Mitochondrial nucleoid distribution and its relation to presynaptic activity investigated by correlative light microscopy
von Axel Rösch
Datum der mündl. Prüfung:2022-11-11
Betreuer:Prof. Dr. Stefan Jakobs
Gutachter:Prof. Dr. Michael Müller
Gutachter:Prof. Dr. Michael Meinecke
EnglischMitochondria are essential for eukaryotic life, because they provide most of the energy for a variety of cellular processes. Especially high energy demanding cells and tissues like the muscles and the neuronal cells of the brain depend on a reliable supply of energy. It is of no surprise that impairments of this organelle manifest in pathophysiological effects. To perform their task as the "power plant of the cell", mitochondria rely on the import of proteins encoded in the nucleus. Together with thirteen proteins encoded in their own genome (mitochondrial DNA, mtDNA), they assemble the oxidative phosphorylation system for energy production. Although many studies investigated neuronal mtDNA, little is known about the relationship between the presence of mtDNA in axonal mitochondria, mitochondrial function and presynaptic activity. The aim of this work was to investigate the nucleoid (Protein-mtDNA complex) distribution in neurons and to study the interplay between neuronal activity and the mtDNA content of neuronal mitochondria. This work shows that the nucleoid distribution is not homogeneous across the neuronal cell. Rather, the majority of mitochondria in the axon do not harbor a nucleoid, whereas 90 % of dendritic mitochondria do. Additionally, it was found that the mitochondria carrying mtDNA in the axon are mainly accumulated in the presynapse. This raised the question if there is a relationship between synaptic activity and the presence of mitochondria with nucleoids. . A single microscopy method such as immunofluorescence microscopy or live cell imaging alone would not be sufficient to answer this question, but a combination of both imaging modalities would is required to gain new insights into the relationship between neuronal activity and mitochondrial dynamics. This is why a new correlative light microscopy workflow was developed that allows functional live cell microscopy to be combined with subsequent immunofluorescence imaging on other microscopes. For this, three major obstacles had to be addressed: the technical requirements of stable on-stage fixation, the adaptation of fixation conditions and the relocation of a predefined region of interest when the sample is moved from one microscope to another. The main obstacle of stable on-stage fixation was overcome by designing a novel live cell imaging chamber. Together with an optimized fixation protocol, this set-up allows for a rapid and homogenous fixation of the cells during live cell image acquisition. Subsequent immunofluorescence staining and transfer of the sample to another microscope presented the challenge of locating the same cells and regions of interest identified during live cell imaging. To address this problem, a combination of a unique label on the coverslip and an image registration script was developed to store regions of interest and relocate them relative to the label. The correlative live cell imaging system was used to study the relationship between presynaptic activity and the presence of mitochondrial DNA in neuronal mitochondria. The results revealed that about 70 % of the mitochondria located in active presynapses harbor nucleoids, whereas 36 % of non-synaptic axonal mitochondria carry nucleoids. This finding led to the question if there is an active selection of mtDNA-loaded mitochondria driven by the V specific demand at their cellular location. The idea of an active selection was supported by silencing neuronal activity with the reversible neurotoxin Tetrodotoxin (TTX), which resulted in a significant reduction to ~44 % mitochondria with mtDNA in presynapses compared to the untreated presynapses (~59 %). Furthermore, after a period of 24 h of toxin washout, the level of nucleoid positive mitochondria recovered fully. Taken together, these results suggest a selective accumulation of mtDNA-positive mitochondria driven by the activity of the presynapse and the associated increased demands on the mitochondria.
Keywords: correlative light microsocpy; Mitochondria; Neurons; Nucleoid