Characterization of the fast and slow components of axonal transport in retinal ganglion cells

The constituent proteins of the fast (110–150 mm/day) and slow (1.5–2 mm/day) components of axonal transport in the retinal ganglion cells of the rabbit were investigated. The fast and slow components were labelled by intraocular injection of (³H)- and (¹⁴C)-leucine, respectively. Subcellular fractionation of the optic nerve and tract and subsequent gel electrophoresis of the fractions showed that most of the soluble proteins moved with the slow phase of axonal transport, whereas only some of the soluble proteins were transported with the rapid phase. Extraction of the microsomal fraction with triton X-100 resulted in the solubilization of highly labelled proteins belonging to the rapid phase. These proteins showed a relatively low electrophoretic mobility.     

Acrylamide impairs fast and slow axonal transport in rat optic system

Effects of single and repeated doses of acrylamide on fast and slow axonal transport of radio labeled proteins following the injection of L-[4,5-³H] leucine have been studied in the optic system of male Sprague-Dawley rats. A single dose of acrylamide (100 mg/kg) had no effect, but higher concentrations (200–300 mg/kg) altered the distribution of fast axonally transported materials in optic nerves and optic tracts. Repeated doses of acrylamide (30 mg/kg/day, 5 days per week for 4 weeks) produced degeneration of tibial nerves but spared optic nerves and optic tracts. Fast axonal transport rate in optic axons was reduced by 50% (reduced to 4 mm/h from 8 mm/h) in acrylamide treated animals. Acrylamide also slowed the velocity of slow axonal transport of labeled proteins in optic axons to 1.0 mm per day from 1.3 mm per day. Since acrylamide impaired the rate of both fast and slow axonal transport in the absence of overt morphological damage, it can be concluded that deficit in axonal transport is an important factor in the pathogenesis of axonal degeneration in acrylamide neuropathy.     

Axonal transport of membranous and nonmembranous cargoes: a unified perspective

Membranous and nonmembranous cargoes are transported along axons in the fast and slow components of axonal transport, respectively. Recent observations on the movement of cytoskeletal polymers in axons suggest that slow axonal transport is generated by fast motors and that the slow rate is due to rapid movements interrupted by prolonged pauses. This supports a unified perspective for fast and slow axonal transport based on rapid movements of diverse cargo structures that differ in the proportion of the time that they spend moving. A Flash feature (http://www.jcb.org/cgi/content/full/jcb.200212017/DC1) accompanies this Mini-Review.     

TRANSPORT AND TURNOVER OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Axonal transport and turnover rate of proteins in the supraoptico-neurohypo-physial tract were studied after injection of ³⁵S cysteine into the region of the supraoptic nucleus. The proximo-distal migration of labelled proteins from the nerve cell bodies to the axon terminals in the neurohypophysis was followed by measuring the radioactivity of neurohypophysial proteins at various time intervals (4 h to 30 days) after isotope injection. A rapidly transported phase of proteins with a minimal transport rate of approximately 60 mm/day was demonstrated. An accumulation of protein-bound radioactivity was also observed in the neural lobe at 9 days after isotope injection, representing slowly transported proteins (0-5 mm/day). In addition, an intermediate phase of axonal transport (1-5 mm/day) was found. Fractionation of neurohypophysial proteins by polyacrylamide gel disc electrophoresis revealed that a predominating portion of the radioactivity was recovered in a single protein component (fraction A) at 4 h as well as at 30 days after isotope injection. This protein component was shown to be a constituent both of the rapid and the slow phase of axonal transport. With time an increasing amount of radioactivity was found in another protein component (fraction B), which reached a maximum at 14 days after injection and then remained fairly constant up to 30 days. When the turnover rates of neurohypophysial proteins were estimated, a half-life of 1-2 days and 8 days was calculated for the rapidly and slowly transported proteins, respectively.     

SLOW AXONAL TRANSPORT OF LABELLED PROTEINS IN SENSORY FIBRES OF RABBIT VAGUS NERVE

Both rapid (415 mm/day) and slow (24 mm/day) rates of axonal transport of proteins were found in sensory fibres of rabbit vagus nerve after injection of [³H]leucine into the nodose ganglion in vivo. The slow phase of transport was dependent on contact between the cell bodies and the nerve trunk, and did not continue under in vivro conditions. The results suggest some difference between the mechanisms of fast and slow transport.     

Components of fast and slow axonal transport in the goldfish optic nerve

The composition of the fast and slow components of axonal transport in the goldfish optic nerve was investigated, using specific radioactive precursors injected into the eye. Tritiated glucosamine and fucose label macromolecules, presumably glycoproteins, which are rapidly transported from the eye to the optic tectum. Material labeled with these precursors is not evident in the slowly transported component. Glucosamine and fucose incorporation are blocked when a protein synthesis inhibitor, acetoxycycloheximide, is injected into the eye concurrently with the precursors. As well as labeling macromolecules, ³H-glucosamine and ³H-N-acetylmannosamine ( a precursor of sialic acids) also label rapidly-transported chloroform-methanol-extractable material which may contain transported glycolipids. Two procedures were used to show that the slow component of axonal transport contains tubulin, a protein characteristic of the microtubules:

• (a) Tracer doses of tritiated colchicine injected into the eye label a wave of radioactivity which moves 0.5 mm/day, the rate of slow axonal transport in the goldfish optic nerve. We believe this wave represents the movement of colchicine which is bound to colchicine-binding protein moving in the slow component of axonal transport.
• (b) Tritiated proline labels a slowly transported protein which is precipitated by vinblastine and has a mobility on polyacrylamide gels comparable to authentic tubulin. These results indicate that the fast and slow components of axonal transport each provide specific chemical substances to the nerve endings.

     

Multiple-rate components of axonally transported proteins in the hypothalamo-neurohypophysial system of the rat

The transport of labeled proteins from the hypothalamus to the neurohypophysis following 35S-methionine injection into the rat supraoptic nucleus was studied using a unique approach adapted for the study of short-axon systems. Multiple-rate components to those found in other neuronal systems were demonstrated. Neurosecretory vesicle-containing proteins (e.g., neurophysins) were transported at fast rates (greater than 120 mm/day), whereas the cytoskeletal protein, actin, moved principally in the slow component of transport. Two-dimensional gel electrophoresis was used to analyze the diverse patterns of labeled proteins found in the various rate components of axonal transport in this system.     

Changes in the axonal protein transport under conditions of elevated functional activity of the neurons

The prolonged nonintensive physical activity by swimming without load (12 +/- 2 h.) has no effect on the overall amount of fast and slow transported proteins of transport velocity in rat central and peripheral sensory fibres of the sciatic nerve. However, the rate of fast axonal transport in the motor fibres decreases by 18% and the amount of proteins by a factor of 2 as compared with control. The rate of slow axonal transport does not change, but the mean level of transported labeled proteins decreases by 1.9 times. The relatively short-term but more intensive activity (swimming with the load during 60 +/- 10 min.) provokes an increase of the rate by 10% and the overall amount of fast transported proteins by 2 times. The rest of the animals during 6 h. returns the above parameters to control values. A suggestion is made that the rate and the amount of transported proteins depend on the variations in functional state of the neurons and their axons.     

Anterograde Components of Axonal Transport in Motor and Sensory Nerves in Experimental 2,5-Hexanedione Neuropathy

Anterograde slow and fast axonal transport was examined in rats intoxicated with 2,5-hexanedione (1 g/kg/week) for 8 weeks. Distribution of radioactivity was measured in 3-mm segments of the sciatic nerve after labelling of proteins with [35S]methionine or [3H]leucine and glycoproteins with [3H]fucose. The axonal transport of the anterograde slow components was examined after 25 (SCa) and 10 days (SCb), in motor and sensory nerves. SCa showed an increased transport velocity in motor (1.25 +/- 0.08 mm/day versus 1.01 +/- 0.05 mm/day) and in sensory nerves (1.21 +/- 0.13 mm/day versus 1.06 +/- 0.07 mm/day). The relative amount of labelled protein in the SCa wave in both fiber systems was also increased. SCb showed unchanged transport velocity in motor as well as in sensory nerves, whereas the amount of label was decreased in the motor system. Anterograde fast transport in motor nerves was examined after intervals of 3 and 5 h, whereas intervals of 2 and 4 h were used for sensory nerves. Velocities and amounts of labelled proteins of the anterograde fast component remained normal. We suggest that the increase in protein transport in SCa reflects axonal regeneration.     

Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins

Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000-dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations.     

Fast axonal transport in motor nerve regeneration

This report describes the fast axonal transport of [³H]-leucine-labeled proteins in regenerating rat sciatic motor nerves. A normal rate of fast transport (383 ± 33 mm/day) was present in the regenerating sprouts, as well as in the central stumps. The rapidly transported proteins passed the level of axotomy without impediment, and accumulated in the endings of the regenerating sprouts, as shown by electron microscope autoradiography. In addition, transported proteins accumulated in terminal neuromas. The relative amount of protein-incorporated radioactivity in the crest of fast transport in the regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating sprouts as in normal axons; during regeneration fast transport appears to add newly synthesized materials to the growing tip.     

Rate of Movement and Composition of Rapidly Transported Proteins in Regenerating Olfactory Nerve

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.     

Slow components of axonal transport: two cytoskeletal networks

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.     

Protein synthesis in axons and its possible functions

Brain Cell Biology - Proteins synthesized in neuronal cell bodies are transported along axons by fast and slow axonal transport. Cytoskeletal proteins and cytosolic proteins that travel by slow...     

Simulation of axoplasmic transport

We have analysed a kinetic model of axonal transport by simulating experimental tracer profiles. The existence of three phases of axoplasmic transport is assumed: fast anterograde, slow anterograde and retrograde. Each phase has its characteristic velocity. Transported materials are postulated to shift between these phases. Also catabolism and sequestration of material is allowed for in our model. Thus, we have set up equations which contain axonal transport, diffusion and cross-over terms. The rate constants of material shifts were determined by computer fitting to experimental data. Best-fitted values of the rate constants for transfer of material between the fast and slow phases were both 2 X 10(-5) sec-1, while the rate constants for transfer between the fast and retrograde phases were both 1 X 10(-5) sec-1. The rate constant of material loss from the slow phase to the extracellular space was 1 X 10(-6) sec-1. The material shift between the slow and retrograde phases was negligibly small. These data show that there is exchange of material between the fast and slow phases and between the fast and retrograde phases. However, there is no significant exchange between the slow and retrograde phases. Diffusion was found to have only a minor effect on the profiles. The velocity of the fast anterograde track in cold-blooded animals was predicted to be around 200 mm/day, or, in other words, to be close to experimentally observed values of the fast anterograde component of axonal transport.     

Linkers, packages and pathways: new concepts in axonal transport

The molecular mechanisms that generate efficient and directed transport of proteins and organelles in axons remain poorly understood. In the past year, many studies have identified specific transmembrane or scaffold proteins that might link motor proteins to their cargoes. These studies have also identified previously unsuspected pathways and raised the intriguing possibility that pre-packaged groups of functionally related proteins are transported together in the axon. Evidence suggests that fast molecular motor proteins have a role in slow axonal transport, and the axonal transport machinery has been implicated in the genesis of neurodegenerative diseases.     

Quantitative Analysis of Axonal Transport of Cytoskeletal Proteins in Chicken Oculomotor Nerve

We studied the axonal transport characteristics of major cytoskeletal proteins: tubulin, the 69,000 molecular weight protein of chicken neurofilaments, and actin. After intracerebral injection of [35S]methionine, we monitored the specific radioactivity of these proteins as they passed through a very short nerve segment of the chicken oculomotor nerve. Specific radioactivities were assessed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The transport patterns obtained for tubulin and the neurofilament protein were very similar, corresponding to transport rate ranges of 1-15 and 1-10 mm/day, respectively. A narrower velocity range of 3 to 4.3 mm/day was found for actin. Tubulin and the neurofilament protein appeared to be largely dispersed during the course of their transit along the nerve. The radioactivity associated with the proteins studied persisted in the nerve segment for a long time after the bulk of the labeled molecules had swept down. Finally, none of these proteins was observed to be transported with the fast axonal transport.     

EFFECTS OF COLCHICINE ON AXONAL TRANSPORT IN PERIPHERAL NERVES

—Colchicine injected intracisternally markedly inhibited the rapid migration (300-400 mm/day) of labelled proteins in the hypoglossal and vagus nerve of the rabbit. The transport of acetylcholinesterase (EC 3.1.1.7) and choline acetyltransferase (EC 2.3.1.6) previously shown to move with the slow (5-26 mm/day) phase of axoplasmic transport in these nerves, was only partially blocked. In view of this differential effect on axonal flow, we suggest that the neurotubules, on which colchicine acts preferentially, are primarily involved in the rapid (300-400 mm/day) axoplasmic flow. After local injection of colchicine into the nerves both the rapidly migrating labelled proteins and the enzymes (AChE and ChAc) accumulated above the site of injection to the same degree as they accumulate above a nerve ligation. Since this blockage of enzyme transport occurred after concentrations of colchicine much higher than those used for intracisternal injections these findings after local injection may represent more severe effects on axonal transport systems.     

Nerve-specific enolase and creatine phosphokinase in axonal transport: soluble proteins and the axoplasmic matrix

The axonal transport of two soluble enzymes of intermediary metabolism was evaluated: the nerve-specific form of the glycolytic enzyme enolase (NSE) and the brain isozyme of creatine phosphokinase (CPK). Previously, little was known about the intracellular movements of the soluble proteins of the cell. Although the soluble enzymes of glycolysis and other pathways of intermediary metabolism have been thought to be freely diffusing in the cytosol, many are required in the axonal extremities of the neuron and must be transported to the sites of utilization. Comigration of purified enzymes with radioactive polypeptides associated with specific rate components of axonal transport in two-dimensional gel electrophoresis indicates that both NSE and CPK move in the axon solely as part of the group of proteins known as slow component b (SCb) at a rate of 2 mm/day. Peptide mapping following limited proteolysis confirmed identification of NSE and CPK in SCb. Materials associated with SCb have been shown to move coherently along the axon and to behave as a discrete cellular structure, the axoplasmic matrix. Association of two soluble enzymes, NSE and CPK, with the SCb complex of proteins requires a reevaluation of the assumption that these and other soluble proteins of the axon are freely diffusible.     

Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons

Abstract: Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish ( Procambarus clarkii ) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.