Differenzierte Motoneurone als Modell zur Untersuchung von Stoffwechselvorgängen im Zusammenhang mit der Charcot-Marie-Tooth Erkrankung

Abstract

MFN2 is a mitochondrial protein involved in the pathogenesis of a specific subtype of the neurodegenerative disorder Charcot-Marie-Tooth (CMT). The aim of this study was to differentiate mouse embryonic stem cells to motoneurons and to use these motoneurons as a tool to investigate metabolic changes in cells overexpressing the CMT2A-related mutation MFN2R94Q. Differentiation to MNs was based on a differentiation module comprising three transcriptional factors responsible for neuronal development that was under the control of a Tet-on system. The results of this study have confirmed that the administration of doxycycline and the respective patterning factors to ES cells leads to the generation of postmitotic motoneurons. Neuronal identity was phenotypically confirmed by expression of common neuronal markers. I further showed that a DIO expression system under the control of a neuron specific Hb9-promoter is sufficient to induce selective overexpression of MFN2 in neuronal cells. Among the tested reagents Lipofectamine 2000 was most efficient for transfection. The impact of mutated MFN2 on metabolic behavior, in particular on lactate and ATP levels, was monitored using FRET based genetically-encoded indicators. The successful expression of genetic indicators was confirmed by confocal microscopy. In contrast to previous suppositions, the indicators could not detect a significant difference in lactate and ATP concentration. These findings suggest that as a basic principle, FRET indicators are a suitable tool to investigate metabolic behavior in motoneurons. However the number of cells included in this analysis might have been too low to detect a significant difference in metabolism. In order to generate more reliable results it might be necessary to further improve transfection efficiency and to establish a method that allows the easy analysis of a larger number of cells.