The mobility of neurofilaments in mature myelinated axons of adult mice.

Studies in cultured neurons have shown that neurofilaments are cargoes of axonal transport that move rapidly but intermittently along microtubule tracks. However, the extent to which axonal neurofilaments move in vivo has been controversial. Some researchers have proposed that most axonally transported neurofilaments are deposited into a persistently stationary network and that only a small proportion of axonal neurofilaments are transported in mature axons. Here we use the fluorescence photoactivation pulse-escape technique to test this hypothesis in intact peripheral nerves of adult male hThy1-paGFP-NFM mice, which express low levels of mouse neurofilament protein M tagged with photoactivatable GFP. Neurofilaments were photoactivated in short segments of large, myelinated axons and the mobility of these fluorescently tagged polymers was determined by analyzing the kinetics of their departure. Our results show that >80% of the fluorescence departed the window within 3 hours after activation, indicating a highly mobile neurofilament population. The movement was blocked by glycolytic inhibitors, confirming that it was an active transport process. Thus, we find no evidence for a substantial stationary neurofilament population. By extrapolation of the decay kinetics, we predict that 99% of the neurofilaments would have exited the activation window after 10 hours. These data support a dynamic view of the neuronal cytoskeleton in which neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon, even in mature myelinated axons. The filaments spend a large proportion of their time pausing, but on a time scale of hours, most of them move. SIGNIFICANCE STATEMENT Neurofilaments are space-filling structural polymers that support the growth of axon caliber, which is an important determinant of axonal conduction velocity. Neurofilaments are also cargoes of axonal transport, but the extent to which they move in adult axons has been controversial. The present study resolves this controversy by demonstrating that most, if not all, neurofilaments are mobile in mature myelinated axons of adult mice. Thus, the neurofilament network in axons is remarkably dynamic. This has important implications for the mechanisms by which neurofilament transport and axon morphology are regulated in health and disease.