Autophagy and apoptosis contribute to neuronal survival in a model system of oxytosis in vitro

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 s...

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Bibliographic Details
Main Author: Neunteibl, Stefanie
Contributors: Culmsee, Carsten (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2014
Pharmakologie und Toxikologie
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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.