Why Oligodendrocytes Keep Axons Alive, but Easily Die

In vertebrates, myelin is required for fast and reliable action potential propagation and long-term survival of axons. Unfortunately the oligodendrocytes that myelinate axons in the brain are easily damaged during energy deprivation. It is possible that mitochondria, which are important for several cell functions including ATP production and lipid metabolism, are involved in the high vulnerability of oligodendrocytes to energy deprivation. In neurons, these organelles constantly move, divide or fuse with each other, and disorders affecting the mitochondrial dynamics cause neuropathology. The dynamics of mitochondria in oligodendrocytes in situ have never been studied, and it is not known whether mitochondria would be able to enter, and move within the tightly wrapped myelin sheath. We have studied the localization and mobility of mitochondria in mature myelinating oligodendrocytes of organotypic brain slices from mouse cortex using confocal microscopy combined with specific expression of fluorescent proteins in oligodendrocyte mitochondria. We show that mitochondria are present not only in the oligodendrocyte somata and primary processes, but also in the myelin sheath, mainly in what appears to be the outer cytoplasmic ridge. We find that mitochondria in primary processes and in the myelin sheath move slower and less often compared with mitochondria in neurons. Previous studies have shown that mitochondria in neurons are stopped by increased intracellular calcium concentrations, for example during activation of NMDA receptors at postsynaptic spines. In oligodendrocyte processes however, we found the opposite: Glutamate increased the mobility of mitochondria, whereas inhibition of glutamate receptors or incubation in calcium free medium reduced mitochondrial mobility. Our data suggest that mitochondrial mobility may be regulated by different mechanisms in oligodendrocytes and neurons. Furthermore, the results indicate that glutamate signaling could play a role in regulating the mitochondrial mobility and thereby the local metabolic activity in oligodendrocytes.By electron microscopy, we show that myelin sheath mitochondria have an atypical morphology with few cristae. Since the cristae surface area is related to the rate of ATP production, our data suggest that myelin sheath mitochondria have a low ATP production. Instead, these mitochondria may be specialized for other tasks such as lipid metabolism.

BACKGROUND AND HYPOTHESES: The brain white matter is composed of nerve cell axons wrapped in myelin, a specialized extended plasma membrane synthesized by oligodendrocytes. Oligodendrocytes and myelin are important for proper brain function, but are rapid ly damaged in low energy conditions. Mitochondria are the main producers of the high-energy molecule adenosine triphosphate (ATP) and are therefore important in cell energy metabolism, but mitochondria have never been shown in CNS myelin. I hypothesize th at mitochondria in CNS myelin are sparse with restricted mobility. This could explain the vulnerability of oligodendrocytes to energy deprivation. I further hypothesize that lactate, the product of glycolysis, is released from myelin and taken up by axons . MAIN OBJECTIVES AND METHODS: I will use structured illumination microscopy (SIM) of organotypic mouse brain slice cultures transfected with a fluorescent mitochondrial marker to study the localization and mobility of mitochondria in myelin. By use of c onfocal time-lapse pH-imaging, I will test whether there is a transfer of lactate from myelin to axons. Finally, I will use electrophysiological methods to check whether lactate uptake into axons is important for action potential propagation. MOST CRITIC AL R&D CHALLENGES TO BE FACED: SIM is a newly developed super-resolution imaging technique that will provide the required spacial and temporal resolution for studying mitochondrial localization and mobility in myelin. So far, SIM has only been used for im aging of single cells in culture, but not on organotypic slice cultures. The SIM imaging on slice cultures is the most critical methodological challenge in my project. I will spend one year in a lab that is at the forefront of super-resolution imaging to perform SIM. Together with experts in the lab, I will develop a technique that enables imaging on the slice cultures.