On the role of superoxide dismutase and protein phosphatase type-2C beta in neuronal cell death

The purpose of this study was to elucidate the role of enzymes, the endogenous free radical scavengers superoxide dismutase 1 and 2 (SOD1/2) as well as protein phosphatase type 2C (PP2C), in different models of neuronal cell death. This should improve the understanding of cellular mechanisms after neuronal damage like stroke, trauma, or neurodegenerative diseases like Huntington’s disease in order to reveal new therapeutic targets. Two different in vivo-models of neuronal damage, cold injury-induced brain trauma (CIBT) and intrastriatal injection of the toxin 3-nitropropionic (3-NP) acid in mice, were employed to study the influence of quantitative modulation of SODs on brain damage. The possible role of PP2C, an enzyme participating in the reversible phosphorylation of proteins, in the induction of apoptosis was elucidated in cultured rat neurons. In the first part of this study it was demonstrated that the cytosolic Cu/Zn-superoxide dismutase (SOD1) influences the development of the lesion after intrastriatal injection of 3-NP. Overexpression of SOD1 in transgenic mice was neuroprotective in this predominantly necrotic model of 3-NP injection, reducing lesion size and edema development, while diminished expression of this enzyme in knock-out mutants only significantly enhanced edema formation. The results of this study support the notion that also in this model of intrastriatal injection free oxygen radical production is a crucial factor of 3-NP intoxication as had been previously demonstrated in milder, systemic models of 3-NP intoxication. Understanding neuronal cell death due to lack of energy by 3-NP intoxication allows insight into neurodegenerative diseases like Huntingtons disease and guides the search for therapeutic targets to treat these diseases. As a next step, the role of mitochondrial Mn-superoxide dismutase (SOD2) in mice was examined using a model of cold injury-induced brain trauma (CIBT). The results of these experiments elucidate, on the one hand, the role of mitochondrial free oxygen radical production and, on the other hand, the development of the lesion after cold injury brain trauma (CIBT) at later time points, especially BBB breakdown and inflammatory response. Reduced expression of SOD2 in knock-out mutants did not influence the development of damage after CIBT. However, increased free oxygen radical production inside the lesion of CIBT was detected by hydroethidine assay. This result supports findings of previous publications postulating reactive oxygen species (ROS) production to play a crucial role in the damage after CIBT. Since SOD2 is localized in the mitochondria, this result allows to draw the conclusion that either the production of free radicals did not co-localize with SOD2 in the mitochondria, in which case SOD2 could not have an influence on the ROS level, or the amounts of ROS were already too devastating in wild type animals to allow an exacerbation of the lesion after reduction of SOD2. By observing the development of vasogenic edema and finding hemorrhagic transformations and inflammation at later time points, this study proved CIBT to be a reproducible model of a biphasic breakdown of the BBB. Stroke as well as trauma research might benefit from this model, since biphasic BBB breakdown and inflammation are factors contributing to the deterioration of these brain damages. Augmented knowledge gained from this study can contribute to the development of new therapies for improved prognosis of patients. The involvement of PP2C’s role in neuronal apoptosis was the third focus of research of this thesis. Protein kinases and phosphatases, including PP2C, regulate numerous cellular mechanisms by reversible phosphorylation of proteins. The results of this study support the hypothesis of PP2C being involved in programmed neuronal cell death (apoptosis). Activators of PP2C, such as oleic acid or ginkgolic acids, were shown to induce apoptosis in a time- and concentration-dependent manner in cultured rat neurons while using the enantiomer “trans-oleic acid” (elaidic acid), known not to activate PP2C in vitro, instead of cis-oleic acid, did not induce apoptosis. A possible apoptotic mechanism, the induction of programmed cell death by dephosphorylation of the proapoptotic protein Bad by activated PP2C, was analyzed by Western blotting experiments. Bad phosphorylated at Ser155 and total Bad (phosphorylated + unphosphorylated) were detected in cultivated rat neurons which had been treated with oleic acid or ginkgolic acids. By the Western blotting method, changes in the amount of unphosphorylated Bad were not detectable since no antibody against unphosphorylated Bad was available. Without knowledge of the ratio of phosphorylated Bad to total Bad in non-treated cultured neurons, the proportion of phospho-Bad/total Bad in oleic acid-treated cells did not allow to draw conclusions about quantitative changes of unphosphorylated Bad.