Axon myelination is essential for neuronal function. Central nervous system (CNS) demyelinating diseases include conditions that result in damage to the myelin sheath that surrounds nerve fibers. MS represents the most common and investigated condition. It affects more than 2 million people worldwide, greatly impacting patient’s quality of life and representing strong economic and health concerns for society. Nevertheless, studies addressing alterations in mitochondrial biology in demyelinated axons are rare and, so far, do not provide a clear view on the degree of organelle dysfunction. Using primary neurons from hippocampus and spinal cord treated with lysolecithin to induce axon demyelination, we will combine biochemical and molecular biology approaches, and functional methodologies by live-cell fluorescence microscopy, to determine the consequences of axon demyelination in mitochondria biology. This will likely provide clear insights into the mechanisms that drive axon degeneration in CNS demyelinating diseases and the pathogenic mechanisms of other common adult-onset neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, which also show myelin pathology.