Onderzoek van Axonaal Mitochondriaal Transport: Wisselwerking met Neuronale Activiteit & een Mogelijke Nieuwe (Bij)Werking van Anesthetica

Due to their large scale and uniquely branched architecture, neurons critically rely on active transport of mitochondria in order to match energy production and calcium buffering to local demand. Consequently, defective mitochondrial trafficking is implicated in various neurological and neurodegenerative diseases. A key signal regulating mitochondrial transport is intracellular calcium. Elevated Ca2+ levels have been demonstrated to inhibit mitochondrial transport in many cell types, including neurons. However, it is currently unclear to what extent calcium-signaling regulates axonal mitochondrial transport during realistic neuronal activity patterns. We created a robust pipeline to quantify with high spatial resolution, absolute Ca2+ concentrations. This allows us to monitor Ca2+ dynamics with pixel precision in the axon and other neuronal compartments. We found that axonal calcium levels scale with firing frequency in the range of 0.1 - 1 μM, whereas KCl-induced depolarization generated levels almost a magnitude higher. As expected, prolonged KCl-induced depolarization did inhibit axonal mitochondrial transport in primary hippocampal neurons. However, physiologically relevant neuronal activity patterns only inhibited mitochondrial transport in axonal segments which made connections to a target neuron. In 'non-connecting' axonal segments, we were unable to trigger this inhibitory mechanism using realistic firing patterns. Thus, we confirm that neuronal activity can indeed regulate axonal mitochondrial transport, and reveal a spatial pattern to this regulation which went previously undetected. Together, these findings indicate a potent, but localized role for activity-related calcium fluctuations in the regulation of axonal mitochondrial transport. General anesthetics (GAs) induce a reversible state of 'central nervous system depression' by enhancing inhibitory neurotransmission or reducing excitatory neurotransmission, enabling invasive medical procedures. However, despite a growing body of research on the interaction between GAs and ion channels, a unifying theory of anesthetic action is currently lacking. Therefore, GAs likely target other facets of neuronal functioning, contributing to the (un)desired effects of these drugs. Interestingly, recent reports suggest that GAs can affect key components of the mitochondrial transport system, like the tracks and motors. However, it is currently unclear whether GAs actually interfere with mitochondrial transport dynamics in living neurons. To investigate this, we performed a pilot study on propofol, one of the most commonly used drugs for induction and maintenance of anesthesia globally. Two independent sets of experiments demonstrate that propofol indeed inhibits axonal mitochondrial transport in primary hippocampal neurons. The main effect of propofol is a reduction in the number of mobile mitochondria and a shift towards lower transport velocities. The effects were concentration-dependent and readily detectable, even at clinically relevant concentrations. Our next aim was to determine whether transport obstruction is a common action shared among other GAs. To screen a panel of GAs, however, a new method was needed since kymograph-based quantification of transport dynamics is too laborious and time-consuming to enable high-throughput testing. Therefore, we created a new method to automatically track and analyze (axonal) mitochondrial transport in dense samples, by combining image pre-processing steps, a pre-existing toolbox for single-particle tracking, and a number of custom-written MATLAB scripts. A first validation of this method was performed using temperature-modulation to mimic drug-induced defects in transport. In two separate experiments, our auto-tracking method successfully detected the alterations in transport dynamics induced by temperature modulation, indicating its potential value as a screening tool. Future research will focus on the application of this new method for the purposes of screening GAs, and the mechanism(s) of action behind their obstruction of axonal mitochondrial transport. This line of research will offer new insights in the pharmacodynamics of GAs as well as broaden our understanding of transport regulation.