Doctoral thesis

Nuclear translocation of components of the neuronal plasma membrane oxidoreductase after oxidative stress and its effects on induction of apoptosis

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Thèse de doctorat: Université de Fribourg, 2000

English German The plasma membrane oxidoreductase (PMO) complex may exert important functions in cellular defence mechanism against oxidative stress and in apoptosis. Thus, we aimed to investigate the function of the five proteins GAPDH, Hsc70, enolase γ, aldolase C and Ulip, all of them tightly bound members of a neuronal PMO multi-protein complex, previously isolated and purified from synaptic vesicles and synaptic plasma membranes by our group. Earlier reports have shown an altered subcellular distribution of some these members in both apoptosis and in the cellular response to oxidative stress. We have now extended these studies using confocal laser scanning microscopy in the hope of uncovering clues to the elusive role of these five proteins by immunocytochemistry. Furthermore, CNS-derived cells, the mouse neuroblastoma NB41A3, and non-CNS derived cells, R6 fibroblast and an apoptosis-resistant Bcl-2 expressing transfectant (R6Bcl-2), were transiently and stably transfected with GFP- and BFP-fusion constructs containing either GAPDH, Hsc70, enolase γ, aldolase C or Ulip. Expression of endogenous GAPDH, the major protein associated with the PMO complex, was predominantly nuclear in NB41A3 cells and cytoplasmic in R6 and R6-Bcl-2 cells. Induction of apoptosis and oxidative stress by H2O2 or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types. In apoptotic cells, GAPDH expression was 3 times higher as compared to non-apoptotic cells. Consistent with a role of GAPDH in apoptosis, cells transfected by a vector coding for a GAPDH-GFP hybrid increased nuclear import of GAPDH-GFP and conferred a higher sensitivity to induction of apoptosis caused by exposure to staurosporine or MG132. Per se 10% of cells overexpressing GAPDHGFP are apoptotic. Bcl-2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells overexpressing GAPDH-GFP fusion protein. Hsc70 is localised in the cytoplasm of neuroblastomas and fibroblasts. Partial translocation of endogenous Hsc70 into the nucleus is induced by oxidative stress, but not by apoptosis. The predominantly cytoplasmic localisation of overexpressed Hsc70-GFP in unstressed cells and its subsequent translocation into the nucleus after oxidative stress is similar to what is observed with endogenous Hsc70. Overexpression of Hsc70 makes cells more resistant to oxidative stress. However, in cells exposed to MG132 or staurosporine, no nuclear translocation of Hsc70-GFP is observed and no indications of a protective function of Hsc70 against apoptosis are found. Aldolase and overexpressed aldolase-GFP, which are present both in a soluble and in a cytoskeleton bound form, are expressed predominantly in the cytoplasm of unstressed cells. The expression is highest at the perinuclear area and in the somal area at the bases of axons. Induction of oxidative stress leads to enhanced nuclear expression of endogenous aldolase in NB41A3 cells. The cellular response to apoptosis induced by MG132 or staurosporine is not influenced by overexpression of aldolase-GFP, suggesting that aldolase C does not have a crucial function during apoptosis. Translocation of endogenous Ulip from the cytoplasm to the nucleus in response to oxidative stress is observed in NB41A3 and in R6 cells. In transfected cells overexpressed Ulip-GFP is also imported into the nucleus during oxidative stress, but it is still expressed predominantly in the cytoplasm and often concentrated in Ulip-GFP-positive speckles. 5 Enolase shows the least oxidative stress-induced changes among the five proteins of the PMO complex. The typical cytoplasmic expression of enolase is not significantly altered, except for a slight rise in nuclear enolase-GFP in transfected NB41A3 cells. Finally we were able to show that many combinations between two of the five PMO proteins in double-transfection experiments, enhance differential translocation into the nucleus or into speckles in response to oxidative stress. From this study we conclude, that the translocation of these PMO associated proteins, especially of GAPDH and Hsc70, may be an important event in apoptosis and oxidative stress. Our results also indicate that the effect of these proteins, when they are overexpressed, cannot be counteracted by Bcl-2. Mittels Immunocytochemie untersuchten wir die Rolle der fünf Komponenten des Plasmamembran- Oxidoreduktase (PMO) Komplexes, GAPDH, Hsc70, Enolase γ, Aldolase C und Ulip, während des Oxidativen Stresses und in der Apoptose. Um die Rolle dieser fünf Proteine, welche von unserer Forschungsgruppe isoliert worden waren, zu erforschen, machten wir uns die Konfokal Laser Scanning Microscopie zu nutze. Darüberhinaus wurden Zellen aus dem Zentral-Nervensystem (ZNS), Maus NB41A3 Neuroblastomen, und Nicht-ZNS Zellen, namentlich R6 Fibroblasten und eine Apoptoseresistente Bcl-2 exprimierende Transfektanten (R6Bcl-2), sowohl transient als auch stabil mit GFP- und BFP-Fusions Konstrukten, die entweder GAPDH, Hsc70, Enolase γ, Aldolase C oder Ulip enthalten, transfektiert. Expression von GAPDH, dem wichtigsten Protein des PMO Komplexes, war vorwiegend im Zellkern in NB41A3 und cytoplasmisch in R6 and R6-Bcl-2 Zellen. Induktion von Apoptose und Oxidativem Stress erhöhte den Import von zelleigenem GAPDH in den Kern. In apoptotischen Zellen war die GAPDH-Expression 3 mal höher als in nicht-apoptotischen. Zellen, in denen GAPDH gekoppelt an GFP überexprimiert wurde, waren per se zu 10% apoptotisch und erhöhten den Zellkern-Import von GAPDH-GFP und und die Sensibilität auf durch Staurosporine oder MG132 induzierte Apoptose. Bcl-2 Ueberexpression vermindert den Kern-Import von GAPDH als auch Apoptose in nicht transfektierten, nicht aber in GAPDH-GFP transfektierten Zellen. Kern-Import von zelleigenem Hsc70 oder überexprimiertem Hsc70-GFP wird nur während des Oxidativen Stresses, nicht aber während der Apoptose beobachtet. Ausserdem zeigen Hsc70-GFP überexprimierende Zellen verminderte Anfälligkeit gegenüber Effekten des Oxidativen Stresses, aber nicht gegenüber der Apoptose. Aldolase und überexprimiertes Aldolase-GFP werden vorwiegend in Kernnähe und am Ansatz von Axonen im Cytoplasma exprimiert. Währendem sich die Aldolase-Expression nach Induktion von Oxidativem Stress im Kern erhöhte, wurde sie durch Apoptose weder in Zellen mit noch ohne Aldolase- GFP Ueberexpression beeinflusst. Bei Oxidativem Stress translosziert Ulip vom Cytoplasma in den Kern, währenddem Ulip-GFP auch häufig in intrazellulären Aggregaten zu finden ist. Von den fünf Proteinen des PMO Komplexes, zeigt die cytoplasmische Exxpression von Enolase die geringsten stress-induzierten Veränderungen. Die gleichzeitige Ueberexpression verschiedener dieser PMO Komponenten steigerte den Kern- Import während des Oxidativen Stresses zusätzlich. Wir schliessen aus dieser Studie, dass die Translokation der PMO assozierten Proteine, speziell von GAPDH und Hsc70, eine bedeutende Rolle während der Apoptose und des Oxidativen Stresses spielen. Zudem können die Effekte dieser Proteine bei Ueberexpression scheinbar nicht durch Bcl-2 verhindert werden.
Faculté des sciences et de médecine
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