Die Rolle von p53 und der Proteinphosphatase 2C in der neuronalen Apoptose

Imbalanced neuronal apoptosis is a cause of many diseases, like cancer, neurodegeneration and stroke. Therefore it is important to study the signaling pathways that are involved and to elucidate the underlying mechanisms to finally find a way to cure those diseases. The first focus of this study was to investigate neuroprotection mediated by Pifithrin alpha, an inhibitor of p53. In a rat model of transient global ischemia, pretreatment with 2 mg/kg Pifithrin alpha reduced damage of hippocampal CA1-neurons significantly. The main purpose of this thesis was to elucidate the role of protein phosphatase type-2C isoforms alpha and beta in oleic acid-induced apoptosis. These two isoforms of the magnesium-dependent serine/threonine phosphatase 2C have been shown to be activated in vitro by fatty acids with characteristic structural features. PP2C-activating oleic acid has additionally been shown to induce apoptosis in various systems. To find out whether or not there is a connection between those two events, a model was established in which oleic acid time- and concentration-dependently induces apoptosis in human neuroblastoma SH-SY5Y cells. Moreover, it was demonstrated, that PP2C-activating fatty acids induced apoptosis in SH-SY5Y cells as well as in cultured embryonical cortical neurons of rats, whereas non-activating ones did not. PP2C alpha can be found in the cytosol of SH-SY5Y cells. PP2C beta can additionally be detected in the nucleus. As shown by Western blotting and immunocytochemistry, oleic acid-treatment did not change the amount of PP2C in the cells. Proapoptotic Bad, which is an in vitro substrate of PP2C, is also located in the cytosol and may therefore be also an in vivo-substrate. Oleic acid-treatment did not change the amount of Bad protein in SH-SY5Y cells. Detection of phosphorylated Bad after oleic acid-induced apoptosis and subsequent immunoprecipitation did not show any change in protein levels. Thus, to show participitation of Bad in fatty acid-mediated apoptosis, further effort is necessary. As there are no specific inhibitors of PP2C available, RNA interference (RNAi) was used to knock down PP2C. Using this model it was possible to specifically reduce mRNA as well as protein levels for PP2C isoforms alpha and beta at the same time. Oleic acid-treatment of SH-SY5Y cells during downregulation of PP2C alpha and beta, respectively, led to less severe apoptotic damage as compared to cells with normal PP2C levels. These results suggest that PP2C is involved in oleic acid-induced apoptosis of SH-SH-SY5Y cells: Its downregulation attenuates cell death. Thus, inhibition of PP2C via knockdown or inhibitors might be a possibility to interfere in disesases based on increased apoptotic cell death.