Investigations of slow axonal transport reveal variation in both protein composition and the rate of movement. However, these studies involve a variety of nerve preparations in different species, and most lack the resolution needed to determine the kinetics of identified proteins. We have compared the axonal transport of slow-transported proteins in retinal ganglion cells and spinal motor neurons of young rats. Nine proteins that contribute to axonal structures were examined: the neurofilament triplet (NFT), alpha and beta tubulin, actin, fodrin, calmodulin, and clathrin. Axonally transported proteins were pulse-labeled by intraocular or intracord injections of 35S-methionine. After allowing sufficient time for labeled slow-component proteins to enter the spinal or optic nerves, consecutive 2-3 mm nerve segments were subjected to SDS-PAGE. Fluorographs were used as templates for locating the gel regions containing the above polypeptides, and the radioactivity in these regions was measured by liquid-scintillation spectrometry. In retinal ganglion cells, the peak of tubulin labeling advanced at 0.36 mm/d in association with the NFT and fodrin. The cotransport of tubulin and the NFT identified this complex as the slower subcomponent of slow transport, termed slow component a (SCa) and representing the movement of the microtubule-neurofilament network. The peaks of actin and calmodulin labeling were cotransported at 2.3 mm/d in near-register with peaks of fodrin and clathrin labeling. These 4 proteins, moving ahead of the NFT, identified this complex as SCb--the faster subcomponent of slow transport, which represents the movement of the cytoplasmic matrix and microtrabecular lattice. Both subcomponents had the same composition and rate as that reported for the optic axons of guinea pigs and rabbits, establishing a basic mammalian pattern. In spinal motor axons, the SCa tubulin peak advanced at 1.3 mm/d, and the SCb actin and calmodulin peaks were cotransported at 3.1 mm/d. Unlike optic axons, SCa in motor axons was more heavily labeled than SCb, and included labeled peaks of actin, clathrin, and calmodulin moving in register with the SCa tubulin peak. Actin was the most heavily labeled of these SCb proteins moving with SCa, and it left a higher plateau of radioactivity behind the advancing SCa peak. The SDS-PAGE labeling pattern for SCb did not differ from that seen in optic axons, except that some tubulin was found to form a peak that advanced in register with the actin and calmodulin peaks.(ABSTRACT TRUNCATED AT 400 WORDS)

Many of the structural and functional differences between axons are thought to reflect underlying differences in the biochemical composition and dynamic aspects of the axonal cytoskeleton and cytomatrix. In this study we investigated how the composition of the 2 slow components of axonal transport, SCa and SCb, which convey the cytoskeleton and cytomatrix, differs in axons that are structurally and functionally distinct. For this comparison we analyzed axons of retinal ganglion cells in the optic nerve (ON), axons of dorsal root ganglion (DRG) cells, and axons of ventral motor neurons (VMN) in adult rats. 35S-Methionine-labeled proteins transported with the peak of SCa and SCb were analyzed using high-resolution 2-dimensional polyacrylamide gels (2D-PAGE) and fluorography, and the amounts of major SCa and SCb proteins were quantified. The polypeptide composition of both SCa and SCb was found to be largely similar in DRG and VMN axons, but major qualitative as well as quantitative differences between these axons and ON axons were found. Notable among these were higher ratios of neurofilament protein to tubulin in SCa in DRG and VMN axons compared to ON axons, and significantly larger amounts of 2 microtubule-associated proteins relative to tubulin in SCa of ON axons than in both VMN and DRG axons. Tubulin was the major SCb protein in VMN and DRG axons, but it was not present in SCb in ON axons. Additionally, relatively larger amounts of 2 metabolic enzymes, creatine phosphokinase and nerve-specific enolase, were present in SCb in ON axons than in DRG or VMN axons. The results indicate that significant biochemical heterogeneity among different types of axons can be identified by examining the slow components of axonal transport.

The kinetics of slow axonal transport in newly regenerating axonal sprouts were compared with those in nonelongating axons. The slowly transported cytoskeletal proteins of ventral motor axons were prelabeled by microinjection of 35S-methionine into the spinal cord. Pulse-labeled slow transport "waves" were observed as they progressed from the surviving "parent" axon stumps (located proximal to a crush lesion) into regenerating "daughter" axon sprouts (located distal to the lesion). Prelabeled cytoskeletal elements of the parent axons were transported into daughter axons, to become distributed into 2 transport waves, "a" and "b." The rate and composition of these waves corresponded to the slow transport subcomponents, SCa and SCb. The shapes of the "a" and "b" waves suggested that the cytoskeletal elements had been reorganized at the junction between the parent and daughter axons. This hypothesis was supported by quantitative analyses of the transport distribution for individual radiolabeled cytoskeletal proteins (actin, spectrin, a 58-67 kDa group that includes microtubule-associated proteins, calmodulin, and tubulin). Specifically, during the first week of outgrowth, the amounts of radiolabeled calmodulin and 58-67 kDa proteins were greater in daughter axons than in nonregenerating control axons. These results support Paul Weiss's "conservative" model of axonal regeneration, which holds that the preexisting transported cytoskeletal elements that continually maintain axonal structure can also provide the cytoskeletal elements required for axonal regeneration. In addition, the results elucidate some of the reorganizational changes in cytoskeletal elements that occur when these are recruited from the parent axon to form daughter axons.

The slow component (SC) of axonal transport conveys structural proteins, regulatory proteins, and glycolytic enzymes toward the axon tip at 1–6 mm/day. Following axon interruption (axotomy), the rate of outgrowth corresponds to the rate of SCb—the fastest subcomponent of SC. Both axonal outgrowth and SCb accelerate 20–25% after axotomy. Tubulin and actin are the major proteins being carried by SCb. To further characterize the acceleration of SCb, we measured the equilibrium between subunits and polymers for both actin and tubulin. We radiolabeled newly synthesized proteins in rat motor neurons by microinjecting [35S]methionine into the spinal cord 7 days after crushing the sciatic nerve (85 mm from the spinal cord). Nerves were removed 7 days later for homogenization in polymer‐stabilizing buffer (PSB) and centrifugation, followed by SDS‐PAGE of supernatants (S) and pellets (P). We removed β‐tubulin, actin, and the medium‐weight neurofilament protein (NF‐M) from each gel by using the fluorogram as a template. After solubilizing gel segments for liquid scintillation spectrometry, we expressed counts as a polymerization ratio: P/[S + P]. In the nerve segments that contained radiolabeled SCb proteins, located 24–36 mm from the spinal cord, axotomy increased the polymerization ratio of SCb actin from 0.23 to 0.36 (P < 0.05) but had no effect on SCb β‐tubulin. In a separate experiment, we added 12 μM taxol to PSB to stabilize newly assembled microtubules. Adding taxol did not alter the polymerization ratio for SCb β‐tubulin in sham‐axotomized nerves but did increase the ratio in axotomized nerves, from 0.44 to 0.63 (P < 0.05); polymerization ratios for SCb actin were unaffected. We conclude that the assembly of microfilaments and microtubules increases to provide cytoskeletal elements for axon sprouts. The resulting loss of actin and tubulin subunits may play a role in the acceleration of SCb. © 1996 Wiley‐Liss, Inc.