Assessment of Mitochondrial Trafficking as a Surrogate for Fast Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Spinal Motor Neurons

Axonal transport is essential for the development, function, and survival of the nervous system. In an energy-demanding process, motor proteins act in concert with microtubules to deliver cargoes, such as organelles, from one end of the axon to the other. Perturbations in axonal transport are a prominent phenotype of many neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe a simple method to fluorescently label mitochondrial cargo, a surrogate for fast axonal transport, in human induced pluripotent stem cell-derived motor neurons. This method enables the sparse labeling of axons to track directionality of movement and can be adapted to assess not only the cell autonomous effects of a genetic mutation on axonal transport but also the cell non-autonomous effects, through the use of conditioned medium and/or co-culture systems.