Neuronal energy metabolism is crucial for axonal transport processes and maintenance of axonal membrane potential. These transport processes are driven by mitochondrial ATP synthesis using pyruvate, lactate, or ketone bodies. Disruption of these mechanisms has been linked to the development of neurodegenerative diseases and axon degeneration. Our project aims to investigate the effects of several of the aforementioned substrates on fast axonal transport (FAT) of autophagosomes, mitochondrial motility, and maintenance of mitochondrial and axonal membrane potentials. We will explore how neuronal cells and primary oligodendrocytes cope with reduced glycolytic support in vitro and also ex vivo in acutely dissected (myelinated) optic nerves. To this end, we will apply stable isotope labeling in combination with mass spectrometry and computational mechanistic modeling to determine metabolic fluxes under different experimental conditions and nutritional states. In addition, we will record metabolomics profiles to determine the effects on metabolite levels. Axonal mitochondrial function in terms of membrane potential and motility will be assessed by high resolution microscopy along with TP4. We will utilize Galloway-Movat syndrome (GAMOS) and spinocerebellar ataxia type 13 (SCA13) models. Mutations in these diseases lead to defects in autophagosome formation and degradation and thus will affect axonal energy homeostasis. In addition, we will investigate how these energy metabolites distribute between neuronal subcompartments and thus investigate metabolic interactions between them. To this end, neuronal cultures will be established in microfluidic chambers, established in TP1). We will add metabolite mixtures to different sites of axonal and somatodendritic compartments and measure the migration rate of autophagosomes. Mitochondrial motility and mitochondrial membrane potential will be determined by TMRM fluorescent labeling and in-device live cell imaging. Finally, using compartment-specific sampling of isotope-labeled compounds, we observe the distribution of metabolites between subcompartments and how cross-talk is affected in disease models.
Prof. Dr. Karsten Hiller
Technische Universität Carolo-Wilhelmina zu Braunschweig
BRICS - Braunschweig Integrated Centre of Systems Biology