissenschaften
Publikationsdatum:2014-08-07
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Apoptosis, Apoptose, Autophagie, Autophagy

Summary:
Autophagy and apoptosis play major roles in determining the cellular fate. Accordingly, they participate in development, cellular homeostasis, and both in physiological as well as in pathological processes. Apoptosis is executed by activated caspases, which are specific enzymes that participate in signaling cascades that culminate in the rapid removal of organelles and other cellular structures. Autophagy is a highly conserved cytoprotective process whereby cytoplasmic contents are sequestered, transported via double-membrane autophagosomes to lysosomes, and degraded. Along with regulated necrosis and other forms of programmed cell death, pathological mechanisms of autophagy and apoptosis have been detected in neurodegenerative diseases, such as Parkinson’s disease or Alzheimer’s disease and acute brain injuries. The aim of this thesis was to investigate the role of autophagy and apoptosis for neuronal resilience versus neuronal cell death in model systems of glutamate toxicity in vitro. The role of autophagy was investigated in the model system of glutamate-induced oxidative stress, i.e. oxytosis in neural HT-22 cells. The objectives were to determine the effect of oxidative glutamate toxicity on autophagic flux and to investigate if oxytosis involves autophagy pathways of cell death. Moreover the neuroprotective effect of 3-Methyladenine (3-MA), a widely used autophagy inhibitor, was explored in the model systems of glutamate-induced oxytosis and excitotoxicity in neural HT-22 cells and primary cortical neurons, respectively. Glutamate clearly enhanced autophagy markers and induced cell death in HT-22 cells and PCN. Cell death was prevented by 3-MA, a widely used inhibitor of autophagy. Interestingly, 3-MA itself induced autophagy in HT-22 cells. A gene silencing approach targeting key regulators of autophagy reduced the autophagic flux, but failed to prevent cell death persistently. 3-MA prevented the glutamate-induced ROS formation, the loss of ATP, and preserved the mitochondrial membrane potential as well as mitochondrial morphology. The activation of the PI3K/Akt pathway and the MAPK/Erk-1/2 pathway do not play a role in 3-MA mediated neuroprotection. In conclusion, these data suggest that glutamate toxicity is associated with increased markers of autophagy. However, specific gene silencing of key regulators of autophagy did not provide protective effects, suggesting that the increased autophagic flux was dispensable for cell death induced by glutamate. The induction of autophagy can rather be interpreted as the ultimate attempt to adapt to lethal stress after glutamate challenge und is rather prosurvival and contributes to cellular homeostasis in HT-22 cells. Further, the findings clearly demonstrate that protective effects by 3-MA occur independently of autophagy inhibition, although the compound is widely used as an inhibitor of autophagy. Nowadays, 3-MA is used frequently in experimental studies in vivo and in vitro. Based on the present findings, caution is recommended in the interpretation of data obtained with 3-MA in the context of autophagy studies, in particular, if mitochondrial alterations are connected. The contribution of apoptosis to neuronal survival through intercellular signaling between dying cells and neurons was investigated using conditioned medium of neural progenitor cells (NPC) and HT-22 cells. Stem cells as well as progenitor cells have been widely used in model systems of neurodegenerative diseases and acute brain injuries where transplantation of these cells into the brain improved neuronal survival and brain functions in experimental settings. However, most transplanted cells die after transplantation in vivo, and the exact mechanism of action of stem cell or progenitor cell transplantation still remains unknown. To investigate mechanisms of intercellular signaling underlying the protective effects of transplanted stem cells, the transplantation conditions were mimicked in vitro by preparation of conditioned medium (CM) obtained from dying neuronal progenitor cells. This CM should contain similar cellular components as released during cell death of the progenitor cell transplants, and this CM should therefore also provide neuroprotective effects. Thus, the CM of dying/apoptotic cells was applied in model systems of cell death in vitro for to test its potential to mediate neuroprotection. Further, the composition of the conditioned medium obtained from the dying progenitor cells was analysed for identifying the most potent protective components that may be applied as neuroprotectants in vitro and in vivo instead of the CM or the cellular transplants. In order to mimic the conditions of transplantation, NPC were exposed to medium lacking growth factors such as FGF and EGF. Such growth factor deprivation induced caspase-dependent cell death in NPC in a time-dependent manner. The conditioned medium obtained from apoptotic NPC significantly attenuated cell death induced by growth factor withdrawal and glutamate exposure in HT-22 cells and cortical neurons in a dose-dependent manner. The protective effect of NPC CM against glutamate neurotoxicity was abolished by heat inactivation at 95°C for 30 min. Further, NPC CM enhanced phosphorylation of PKB/Akt and Erk 1/2 in neurons in a similar time frame as the neurotrophin BDNF. Inhibition of autophagy did not diminish the protective effect of NPC CM. Also the use of spermidine conditioned medium (Sp CM) from HT-22 cells, whose production aimed on the specific activation of autophagy, indicated that the induction of autophagy is not essential for the protective effect of conditioned medium. In total, these findings suggest that NPC secrete neuroprotective factors that stimulate neurotrophin-like survival signaling thereby providing protective effects against growth factor withdrawal and glutamate neurotoxicity. The results obtained from the in vitro model system in this thesis are transferable to the adult organism. Thus, the results are the basis for the development of a highly potent, standardized composition for the therapy of neurodegenerative diseases and acute brain injury. Therefore, CM and its active components could serve as an alternative therapeutic option to stem cell transplantation.

Zusammenfassung:
Autophagie und Apoptose sind hochregulierte, komplexe Mechanismen, die eine bedeutende Rolle in der Entwicklung, Funktion und Homöostase von Zellen und Geweben eines Organismus einnehmen. Apoptose ist durch die Aktivität spezifischer Enzyme, der Caspasen, gekennzeichnet, die festgelegte programmierte Signalkaskaden in Gang setzten, was u.a. einen schnellen Abbau von Organellen und anderen zellulären Strukturen zur Folge hat. Autophagie ist ein hoch konservierter und in der Regel protektiver Prozess, bei dem zytoplasmatische Bestandteile in so genannte Autophagosomen eingeschlossen und transportiert werden und schließlich in den in Lysosomen degradiert werden. Bei neurodegenerativen Erkrankungen, wie Morbus Parkinson, Morbus Alzheimer und akuten Gehirnerkrankungen sind in den letzten Jahren zunehmend auch Mechanismen der Autophagie und Apoptose detektiert worden. Diese Befunde führten zu der Schlussfolgerung, dass bei diesen Erkrankungen die erhöhte Aktivität von Apoptose und Autophagie zum fortschreitenden Zelltod der Neurone und dem progressiven Verlust der Hirnfunktionen essentiell beitragen. Das Ziel dieser Arbeit war es daher, die möglichen Funktionen von Autophagie und Apoptose in Modellsystemen von Glutamat-Toxizität in kultivierten Neuronen in vitro zu untersuchen, um die Rolle dieser Mechanismen beim neuronalem Zelltod zu klären. Die Rolle von Autophagie wurde im Modellsystem von Glutamat-induziertem oxidativem Stress in neuronalen HT-22 Zellen untersucht. Eine Zielsetzung war, den Effekt von oxidativer Glutamat-Toxizität auf die Aktivierung von Autophagie-Mechanismen in den Neuronen zu bestimmen und so zu klären, ob und in welchem Ausmaß hier Autophagie zum Zelltod beiträgt. Zudem wurde in Modellsystemen der Glutamat-Toxizität auch der neuroprotektive Effekt des vielfach verwendeten Autophagie-Inhibitors 3-Methyladenin (3-MA) untersucht. In HT-22 Zellen und primären kortikalen Neuronen (PCN) war die Glutamat-induzierte Toxizität von einem deutlichen Anstieg von Autophagie-Markern begleitet. Der Glutamat-induzierte Zelltod wurde durch den Autophagie-Inhibitor 3-MA signifikant vermindert. Allerdings induzierte 3-MA selbst Autophagie in HT-22 Zellen. Im Gegensatz dazu gelang es nicht, die Zellen mit Hilfe von genetischer Regulation der Autophagie gegen die Glutamat-Schädigung zu schützen. Die siRNA-vermittelte Expressionshemmung von Schlüsselregulatoren der Autophagie reduzierte zwar erwartungsgemäß die autophagische Aktivität, war aber nicht in der Lage, den Zelltod dauerhaft aufzuhalten. Der Inhibitor 3-MA verhinderte zudem die Glutamat-induzierte Bildung von reaktiven Sauerstoffspezies und den Verlust von ATP, wobei sowohl das Mitochodrienmembranpotential als auch die mitochondriale Morphologie erhalten blieben. Die Aktvierung der PI3K/Akt- und der MAPK/Erk-1/2 Signalwege sind offenbar nicht an der 3-MA vermittelten Neuroprotektion beteiligt. Schlussfolgernd zeigen diese Daten, dass Glutamat-Toxizität mit einer Aktivierung der Autophagie einhergeht. Allerdings lieferte die siRNA-vermittelte spezifische Hemmung der Expression von Schlüsselregulatoren der Autophagie keine protektiven Effekte, so dass die gemessene autophagische Aktivität nicht entscheidend zum Glutamat-induzierten Zelltod beiträgt. Die Induktion von Autophagie ist in diesem Zusammenhang offenbar eine Gegenregulation der Zellen, über die eine Anpassung an den Stress vermittelt wird. In den untersuchten Neuronen trägt die Autophagie eher zum Überleben und zur zellulären Homöostase bei und ist nicht als Mechanismus des Zelltodes zu verstehen. Weiterhin zeigen die Ergebnisse, dass der protektive Effekt von 3-MA in den Mitochondrien der Neurone unabhängig von einer Inhibition der Autophagie erfolgt, obwohl die Substanz vielfach als Inhibitor der Autophagie verwendet wird. Basierend auch auf den vorliegenden Ergebnissen sind also Daten, die mit 3-MA in Autophagiestudien erhoben werden, zurückhaltend zu interpretieren, insbesondere dann, wenn mitochondriale Schädigungen den Zelltod vermitteln. Der Beitrag von Apoptose zu neuronalem Überleben durch interzelluläre Signale zwischen sterbenden (Stamm) Zellen und Neuronen wurde in Modellen des Nährstoffentzugs in neuronalen Vorläuferzellen und im Glutamat-Schädigungsmodell in den HT-22 Zellen untersucht. Therapeutische Effekte von Stammzellen und neuronalen Vorläuferzellen sind in Modellsystemen von akuten und chronischen neurodegenerativen Erkrankungen umfangreich nachgewiesen worden. In experimentellen Studien steigerten beispielsweise entsprechende Zelltransplantate das neuronale Überleben und die Erhaltung und Regeneration von Gehirnfunktionen. Jedoch sterben die meisten Zellen nach der Transplantation in das Gehirngewebe schnell ab und auch der exakte Mechanismus der therapeutischen Effekte von solchen Zelltransplantationen ist bislang weitgehend unbekannt. Die vorliegende Arbeit untersuchte, inwieweit das Absterben von neuronalen Vorläuferzellen für die beobachteten protektiven Effekte von transplantierten Zellen von Bedeutung ist. Zu diesem Zweck wurden die Transplantationsbedingungen in vitro nachgeahmt indem aus dem Medium von neuronalen Vorläuferzellen (NPC) die Wachstumsfaktoren EGF und FGF entfernt wurden, um so apoptotischen Zelltod zu induzieren. Das konditionierte Medium von diesen apoptotischen Zellen sollte ähnliche zelluläre Komponenten enthalten, die auch nach der Transplantation von den sterbenden Stamm- oder Vorläuferzellen im Gehirn in vivo freigesetzt werden und die möglicherweise die neuroprotektiven Effekte vermitteln. Das konditionierte Medium der apoptotischen neuronalen Vorläuferzellen wurde im Modell der Glutamatschädigung in HT-22 Zellen auf neuroprotektive Effekte untersucht. Weiterhin wurde die Zusammensetzung des konditioniertem Mediums der sterben Progenitorzellen analysiert, um die wirksamsten protektiven Komponenten zu identifizieren, die dann vielleicht in vitro und in vivo an Stelle des konditionierten Mediums oder zellulärer Transplantate appliziert werden könnten. Der Wachstumsfaktorentzug induzierte in den NPC einen Caspase-abhängigen Zelltod. Das konditionierte Medium (CM) von den apoptotischen NPC zeigte deutliche dosisabhängige protektive Effekte und verhinderte in HT-22 Zellen und kortikalen Neuronen den Zelltod nach Wachstumsfaktorentzug und Glutamatbehandlung. Der protektive Effekt des konditionierten Mediums wurde durch Hitzeinaktivierung bei 95°C für 30 Minuten aufgehoben. Weiterhin verstärkte das CM die Phosphorylierung von Erk 1/2 und PKB/Akt in primären Neuronen in einem ähnlichen Zeitrahmen wie das Neurotrophin BDNF. Autophagieinhibition in NPC schwächte die beschriebene neuroprotektive Wirkung nicht ab. Konditioniertes Medium, das von den NPC nach Induktion von Autophagie durch Spermidin gewonnen wurde, zeigte keinerlei protektiven Effekte in dem Modell der Glutamat-Schädigung in HT-22 Zellen. Die Aktivierung von Autophagie in NPC ist also im Gegensatz zur Apoptose offenbar nicht ausreichend, um protektive Faktoren zu bilden und in das konditionierte Medium abzugeben. Zusammenfassend deuten diese Ergebnisse deuten darauf hin, dass apoptotische NPC neuroprotektive Faktoren sezernieren, die neurotrophin-ähnliche Überlebenssignalwege stimulieren und so protektive Effekte gegen Nährstoffentzug und Glutamat-Toxizität vermitteln. Die Resultate des in vitro Modellsystems in dieser Arbeit sind übertragbar zum adulten Organismus. Die vorliegenden Erkenntnisse sind die Basis für die Entwicklung einer zellfreien, standardisierten und potenten neuroprotektiven Komposition, die eine Alternative zur Stammzelltransplantation darstellt und neue therapeutische Optionen für die zukünftige Behandlung von akuten und chronischen neurodegenerativen Erkrankungen bieten kann.

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