Analyse funktioneller und struktureller Mitochondrienveränderungen in einem Maus-Modell für das Rett-Syndrom mittels 2-Photonen-Mikroskopie
Functional and structural analysis of mitochondria changes in a mouse model for the Rett syndrome by means of 2-photons microscopy
by Dörthe Friederike Bebensee
Date of Examination:2017-01-25
Date of issue:2017-01-09
Advisor:Prof. Dr. Michael Müller
Referee:Prof. Dr. Michael Müller
Referee:Prof. Dr. Stefan Jakobs
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Abstract
English
Rett syndrome is a neurodevelopmental disorder that is caused by de novo mutations in the X-chromosomal MECP2 gene which encodes for the transcriptional modulator methyl CpG binding protein 2 (MeCP2). Almost girls are being affected by Rett syndrome. First symptoms occur at an age of typically 6-18 months, when normal development is followed by loss of speech, cognitive impairment, epilepsy as well as severe breathing disturbances. Various indications suggest that also mitochondria are impaired in Rett syndrome. For example, ATP levels seem to be reduced in MeCP2-deficient brain tissue, the inner mitochondrial membrane is leaking protons, and parts of complex III of the respiratory chain are potentially dysregulated. Based on the increased oxidative damage detected in blood samples of Rett patients also oxidative stress has been proposed. In acute and cultured hippocampal slices we have already verified alterations in mitochondrial metabolism and mitochondrial membrane potential as well as an increased formation of reactive oxygen species. We therefore performed high-resolution multiphoton imaging to screen for changes in the morphology, mass and mitochondrial membrane potential of individual mitochondria in MeCP2-deficient mice (Mecp2-/y). As a marker for mitochondrial membrane potential we chose the ratiometric indicator JC-1. Depending on the mitochondrial membrane potential, JC-1 forms either green fluorescing monomers (depolarized mitochondria) or red fluorescing J-aggregates (hyperpolarized mitochondria). Accordingly, the ratio of red/green JC-1 fluorescence reports the membrane potential of individual mitochondria. In primary hippocampal cell cultures, individual mitochondria could be easily identified especially in flatly grown astrocytes. Deconvolution of image stacks followed by semi-automated analyses yielded the number of clearly identifiable mitochondria per cell, their morphological parameters and mitochondrial membrane potential. Astrocytes obtained from Mecp2-/y hippocampus were found to contain a larger number of mitochondria. The size of the individual mitochondria, however, did not differ, which suggests an increased mitochondrial mass in Mecp2-/y. The mitochondrial membrane potentia of WT and Mecp2-/y mitochondria did not differ significantly. Mitochondrial inhibition by CN- or uncoupling by FCCP caused similar responses in Mecp2-/y and WT astrocytes. Incubation with the radical scavenger Trolox did not markedly affect the mitochondrial membrane potential or size of individual mitochondria. Yet it decreased the number of mitochondria per cell and abolished the genotypic differences observed earlier among WT and Mecp2-/y astrocytes. These studies show that on the level of individual organelles, mitochondria of Mecp2-/y hippocampus are more numerous. This increase in mitochondrial mass may contribute to the earlier observed genotypic differences in cellular redox homeostasis and the more oxidizing conditions in FAD/NADH ratio found in acute and/or organotypic slices from Mecp2-/y hippocampus. Trolox treatment, which is known to counteract oxidative stress and an increased formation of reactive oxygen species, abolishes the difference in mitochondrial density among WT and Mecp2-/y hippocampus. Adverse effects of Trolox treatment on the function of mitochondria were not observed.
Keywords: Rett syndrome; MECP2; mitochondria; JC-1; astrocytes
Schlagwörter: Rett-Syndrom; Astrozyten; JC-1; Mitochondrien; MECP2