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.     

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.     

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.     

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.     

Slowly migrating axonal polypeptides. Inequalities in their rate and amount of transport between two branches of bifurcating axons

Polypeptides in the dorsal root ganglion (L5) of the adult rat were radioactively labeled, and components slowly migrating in the sciatic nerve (peripheral axons) and dorsal root (central axons) were analyzed, using SDS-polyacrylamide slab gel electrophoresis and fluorography. In particular, the transport rates and amounts of six major polypeptides, i.e., the triplet (reference 15; with mol wts of 200,000, 160,000, and 68,000 daltons), alpha- and beta-tubulins and actin were compared between the two axon branches. In peripheral axons, fronts of the triplet, tubulins, and actin migrate at 2-3 mm/d, 9-13 mm/d and approximately 19 mm/d, respectively. The corresponding values in central axons are 1-2 mm/d, 3-4 mm/d, and approximately 4 mm/d, indicating an obvious asymmetry in the transport rate between the two branches of bifurcating axons. A greater amount of labeled triplet, tubulins, and actin each is found to migrate in peripheral than in central axons. Another striking aspect of asymmetry between the two branches relates to the tubulins/triplet ratio which is significantly higher in the peripheral branch. Considerable proportions of radioactivities associated with tubulins and actin in the ganglion are nonmigratory, which are thought to derive mostly from periaxonal satellite cells. In contrast, most if not all of the labeled triplet is migratory, suggesting a virtual absence of triplet polypeptides in satellite cells. The possible significance of peripheral-central inequalities in slow axoplasmic transport is discussed from the viewpoints of axon volume and axonal outgrowth.     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY 1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

Transport complexes associated with slow axonal flow

Cytoskeletal proteins-neurofilament polypeptides, tubulin and actin-are transported along axons by slow transport. How or in what form they are transported is not known. One hypothesis is that they are assembled into the cytoskeleton at the cell body and transported as intact polymers down the axon. However, recent radiolabeling and photobleaching studies have shown that tubulin and actin exist in both a mobile phase and a stationary phase in the axon. Consequently, it is more likely that cytoskeletal proteins move along the axon in some form of transport complex and are assembled into a cytoskeleton which is stationary. In this overview we discuss these topics and consider the evidence for the existence of transport complexes associated with slow axonal flow. Such evidence includes the slow transport of particulate complexes containing tubulin and neurofilament polypeptides along reconstituted microtubules in vitro, and the coordinate slow transport of actin with actin-binding in vivo.Special issue dedicated to Dr. Lawrence Austin.     

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 Slow Component a in Sciatic Nerves of Hypo-and Hyperthyroid Rats

Axonal transport of slow component a was studied in dorsal root afferents of the sciatic nerves of hypo- and hyperthyroid rats. Three experimental groups of rats were made hypothyroid at the age of 12 weeks by the administration of 131I. From the age of 22 weeks to the end of the study, the groups were treated with daily subcutaneous injections of thyroxine in various doses to make them hypo-(0 microgram/100 g), normo- (1 microgram/100 g), and hyperthyroid (6 micrograms/100 g), respectively. The hypothyroid group had a moderate thyroid hormone deficiency (a serum triiodothyronine level of 0.19 +/- 0.10 nmol/L and a heart/body weight ratio of 1.87 +/- 0.09 g/kg at time of killing compared with 0.60 +/- 0.09 nmol/L and 2.18 +/- 0.06 g/kg, respectively, for the control group). The hyperthyroid group was severely deranged, with serum triiodothyronine being 3.30 +/- 0.37 nmol/L and a heart/body weight ratio of 3.11 +/- 0.16 g/kg. The hypothyroid rats showed a reduction in mean velocity for the transport of slow component a (0.80 +/- 0.07 mm/day compared with 0.91 +/- 0.05 mm/day in the controls). The width of the wave of activity was smaller for the hyperthyroid group than for the control group (6.6 +/- 0.7 mm compared with 8.1 +/- 1.2 mm), suggesting an increased clearance of the axonally transported activity in the proximal axon. A decrease in transport of slow component a in hypothyroidism may be the explanation of peripheral neuropathy with axonal degeneration occasionally seen in patients with severe myxoedema.     

Slow axonal transport: fast motors in the slow lane.

The bulk of neuronally synthesized proteins destined for the axon is transported in a phase of transport approximately 100 times slower (1mm/day) than the vesicular traffic of fast axonal transport (100mm/day). Of late, a number of studies have shed considerable light on the controversies and mechanisms surrounding this slow phase of axonal transport. Along-standing controversy has centered on the form of the transported proteins. One major transport cargo, neurofilament protein, has now been seen in a number of contexts to be transported primarily in a polymeric form, whereas a second cargo tubulin is transported as a small oligomer. The development of techniques to visualize the slow transport process in live cells has demonstrated that instantaneous motions of transported neurofilaments, and presumably other slow transport cargoes, are fast, bidirectional and interspersed with long pauses. This and additional biochemical efforts indicate that traditional fast motors, such as conventional kinesin and dynein, are responsible for these fast motions.     

The Role of the Axonal Cytoskeleton in Diabetic Neuropathy

The neuropathy associated with diabetes includes well documented impairment of axonal transport, a reduction in axon calibre and a reduced capacity for nerve regeneration. All of those aspects of nerve function rely on the integrity of the axonal cytoskeleton. Alterations in the axonal cytoskeleton in experimental diabetes include an insulin-dependent non-enzymatic glycation of actin that is reflected in increased glycation of platelet actin in the clinical situation. There is a reduced synthesis of mRNA for the isoforms of tubulin that are associated with nerve growth and regeneration and an elevated non-enzymatic glycation of peripheral nerve tubulin in both diabetic patients and diabetic animals. mRNAs for neurofilament proteins are selectively reduced in the diabetic rat and the post-translational modification of at least one of the neurofilament proteins is altered. There is some evidence that altered expression of isoforms of protein kinases may contribute to these changes.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.     

Stochastic simulation of neurofilament transport in axons: the "stop-and-go" hypothesis

According to the "stop-and-go" hypothesis of slow axonal transport, cytoskeletal and cytosolic proteins are transported along axons at fast rates but the average velocity is slow because the movements are infrequent and bidirectional. To test whether this hypothesis can explain the kinetics of slow axonal transport in vivo, we have developed a stochastic model of neurofilament transport in axons. We propose that neurofilaments move in both anterograde and retrograde directions along cytoskeletal tracks, alternating between short bouts of rapid movement and short "on-track" pauses, and that they can also temporarily disengage from these tracks, resulting in more prolonged "off-track" pauses. We derive the kinetic parameters of the model from a detailed analysis of the moving and pausing behavior of single neurofilaments in axons of cultured neurons. We show that the model can match the shape, velocity, and spreading of the neurofilament transport waves obtained by radioisotopic pulse labeling in vivo. The model predicts that axonal neurofilaments spend approximately 8% of their time on track and approximately 97% of their time pausing during their journey along the axon.     

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with³H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of³²P into purified neurofilament proteins when they were incubated with³²P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.     

Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase.

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

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.     

The long-term effects of a single injection of beta,beta'-iminodipropionitrile on slow axonal transport in the rat

Following a single intraperitoneal injection of beta,beta'-iminodipropionitrile (IDPN) simultaneously with or 5 weeks after injection of [35S]methionine into the ventral horn area of rat spinal cord, the changes of slowly migrating axonal proteins were analyzed electrophoretically up to 10 weeks after labeling, and the following results were obtained. After a single injection of IDPN, only the transport of neurofilament proteins is inhibited, leaving that of tubulin and actin almost unaffected, though a small portion of the former was retarded through the interaction with neurofilaments. The inhibitory effect of IDPN on neurofilament transport is not a complete blockage, but a slowing of the rate of transport to about a half of the control with a possible short halting period just after IDPN treatment. The dose-response data indicate a threshold between 0.5 and 1.0 g/kg of body weight, increasing the dose above which does not further affect the neurofilament transport. The transport of neurofilaments is uniformly impaired by IDPN along the whole axon.     

Effects of taxol on slow and fast axonal transport

Axonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.     

Electrophoretic Analysis of Axonally Transported Proteins in Toad Retinal Ganglion Cells

As a preliminary step to studying changes in axonal transport in regenerating neurons, we have analyzed the composition and organization of polypeptides normally axonally transported in a neuronal system capable of regeneration, i.e., the retinal ganglion cells of the toad, Bufo marinus. We labeled proteins synthesized in the retina with 35S-methionine and subsequently used one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to analyze labeled, transported proteins in tissues containing segments of the axons (the optic nerve, optic tract, and optic tecta) of the retinal ganglion cells. The transported polypeptides could be divided into five groups according to their apparent transport velocities. Many of the polypeptides of each group were electrophoretically similar to polypeptides of corresponding groups previously described in rabbit and guinea pig retinal ganglion cells, and in some cases, additional properties of the polypeptides indicated that the transported materials of the two vertebrate classes were homologous. These results serve two purposes. First they establish the retinal ganglion cells of the toad Bufo marinus as a model system in which changes in gene expression related to regeneration may be studied. Second they show that the organization and many aspects of the composition of axonal transport in retinal ganglion cells have been conserved in animals as unrelated as amphibians, and mammals.