Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothesis states that cytoskeletal proteins are transported within the axon as polymers. We tested this hypothesis by visualizing individual cytoskeletal polymers in living axons and determining whether they undergo vectorial movement. We focused on neurofilaments in axons of cultured sympathetic neurons because individual neurofilaments in these axons can be visualized by optical microscopy. Cultured sympathetic neurons were infected with recombinant adenovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time lapse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograde directions but with a strong anterograde bias. NF transport in both directions exhibited a broad spectrum of rates with averages of approximately 0.6-0.7 microm/sec. However, movement was intermittent, with individual neurofilaments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of the time moving and rest of the time paused. These results establish that the slow axonal transport machinery conveys neurofilaments.

No abstract

When the cerebral midline is lesioned in the embryo or neonate, the would-be callosal axons form neuromas. We have shown that an untreated Millipore implant inserted between the neuromas in young acallosal animals can support the migration of immature astrocytes that, in turn, support the de novo growth of commissural axons between the hemispheres. Since callosal neuromas persist into adulthood, we asked whether a critical period exists after which reactive glia no longer promote axon growth. We found that a critical period does exist and have documented a variety of changes in reactive gliosis that, in part, may lead to the axon growth-refractory state. In acallosal mouse postnates given untreated implants on or prior to day 8, glial fibrillary acidic protein (GFAP)+, stellate-shaped astrocytes migrated and attached to the implant by inserting foot processes into the pores of the filter. This form of gliotic response established an axon growth-promoting substratum within 24-48 hours after implantation. During this critical stage there was no evidence of scar formation or necrosis at or around the implant surface. However, when acallosal mice were implanted on or later than postnatal day 14, extensive tissue degeneration occurred, and a mixed population of astrocytes and fibroblasts invaded the surface of the filter, producing a dense scar. Reactive cells within the scar did not promote axonal outgrowth. To determine whether glia from neonates can influence the host environment and/or induce axonal regeneration in acallosal animals after the critical period, we harvested immature astrocytes on Millipore from critical-period mouse forebrains and transplanted the glia-coated prostheses into the brains of post-critical-period acallosal animals. Such transplants reduced glial scarring in the host, inhibited extensive bleeding and secondary necrosis, and promoted axonal regeneration. Our studies suggest that when controlled with a prosthesis, gliosis during the critical period is a beneficial process that can promote the reconstruction of malformed axon pathways; that in older animals a variety of changes in reactive glia and the extracellular matrix may work together to hinder axon regeneration after the critical period; and that axonal regeneration in the postcritical CNS may be stimulated by reintroducing an immature glial environment at the lesion site.