Mitochondrial adaptation at the neuronal presynapse
by Felix Lange
Date of Examination:2021-02-25
Date of issue:2021-11-22
Advisor:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Carolin Wichmann
Referee:Prof. Dr. Tiago Fleming Outeiro
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Abstract
English
Synaptic transmission poses a major energy consuming process in the brain, but how neurons maintain a constant energy supply during extended synaptic activity and how presynaptic mitochondria contribute to the energy supply remains elusive. The mitochondria are key organelles to account for the majority of ATP newly synthesised in neuronal cells. Hence, the present study aimed at unravelling the structural adaptation of mitochondria to an increase in presynaptic energy demand. To this end, I first characterized the mitochondrial membrane architecture and how it changes in response to different energy sources using transmission electron microscopy. As a model system, several cancer cell lines were used in addition to primary hippocampal neuronal cultures isolated from rat brain. In all tested cancer cell lines, a long-term metabolic switch to ketosis induced significant changes in the mitochondrial structure reflected by an increase of the mitochondrial diameter as well as a significant increase in the abundance of crista membranes. In contrast, a metabolic switch to glycolysis resulted in a decrease of the mitochondrial diameter and a reorientation of the crista membranes in parallel to the mitochondrial outer membrane in these cells. Likewise, in cultivated hippocampal neurons, the metabolic switch to ketosis induced a 20 % increase of crista membranes. Interestingly, only about 35 % of the neuronal presynapses showed a mitochondrial occupation which was not influenced by chemical depolarization with high concentrations of potassium chloride (KCl), suggesting that induced presynaptic activity does not affect the travelling of axonal mitochondria to the presynapses of hippocampal neurons. Furthermore, I used the cochlear nucleus of mice before and after the onset of hearing as a model system for presynaptic states of low and high-energy demand. Here, the synaptic morphology together with the presynaptic mitochondrial structure using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) was assessed. The volume of the synaptic boutons, the synaptic vesicle pool as well as the post-synaptic density were increased in synapses after the onset of hearing. Beyond that, the mitochondrial volume in these presynapses increased significantly. This suggests that synapses and mitochondria undergo major structural changes to serve the higher energy demands after the onset of hearing. Intriguingly, synapses containing mitochondria displayed an overall larger volume, synaptic vesicle pool and postsynaptic density when compared to synapses lacking mitochondria in mice before and after the onset of hearing. In conclusion, mitochondria play a key role in shaping presynaptic structure and are thus pivotal for synaptic transmission. In order to gain functional insights into the role of mitochondria in synaptic transmission in addition to the structural information obtained by TEM, I developed novel correlative imaging V tools. These tools include live cell 3D correlative light and electron microscopy (3D CLEM), which allows to monitor relevant mitochondrial targets by fluorescent labelling and subsequently correlate the functional information with structural aspects, and high-accuracy CLEM allowing for high spatial resolution of the detected features in both, light and electron microscopy. These protocols built the foundation for an extended correlative approach. There, I combined high-accuracy CLEM with nanoscale secondary ion mass spectrometry (NanoSIMS). This, for the first time, allowed extracting the functional and structural information together with the chemical composition of subcellular areas of the same resin-embedded specimen. These tools could now be applied to study individual players in mitochondrial function by simultaneously getting information of their impact on synaptic structure and chemical composition.
Keywords: Mitochondria; Presynapse; Structure; Neuronal Cells; Microscopy