Axonal Transport Characteristics of Gangliosides in Sensory Axons of Rat Sciatic Nerve

The distribution of axonally transported gangliosides and glycoproteins along the sciatic nerve was examined from 3 h to 4 weeks following injection of[3H]glucosamine into the fifth lumbar dorsal root ganglion of adult rats. Incorporation of labeled precursor into these glycoconjugates reached a maximal level in the ganglion within 6 h. Outflow patterns of radioactivity for glycoproteins showed a well-defined crest with a transport rate of approximately 330 mm/day. In contrast, the crest of transported gangliosides was continuously attenuated, implying a significant deposition along the axon, and an alternative method of calculating velocity was required. Analysis of accumulation of labeled material at double ligatures demonstrated both anterograde and retrograde transport of glycoproteins and gangliosides and allowed for the calculation of an anterograde transport rate of about 270 mm/day for each. Additional evidence of ganglioside transport is provided in that the TLC pattern of transported radioactive gangliosides accumulating at a ligature is significantly different from the pattern seen in the dorsal root ganglion or following intraneural administration of the labeled precursor. These data indicate that gangliosides are transported at the same rapid rate as glycoproteins but are subject to a more extensive exchange with stationary material than are glycoproteins.     

Electrophoretic analysis of axonally transported proteins in rabbit vagus nerve

Proteins synthesized in the nodose ganglia of rabbits were radiolabeled with 35S-methionine and the proteins present in the vagus nerve, at various times later, were analyzed by SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Three major groups of proteins were transported as waves of radioactivity within the nerve at rates of 15-17 mm/h, 12-15 mm/day, and 25-30 mm/day. The front of the fastest wave was composed of two proteins only, of apparent molecular weights 21,000 and 24,000. These were followed after a delay by a number of proteins of higher molecular weight, traveling at the same fast rate. The 25-mm/day wave contained several proteins including a major one of molecular weight 43,000 while the 12-mm/day wave was composed entirely of two proteins of molecular weights 54,000 and 56,000. These groups of slowly transported proteins are therefore similar to those transported much more slowly in other mammalian nerves, with the exception that no proteins with molecular weight similar to the neurofilament proteins could be detected. We have confirmed the dependence of slow transport for both groups of proteins on contact between cell body and axon and suggest that it may be a general phenomenon in all mammalian nerves.     

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: 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.     

Slow Axonal Transport of Structural Polypeptides in Rat, Early Changes in Streptozocin Diabetes, and Effect of Insulin Treatment

The synthesis and transport of slowly transported polypeptides in sciatic nerves of rats was investigated by [35S]methionine pulse labeling and gel electrophoresis in control, diabetic, and insulin-treated diabetic rats. To detect very early changes diabetes was induced by streptozocin only 5 days prior to the labeling of the dorsal root ganglion cells. Fourteen days were allowed for axonal transport. In this experimental system, the neurofilament triplet is transported at an apparent velocity of 1.1 +/- 0.1 mm/day (mean +/- SD). The actin-related complex, including actin and two polypeptides of 87 kilodaltons and 37 kilodaltons, was transported at a velocity of 2.6 +/- 0.2 mm/day. For alpha- and beta-tubulin we found an apparent transport velocity of 2.2 +/- 0.1 mm/day, placing it between actin and the neurofilament triplet. The diabetic rats had a selective 32% decrease in the amount of the heaviest neurofilament subunit: 0.47 +/- 0.19% of trichloroacetic acid-insoluble radioactivity versus 0.69 +/- 0.17% in controls; 2p less than 0.05. This decrease was associated with a proximal accumulation of the two lighter neurofilament subunits. Insulin treatment of a diabetic group failed to normalize the changes of axonal transport and additional changes suggesting a hypoglycemic injury was observed.     

Effect of lectins on the transport of food ingredients in Caco-2 cell cultures

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03 approximately 2 mmol/L) in the presence or absence of lectins (1 approximately 180 microg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway.     

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.     

Position-sensitive detector studies of the axonal transport of a pulse of radioisotope

Axonal transport of a pulse of 35S-methionine-labelled material was studied in vitro in amphibian sciatic nerve using position sensitive detectors. Following formation of a pulse of activity using the cold block technique, the nerve was ligated proximal and distal to the pulse and its movement monitored at room temperature (22.5-23.5 degrees C) for up to 16 h. Material transported in the anterograde direction did so with an average maximum velocity of 147 mm/d. The pulse was found to disperse at an average rate of 0.23 mm/mm travel; however, dispersion was found to vary from preparation to preparation more than would be predicted from experimental error alone. Label was observed to reverse direction at the distal ligature in only 2 of 13 preparations. Reversal of label began within approximately 0.4 h of first arrival, and the most rapidly retrogradely transported material moved at a velocity of 80% that of the most rapidly anterogradely transported material.     

Acrylamide-Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve

The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of approximately 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.     

Differential Axonal Transport of Soluble and Insoluble τ in the Rat Sciatic Nerve

Abstract: Axonal transport of microtubule-associated protein τ was studied in the motor fibers of the rat sciatic nerve 1–4 weeks after labeling of the spinal cord with [³⁵S]methionine. As 60–70% of low molecular weight τ in this system was found to be insoluble in 1% Triton-containing buffer, labeled proteins in 6-mm consecutive nerve segments were first separated into Triton-soluble and insoluble fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-tau antibody confirmed the presence of τ among labeled, transported proteins in both fractions. Isoform composition of labeled τ was similar to that of bulk axonal τ, the most acidic species with apparent molecular mass of 66 kDa being the major component. Transport profiles obtained by measuring radioactivities associated with this major isoform showed that soluble and insoluble τ were transported at different rates. Insoluble τ, which contained the majority of τ-associated radioactivity, was transported at 1.7 mm/day in slow component a (SCa), whereas soluble τ was transported faster, at 3 mm/day, corresponding to the rate of slow component b (SCb). Cotransport of insoluble τ with insoluble tubulin in SCa suggests its association with stable microtubules.     

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.     

Bidirectional axonal transport of 16S acetylcholinesterase in rat sciatic nerve

Axonal transport of the 16S Molecular form of acetylcholinesterase (16S-AChE) in doubly ligated rat sciatic nerves was studied by means of velocity sedimentation analysis on sucrose gradients. This form of AChE was selectively confined to motor, and not to sensory, fibers in the sciatic nerve, where it represented 3--4% of total AChE. Its activity increased linearly with time (4--20 hr) in nerve segments (7 mm) proximal to the central ligature (4.5 mU/24hr) and distal to the peripheral ligature (2.0 mU/24 hr). From the linear rates of accumulation of 16S-AChE, we conclude that the enzyme is conveyed by anterograde and retrograde axonal transport at velocities close to those previously defined for the movement of total AChE (410 mm/day, anterograde; 220 mm/day, retrograde). The transport of AChE molecular forms, other than the 16S form, could not be resolved presumably due to their presence in blood as well as at extraaxonal sites. The present findings are consistent with the view that in rat sciatic nerve most, if not all, of the small portion of total AChE (approximately 3%) which is transported may be accounted for by 16S-AChE.     

Axoplasmic Transport and Turnaround of Neurotoxic Esterase in Hen Sciatic Nerve

We have recently found that there is a proximo-distal delay in the recovery of neurotoxic esterase (NTE) following inhibition along the sciatic nerve of the hen. To determine whether this delay could be due to a requirement for the transport of newly synthesized NTE from the cell body, we investigated the transport of NTE by measuring the rate of accumulation of activity at either one or two ligations. Although rapid turnaround of accumulated protein confounds calculation of the transport rate, it appeared that NTE is transported down the hen sciatic nerve at a rate close to 300 mm/day. Acetylcholinesterase (AChE) was found to be transported at a rate of about 500 mm/day, which is close to the expected rate of fast axoplasmic transport in the chicken. The relatively rapid turnaround of NTE compared with the retrograde transport rate precluded the estimation of a retrograde transport rate. A model is presented that accounts for turnaround as a result of exchange between mobile and stationary transport pools. Exchange of NTE between pools may account for the rapid turnaround of NTE described in this paper and for the proximo-distal delay in recovery as a dilution of newly synthesized NTE in the anterograde fast transport pool by inhibited protein as it travels down the nerve.     

INFLUENCE OF TEMPERATURE ON THE VELOCITY AND ON THE ISOTOPE PROFILE OF SLOWLY TRANSPORTED LABELED PROTEINS

Abstract— Slow intra-axonal flow of [³H]leucine labeled proteins has been studied in the garfish olfactory nerve. Because of the homogeneity of the nerve a very well defined peak of slowly transported radioactivity is observed. The velocity of slow flow increases linearly with temperature. Between 14 and 28°C, the rate of the peak apex increases from 0.26 to 1.57 mm/day and the rate of the leading edge of the wavefront from 0.54 to 2.75 mm/day. Extrapolation of the rate-temperature function indicates that slow flow should stop at 11°C. However, a velocity of 0.1 mm/day was determined for experiments conducted at 10°C. Between 15 and 25°C a Q ₁₀ of 3.7 was determined for the peak apex and of 3.3 for the leading edge of the wavefront. The Q₁₀'s are significantly larger than the value of 2.2 found for fast transport (G ross & B eidler , 1975) and support the possibility of at least partial differences between the mechanisms of fast and slow transport. A very small peak was found to migrate in front of the main peak. The positioning of this peak seems to be similar to one found by L asek & H offman (1976) in rat ventral motor neurons.
A temperature dependent exponential decrease of the slow moving peak height was measured and it can be estimated that only 1% of the slowly transported radioactivity reaches the synapses. Most of the slow radioactivity appears to remain in the axon behind the peak. The plateau height was also found to decrease exponentially with time. The rate of disappearance greatly affects the profile determined by the slowly transported labeled proteins along the nerve.     

Bidirectional axonal transport of free glycine in identified neurons R3--R14 of Aplysia.

The axonal transport of 3H-amino acids was studied in the axons of identified neurons R3--R14 in the parietovisceral ganglion (PVG) of the mollusc Aplysia. The PVG was incubated (3--24 hr) in media containing physiological concentrations of single 3H-amino acids while the isolated nerve was superfused with plain or chemically altered media. The nerve was then sliced into sequential segments for biochemical analyses or fixed for autoradiography. 3H-glucine was transported at 70 mm/day in 6X greater quantities than other amino acids which were transported at less than 40 mm/day. In the 3H-glycine experiments, greater than 80% of the label transported into the nerve remained as free glycine, comigrating with glycine in thin-layer chromatographs. In autoradiographs of sections 4 mm from the ganglion-nerve barrier, greater than 50% of the silver grains were over R3--R14 axons which occupy less than 10% of the nerve cross-sectional area. EM autoradiographs confirmed that grains were within R3--R14 and not in surrounding glia. The selective transport of glycine was inhibited by Hg2+, by vinblastine and Nocodazole, and by low Ca2+ media. Autoradiographs of vinblastine-treated nerves showed a drastic reduction in label over R3--R14 and other axons. Label was also transported retrogradely; this transport rate was similar to the orthograde rate, but 5--10 times less label moved retrogradely. Autoradiographs showed that the retrograde label was localized to R3--R14 axons. This report clearly demonstrates the rapid, selective, and bidirectional transport of a free amino acid and provides further evidence that glycine may be used as a neurochemical messenter by neurons R3--R14.     

Comparison of Axonal Transport of Cytoplasmic- and Particulate-Associated Tubulin in Rat Optic System

Abstract: Adult rats were injected intraocularly with [³⁵S]methionine and killed from 1 to 10 weeks later. Optic nerves, optic tracts, and superior colliculi were dissected and then homogenized and separated into soluble and particulate fractions by centrifugation. Radioactivity coelectrophoresing with tubulin in buffers containing sodium dodecyl sulfate was determined (in cytoplasmic fractions, preliminary enrichment was achieved by vinblastine precipitation). Accumulation of radioactive tubulin along the optic pathway occurred in parallel (and in approximately equal amounts) in cytoplasmic and particulate fractions. Transported tubulin peaked at approximately 2 and 4 weeks in the optic nerve and tract, respectively, corresponding to a transport rate of ～ 0.4 mm/ day. There was little diminution in the amount of transported tubulin between optic nerve and tract, suggesting tubulin was not degraded in the axon. Accumulation in the superior colliculus reached a plateau by 4 weeks at less than 20% of the peak in the optic nerve, indicating turnover of tubulin at the nerve endings. The α/β subunit labeling ratio (radioactivity distribution between the tubulin subunits) was 0.57 for both cytoplasmic- and particulate-transported tubulin. In contrast, this ratio was 0.69 for whole brain tubulin prepared by vinblastine precipitation of soluble material. Isoelectric focusing and two-dimensional gel electrophoresis showed that the subunit compositions (microheterogeneity of the α and β bands) of transported tubulins in the cytoplasmic and particulate fractions were very similar. However, some differences relative to whole brain tubulin were noted; a tubulin subunit not identifiable in whole brain tubulin preparations but present in both soluble- and particulate-transported tubulin was observed. Because of the compositional and metabolic similarities of transported tubulin in the soluble and particulate fractions, we conclude that they form a common metabolic pool. This suggests either that, at least for some membranes, the well-characterized tight association between particulate tubulin and membranes may be artifactual or else that an equilibrium exists between soluble and particulate tubulin.     

Time course and subcellular distribution of the radioactivity in a synaptic terminal after supplying the perikaryon with labelled glutamic acid

Summary The cell bodies of the preganglionic neurons of the chick ciliary ganglion were supplied with ³H-glutamic acid by intracerebral injection. The ciliary ganglia were studied with light and E.M. radioautography at 3, 18, 24 hours, 2, 6 and 16 days after injection. The reaction in the ganglion was intense over the preganglionic structures but very weak over the ganglionic cell bodies. The reaction increased in intensity from the myelinated region toward the ending; within the axon, the radioactivity was rather peripheral during early stages and more evenly distributed from the second day onward. The ending showed two peaks of radioactivity, at 18 hours and at 6 days; these peaks are interpreted as the accumulation of material which arrived in two phases; the first with an average velocity greater than 80 mm/ day and the second with a velocity of 2–5 mm/day. Within the nerve ending, the material transported in the fast phase was associated preferentially with the axoplasm rich in synaptic vesicles, close to the synaptic region, whereas the material transported in the slow phase was associated rather with mitochondria and the axoplasm devoid of synaptic vesicles that lies away from the synaptic surface. In contrast to what is observed with lysine, the density of the reaction on the preterminal axons was much lower than that on the endings. It is suggested that a protein rich in glutamyl residues with a rather long mean-life is transported to the ending in the fast phase.On leave from the Catholic University, Santiago, Chile.Supported by Institut National de la Santé et de la Recherche Médicale, France and IBRO. Acknowledgement. I express my sincere thanks to Dr. B. Droz for his hospitality and for helpful discussions throughout the work.     

Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid axoplasmic transport of free leucine

Axoplasmic transport of free 3H-leucine has been studied in vivo in the pike olfactory nerve following application of labeled leucine to the olfactory mucosa. A considerable amount of free 3H-leucine is transported at constant velocity along the axon in the form of a distinct peak. The maximum transport velocity for free 3H-leucine is the same as for rapidly transported 3H-protein (130 and 135 mm/day, respectively, at 19 degrees C). Microtubule inhibitors block or significantly reduce the amount of free 3H-leucine transported, but do not influence the transport velocity. Disruption of the oxygen supply abolishes free 3H-leucine transport, so that this phenomenon cannot be explained by diffusion. The amount of free leucine in the rapidly moving peak decreases with time and distance along the axon and is not detectable after 5 h or more. The transported 3H-leucine is not derived from the circulation or from proteolysis of rapidly transported proteins. This study may help to resolve the controversy over the axoplasmic transport of free amino acids since it shows that free 3H-leucine is transported rapidly but does not travel by rapid axoplasmic transport to the end of axons longer than about 30 mm.