Axonal Transport of Glycoconjugates in the Rat Visual System

Long-Evans rats at 45 days of age were injected intraocularly with 25 mu Ci of [3H]glucosamine. Incorporation of radioactivity into retinal gangliosides, glycoproteins, and glycosaminoglycans (GAGs) was determined at various times after injection. Portions of all three classes of radioactive macromolecules were committed to rapid axonal transport in the retinal ganglion cells. With respect to gangliosides about 60% of those synthesized in the retina were retained in that structure, 30% were committed to transport to regions containing the nerve terminal structures (lateral geniculate body and superior colliculus), and about 10% were deposited in stationary structures of the axons (optic nerve and tract). With the exception of ganglioside GD3 the molecular species distribution of gangliosides synthesized in the retina matched that committed to transport. In contrast to gangliosides a smaller fraction of newly synthesized retinal glycoprotein (less than 12% of that synthesized in the retina) was committed to rapid transport to nerve ending regions and only about 0.5% was retained in the nerve and tract. The molecular-weight distribution of glycoproteins committed to transport differed quantitatively from that of the retina. With respect to GAGs an even smaller portion (1-2%) of that synthesized in the retina was committed to rapid transport; of this portion almost all was recovered in nerve terminal-containing structures. A constant proportion of each retinal GAG species was transported to the superior colliculus. We suggest that most of the retinal gangliosides are synthesized in neurons and preferentially in ganglion cells (possibly a function of the large surface membrane area supported by these cells). Subcellular fractionation experiments indicated that transported gangliosides, glycoproteins, and GAGs may be preferentially distributed into different subcellular compartments.     

Retrograde Axonal Transport of Gangliosides and Glycoproteins in the Motoneurons of Rat Sciatic Nerve

Axonal transport of glycoconjugates was studied in the motoneurons of rat sciatic nerve following injection of [3H]glucosamine into the lumbosacral spinal cord. After varying time intervals, the sciatic nerve was exposed, and two ligatures were tied for collection of materials undergoing anterograde and retrograde transport. Gangliosides and glycoproteins were found to undergo fast anterograde transport, estimated at 284-446 mm/day. Both classes underwent retrograde transport as well, with labeled glycoproteins returning slightly ahead of labeled gangliosides. Only minor quantities of labeled proteoglycans were detected. Purified gangliosides extracted from nerve segments were fractionated according to sialic acid number on diethylaminoethyl-Sephadex; the distributional pattern tended to resemble that of brain gangliosides. The similarity between anterograde and retrograde patterns suggested absence of metabolic changes in gangliosides entering and leaving the axon-nerve terminal structures.     

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.     

Axonal Transport, Deposition, and Metabolic Turnover of Glycoproteins in the Rat Optic Pathway

Abstract: Following intraocular injection of [³H]fucose in the rat, radioactive glycoproteins are rapidly transported to the nerve terminals in at least two waves, one with a peak at 8 h and a second with a peak at about a week. The molecular weight distribution of radioactive peptides in ach transport wave as determined by gel electrophoresis in buffers containing sodium dodecyl sulfate is very similar. Most of the many glycopeptides in the first wave of rapid transport pass through the optic tract in unison (apparent half-life of about 15 h) and are preferentially destined for the nerve endings. However, two proteins of apparent M. W. 28,000 and 49,000 are preferentially retained in the axons. The remaining proteins, after reaching the nerve endings (superior colliculus), decay with apparent half-lives ranging from 17 to 34 h. During the second wave a large amount of the 28,000 and 49,000 M. W. peptides are again preferentially retained in the axons. The remaining proteins, on reaching the nerve endings, decay with apparent half-lives ranging from 5 to 9 days. Subcellular fractionation of the superior colliculus supports the hypothesis that the 49,000 and 28,000 M. W. peptides are the predominantly labeled glycoproteins present in myelinated axons (representing over 50% of the radioactive glycoproteins 7 days following injection), although they are probably also present in membranes of the nerve endings. A comparison with glycoprotein transport in other tracts (geniculocortical and nigrostriatal tracts) suggests that glycoprotein transport in these pathways has many similarities to glycoprotein transport in the retinal ganglion cells, and that the optic system is a good general model for axonal transport in the CNS.     

Axonal Transport of Polyamines in Intact and Regenerating Axons of the Rat Sciatic Nerve

The axonal transport of putrescine or its polyamine derivatives spermidine or spermine is a subject of some debate. We investigated this question by injecting [3H]putrescine into the lumbar spinal cord of the rat and measuring the accumulation of radioactivity central to ligatures placed on intact and regenerating sciatic nerves. In normal nerves, approximately twice as much radioactivity built up proximal to these ligatures 2 or 3 days after injection than at more distal ligatures used to control for accumulation of radioactivity which might be due to tissue damage alone. In regenerating nerves the amount of radioactivity accumulating at the ligature was approximately five times that at the distal ligature and two to three times greater than in intact nerves. The identity of the radioactivity in regenerating nerves, determined on an amino acid analyzer, was found to be primarily spermidine and an unknown compound that migrated as a frontal elution peak. Autoradiographic analysis showed that the radioactivity was largely confined to axons, but a significant amount of the silver grains was associated with Schwann cells and myelin sheaths surrounding labeled axons in both intact and regenerating nerves. The data indicate that polyamine derivatives of putrescine are transported axonally in rat sciatic nerves, and some of this transported material accumulates in Schwann cells surrounding the labeled axons. These processes are apparently augmented during regeneration of the injured axons.     

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me-^HC]choline and 15 μCi [2-³H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [¹⁴C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.     

A Rapid Anterograde Axonal Transport of Carboxypeptidase H in Rat Sciatic Nerves

Abstract: Using the highly sensitive HPLC-fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative pro-hormone processing enzyme that removes a basic amino acid from the C-terminus of a precursor peptide, was measured 12–72 h after double ligations of rat sciatic nerves. CPH-like activity in rat sciatic nerves was 60-fold lower than that in the pituitary gland. CPH-like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is ∼50 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.     

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [³H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light-microscope autoradiographs, a class of large-caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build-up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.     

Composition and Subcellular Distribution of Glycoproteins and Glycosaminoglycans Undergoing Axonal Transport in Garfish Olfactory Nerves

The study examined the subcellular distribution of [3H]glucosamine-labeled glycoconjugates undergoing axonal transport in 100,000 x g soluble and two membranous subfractions of the garfish olfactory nerve. Analysis was made of intact glycoconjugates and of glycopeptides and glycosaminoglycans derived from these molecules by limit protease digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed labeling of a variety of high-molecular-weight molecules with a lower molecular weight distribution in the soluble fraction than in the membranous fractions. Following protease digestion, nearly two-thirds of transported radioactivity in glycopeptides was recovered in the plasma membrane-enriched subfraction, with the remainder equally divided between soluble and higher density membrane fraction. Comparison of the distribution of glycopeptide radioactivity and chemically assayed hexosamine revealed transport labeling of a large variety of different-sized neutral and acidic glycopeptides in all subfractions. Transport labeling of most glycoprotein carbohydrate chains was in proportion of their hexosamine content. Transported glycosaminoglycan label was most heavily concentrated in the plasma membrane fraction, whereas hexosamine was most concentrated in the higher density membrane fraction. The labeling pattern suggested both transported and nontransported pools of these molecules. The specific glycosaminoglycans chondroitin sulfate and heparan sulfate were recovered in all subfractions, whereas hyaluronic acid was confined to the soluble fraction.     

Retrograde Axonal Transport of Locally Synthesized Phosphoinositides in the Rat Sciatic Nerve

Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo-[3H]inositol into the rat sciatic nerve (50-55 mm distal to L4 and L5 dorsal root ganglia), a time-dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10-12-fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water-soluble label in the dorsal root ganglia still remained approximately 1.0, whereas the corresponding ratio in the chloroform/methanol-soluble fraction was approximately 20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injection site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia.     

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.     

Glycoproteins of Axonal Transport: Affinity Chromatography on Fucose-Specific Lectins

Rapidly transported fucose-labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose-specific lectins. A recently characterized fucose-specific lectin from Aleuria aurantia bound reversibly approximately 60% of the applied protein-bound radioactivity. The lectins from Lotus tetragonolobus and Ulex europaeus bound are very small proportions of the labeled rapidly transported glycoproteins.     

A Comparison of the Effects of Acrylamide and Experimental Diabetes on the Retrograde Axonal Transport of Proteins in the Rat Sciatic Nerve: Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis

Retrogradely transported proteins that accumulated for 18 h distal to a ligature on the sciatic nerve of rats were separated by two-dimensional polyacrylamide gel electrophoresis and visualised with silver stain. A total of 14 proteins were detectable in this way as being retrogradely transported. In rats rendered diabetic 14 days previously by a single intravenous dose of streptozotocin, the accumulation of four of those proteins was noticeably decreased. Administration of acrylamide to a cumulative dose of 150 mg.kg-1 or 350 mg.kg-1 decreased the accumulation of four and eight proteins, respectively. Three of the four protein changes were common to the diabetic and acrylamide-treated animals, and were present before signs of neuropathy could be detected. The results suggest that those alterations may play a causal role in the development of neuropathy.     

Locally Synthesized Phosphatidylcholine, but Not Protein, Undergoes Rapid Retrograde Axonal Transport in the Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phosphatidylcholine in the sciatic nerve has been demonstrated only after injection of lipid precursors into the cell body region. We now report, however, that after microinjection (1 μl) of [methyl-³H]choline chloride into the rat sciatic nerve (35-40 mm distal to the L4 and L5 dorsal root ganglia), time-dependent accumulation of ³H-labeled material occurred in dorsal root ganglia ipsilateral, but not contralateral, to the injection site. The level of radioactivity in the ipsilateral dorsal root ganglia was minimal at 2 h after isotope injection but was significantly increased at 7, 24, 48, and 72 h after intraneural isotope injection (n = 3–8 per time point); at these time points, all of the radiolabel in the chloroform/methanol extract of the ipsilateral dorsal root ganglia was present in phosphatidylcholine. The radioactivity in the water-soluble fraction did not show a time-dependent accumulation in the ipsilateral dorsal root ganglia as compared with the contralateral DRGs, ruling out transport or diffusion of precursor molecules. In addition, colchicine injection into the sciatic nerve proximal to the isotope injection site prevented the accumulation of radiolabel in the ipsilateral dorsal root ganglia. Therefore, this time-dependent accumulation of radiolabeled phosphatidylcholine in the ipsilateral dorsal root ganglia is most likely due to retrograde axonal transport of locally synthesized phospholipid material. Moreover, 24 h after injection of both [³H]choline and [³⁵S]-methionine into the sciatic nerve, the ipsilateral/contralateral ratio of radiolabel was 11.7 for ³H but only 1.1 for ³⁵S. indicating that only locally synthesized choline phospholipids, but not protein, were retrogradely transported.     

Differences in the Kinetics of Axonal Transport for Individual Lipid Classes in Rat Sciatic Nerve

Lipid precursors ([2-3H]glycerol for phospholipids and [3H]acetate for cholesterol) were injected into the L-5 dorsal root ganglion of adult rats. At various times, animals were killed, the ganglion and consecutive 5-mm segments of sciatic nerve were dissected, and lipids were extracted and analyzed by TLC. Individual lipid classes exhibited markedly different transport patterns. The crest of radioactive phosphatidylcholine moved as a sharply defined front at about 300 mm/day, with a relatively flat plateau behind the moving crest. Although some radioactive phosphatidylethanolamine also moved at the same rate, the crest was continually attenuated as it moved so that a gradient of radioactive phosphatidylethanolamine along the axon was maintained for several days. Transported diphosphatidylglycerol exhibited a defined crest, as did phosphatidylcholine, but moved at about half the rate. Labeled cholesterol was transported at a rapid rate similar to that for phosphatidylcholine and phosphatidylethanolamine, but like phosphatidylethanolamine, the initial moving crest of radioactivity was continually attenuated. Relative to the phospholipids, cholesterol showed a more prolonged period of accumulation in the axons and was more metabolically stable. We propose that most labeled phosphatidylcholine, phosphatidylethanolamine, and cholesterol is transported in similar (or the same) rapidly moving membranous particles. Once incorporated into these particles, molecules of phosphatidylcholine tend to maintain associated with them during transport. In contrast, molecules of phosphatidylethanolamine and cholesterol in these transported particles exchange extensively with unlabeled molecules in stationary axonal structures. Diphosphatidylglycerol, localized in a specialized organelle, the mitochondrion, is transported at a slower rate than other phospholipids, and does not exchange with other structures.     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Blood–Brain Transport of Thiamine Monophosphate in the Rat: A Kinetic Study In Vivo

To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi.mmol-1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27-39 pmol.g-1.min-1 and 2.6-4.8 microM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol.g-1.min-1, thus representing a significant component of thiamine supply to nervous tissue.     

Reduced Axonal Transport of the G4 Molecular Form of Acetylcholinesterase in the Rat Sciatic Nerve During Aging

Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40-60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.