Characterization of Axonally Transported Glycoproteins in Regenerating Garfish Olfactory Nerve

Abstract: This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase-generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration-related increase in the proportion of [³H]glucosamine label in lower-molecular-weight Con A-binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose-rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A-binding glycopeptides are enriched in membrane subfractions, particularly in a high-density fraction that morphologically resembles isolated cell surface coat. Regeneration-related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport-labeled glycoproteins disclosed growth-correlated increases in radioactivity associated with 180–200K, 105–115K, and 80–90K components, while a 150–160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50–140K range.     

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.     

Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

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.     

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 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.     

Rapid Migration of Inositol Phospholipids with Axonally Transported Substances in the Rabbit Optic Pathway

Following an intraocular injection of myo-[2-3H]inositol, the axonal transport of labelled water-soluble substances and inositol phospholipids was investigated. Evidence was obtained for a rapid axonal transport of a relatively small amount of labelled inositol phospholipids. In contrast to other axonally transported phospholipids, there was no significant accumulation of labelled, rapidly transported inositol phospholipids in the nerve terminal region at later time intervals following the isotope administration.     

Two Populations of Axonally Transported Tubulin Differentiated by Their Interactions with Neurofilaments

In the sensory fibers of the rat sciatic nerve (fibers of the dorsal root ganglion cells), two components of tubulin transport were observed that differed in the rate of transport, solubility in Triton, and subunit composition. The faster component, migrating ahead of the neurofilament proteins, was soluble in 1% Triton. The slower component, migrating with the neurofilament proteins, was insoluble in 1% Triton and contained a unique polypeptide, "NAP," in the tubulin region that was not present in the faster component. "NAP" was not a subspecies of tubulin as evidenced by peptide mapping. It seems to be a neurofilament-associated protein. When a complete separation of the main tubulin wave from the neurofilament wave was achieved in the motor axons of the same nerve (axons of the ventral motoneurons) under the effect of beta,beta'-iminodipropionitrile, a portion of tubulin was still found associated with the retarded neurofilament wave. The subunit composition of this portion was similar to the slower, neurofilament-associated component in the sensory fibers under normal conditions, i.e., enriched in "NAP" and the most acidic subtype of beta-tubulin. It is suggested that two populations of transported tubulin exist that are differentiated by the extent of their interaction with neurofilaments.     

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.     

Subcellular Fractionation of Rat Sciatic Nerve and Specific Localization of Ganglioside LM1 in Rat Nerve Myelin

Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctaosylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (less than 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were approximately 70-75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (approximately 40%) and GM1 (approximately 20%). LM1 in these fractions was minimal (less than approximately 5%). Significant amounts of GM3 with N-glycolylneuraminic acid (approximately 10%) and GM1b (4-14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.     

Polypeptides in Frog and Rat: Evolutionary Changes in Rapidly Transported and Abundant Nerve Proteins

Abstract: Dorsal root ganglion (DRG) neurons from rat and frog were labeled in vitro with [³⁵S]methionine, and the newly synthesized, rapidly transported proteins were collected at ligatures on the sciatic nerves. The proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. Exposure of x-ray film to dried gels allowed comparison of the labeled, rapidly transported proteins from frog and rat. The gel staining patterns of abundant proteins in the sciatic nerves were also compared. Triolets of gels were examined: one gel from frog, one from rat, and one from frog plus rat combined. Among the transported proteins, some (including A2, A17 and/or A18, B6, B14_a-i, C1, C22, and some members of Al_a-i and B3_a-g) co-migrated on the gels, suggesting that these proteins have been well conserved during evolution. The gel staining patterns of abundant proteins in the sciatic nerves also show some similarities: two forms of actin, serum albumin, and α- and β-tubulin are each in identical positions on the frog and rat gels. Other sciatic nerve and rapidly transported proteins had similar, but not identical, positions on the gels. A number of the rat and frog proteins had no obvious counterpart. We have calculated the magnitude of expected changes in charge and molecular weight of proteins due to accumulation of point mutations during evolution. We conclude that many of the differences between rat and frog protein patterns on the two-dimensional gels could be the result of such point mutations, but we cannot rule out radical changes in polypeptide sequence or abundance between frog and rat for some of these proteins.     

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.     

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.     

Subsets of Axonally Transported and Periaxonal Polypeptides Are Released from Regenerating Nerve

Using two-dimensional polyacrylamide gel electrophoresis to analyze proteins, we have found subsets of periaxonal and fast-transported axoplasmic proteins that are released in vitro from regenerating sciatic nerve into a surrounding bath. Of the fast-transported proteins that are released from nerve, there is a subset of at least five polypeptides that appears in greater relative abundance in the bath than in the nerve. Some of these released, fast-transported proteins are glycosylated. Several periaxonally synthesized polypeptides are released in significantly greater amounts from regenerating nerve, and of these polypeptides, two are released in greater amounts from nerve only at regions of regeneration or distal to regeneration. These released polypeptides do not represent the most abundant of the locally synthesized proteins. The released, fast-transported and periaxonal proteins may play a role in intercellular signaling or in modulation of the extracellular environment during nerve regeneration.     

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.     

Axonal Transport and Metabolism of Glycoproteins in Rat Sciatic Nerve

The distribution of 5'-nucleotidase activity, dopaminergic [3H]spiperone binding sites, and [3H]quinuclidinyl benzilate (QNB) binding sites in different subcellular fractions of bovine caudate nucleus has been studied. Each activity was enriched in a microsomal (P3) preparation from that tissue. The microsomal preparation was further fractionated by different techniques. First, the P3 fraction, or a sonicated P3 fraction, was fractionated on a discontinuous sucrose density gradient. Second, the P3 fraction, or a digitonin pretreated P3 fraction, was fractionated on a continuous sucrose density gradient. The results obtained demonstrate that 5'-nucleotidase activity does not cofractionate with radioligand binding activity, although no difference between the distributions of [3H]spiperone binding and [3H]QNB binding were seen. It is concluded that the two radioligand binding activities are located on nonglial membranes.