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.     

CELL SURFACE PROTEINS OF CULTURED BRAIN CELLS AND THEIR RECOGNITION BY ANTI-CEREBELLUM (ANTI-NS-4) ANTISERUM

Abstract— Surface proteins of cultured young postnatal mouse cerebella and embryonic mouse cerebral hemispheres were identified by Iactoperoxidase-catalysed radioiodination and by their interaction with an anti-mouse cerebellum antiserum (anti-NS-4 serum) which recognizes surface components on brain cells. Several (8 10) iodinated polypeptides are recognized by radioautography after polyacrylamide gel electrophoresis. Their surface location was confirmed by their sensitivity to mild trypsin treatment on intact cells. Iodinated polypeptides from cells of non-nervous tissues showed a different gel pattern. Immuno-precipitates of solubilizcd surface-iodinated cerebellar cells with anti-NS-4 serum contained two prominent labeled proteins with apparent molecular weights of 200 × 10³ and 145 × 10³. These proteins were also biosynthetically labeled with [³H]leucine. The 145 × 10³ molecular weight component was also found in immunoprecipitates prepared from embryonic cerebral cells, but the 200 × 10³ molecular weight component was replaced by a broad peak with an apparent molecular weight of around 250 × 10³.     

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.     

FAST AXOPLASMIC TRANSPORT OF A CALCIUM-BINDING PROTEIN IN MAMMALIAN NERVE

Abstract— Calcium is transported at a fast rate of 410 mm/day in cat sciatic nerve on injection of ⁴⁵Ca²⁺ into the L7 dorsal root ganglia. Nerve segments corresponding to the crest and the plateau regions of transported activity were analyzed by column chromatography on Sephadex G-100 and Biogel A 5m columns and the fast transported ⁴⁵Ca²⁺ found to be bound to a protein of 15,000 dalton. Using [³H]leucine as a precursor, a labeled calcium binding protein (CaBP) was found located at the same position in elution volumes from the columns as was the protein-bound ⁴⁵Ca^{2 +}. The level of [³H]-labeled CaBP in the crest and plateau regions were compared using column chromatography and polyacrylamide gel electrophoresis techniques and approx 3×4 times more [³H]-labeled activity was found in the crest as compared to the plateau. These findings indicate that Ca²⁺ is fast transported in association with the CaBP. The relation of CaBP to the transport filament model of axoplasmic transport and its possible role in nerve are discussed.     

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.     

62K Proteins Constitute the Major Neurofilament Proteins in the Frog Optic Nerve

The frog optic nerve contains a major group of proteins at a molecular weight of 62K. These proteins are insoluble in nonionic detergents, reactive with a general antibody to intermediate filament proteins, and not labeled by ex vivo incubations of optic nerve. They were therefore considered neurofilament proteins. Axonal transport and enucleation studies were performed to characterize further the origin of these proteins. The results show that the 62K proteins are transported into the optic nerve at a very slow rate (0.1 mm/day). After enucleation, these proteins are substantially reduced in concentration to 20% of the control value at 13 weeks. The predominant neurofilament proteins of the frog optic nerve are 62K in molecular weight. These results are discussed in terms of the anatomy of the frog optic nerve and also contrasted to findings obtained for the goldfish optic nerve.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

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.     

Cholesterol Synthesis and Nerve Regeneration

Abstract: In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [³H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25-diazacholes-terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia-zacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled des-mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [³H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro , diazacholesterol did not inhibit optic nerve regeneration in vivo , as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.     

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.     

Amyloid Precursor Protein Is Synthesized by Retinal Ganglion Cells, Rapidly Transported to the Optic Nerve Plasma Membrane and Nerve Terminals, and Metabolized

Abstract: We have investigated the synthesis, axonal transport, and processing of the β-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f_1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [³⁵S]- methionine, [³⁵S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

SDS GEL ELECTROPHORESIS OF RAPIDLY TRANSPORTED PROTEINS IN GARFISH OLFACTORY NERVE

Abstract— Proteins undergoing rapid axonal transport in the garfish olfactory nerve were examined by sodium dodecyl sulphate gel electrophoresis. The distribution of polypeptides and the extent of their labeling by transported molecules was determined in several nerve subfractions including: total particulate, total membrane, mitochondrial and two membrane subfractions rich in axolemma. The polypeptide composition of the various fractions was found to be relatively similar, with each showing a major protein with an estimated MW of 58,000. Specific differences in the concentrations of certain proteins were noted between fractions, including differences between the lower and higher density axolemma rich subfractions. Axonally transported radioactivity was predominantly localized among high molecular weight proteins, with all fractions, except mitochondrial pellet, displaying a major peak of radioactivity centered at 126,000-MW. Several major proteins including the 58,000-MW band were labeled by rapid transport to a much smaller extent. Certain labeled peaks were found to be concentrated in individual fractions, particularly a polypeptide (MW 35,000) more predominantly found in the lower density axolemma rich fraction.
Systemic labeling of the nerve is found to give a general distribution of radioactivity on gels, which is clearly different from the pattern obtained after axonal transport labeling.     

ELECTROPHORETIC ANALYSES OF PROTEINS TRANSPORTED TO THE RAT POSTERIOR PITUITARY

Abstract— [³⁵S]cysteine, [³H]methionine, or [³H]fucose were injected into the supraoptic nuclei (SON) of rats, and the labelled proteins that were transported to and accumulated in the posterior pituitary 24h post-injection were analyzed electrophoretically. The transported, labelled proteins which were soluble in 0.1 m -HCl were primarily of low molecular weight (about 12,000 on SDS gels). However, the selectivity of labelling of these proteins by the three different labelled precursors could be revealed by isoelectric focusing. The 0.1 m -HCl insoluble labelled proteins, presumably reflecting membrane proteins transported from the SON to the pituitary, were more diverse and generally of higher molecular weight (> 43,000 on SDS gels).     

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.     

RAPID TRANSPORT OF FUCOSYL GLYCOPROTEINS TO NERVE ENDINGS IN MOUSE BRAIN

Abstract— Mice were injected intracerebrally with mixtures of [³H]fucose and [¹⁴C]gluco-samine, and incorporation into macromolecules in various subcellular fractions of brain was studied at 1, 2, 3 and 4 h after administration of the precursors. There was a lag of several hours between the incorporation of [³H]fucose into the glycoproteins of the whole brain fractions and of that into the soluble and particulate glycoproteins of the nerve ending fractions. In contrast, no lag was observed between the incorporation of [¹⁴C]glucosamine into the macromolecules of the whole brain fractions and of that into the soluble macro-molecules of the nerve ending fraction. We conclude that fucosyl glycoproteins of the nerve ending fraction were synthesized in the nerve cell bodies and transported to nerve endings by rapid axoplasmic transport, whereas a substantial proportion of the glucosamine in the soluble macromolecules of the nerve ending fraction was incorporated by the nerve endings themselves. In addition, our evidence indicates that cyclobeximide inhibited fucose incorporation into brain glycoproteins by inhibiting the synthesis of acceptor proteins rather than fucosyl transferase.     

Differences in the Structure of A and B Forms of Human Monoamine Oxidase

Abstract: [³H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in human tissues were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [³H]Pargyline was bound to MAO A in a crude mitochondrial fraction from the placental trophoblast of a male newborn and to MAO B in blood platelets from the umbilical vein of the same newborn. [³H]Pargyline was also bound to MAO A and B in a crude mitochondrial fraction from cultured skin fibroblasts of a male adult and to MAO B in blood platelets from the same individual. Specific labeling of proteins associated with type A or type B activity in fibroblast cells was achieved by preincubation with selective B or A inhibitors, respectively. For all tissues, SDS-PAGE of [³H]pargyline-bound samples revealed a labeled protein band of apparent molecular weight 63,000 for MAO A and 60,000 for MAO B. When SDS-solubilized, [³H]pargyline-labeled MAO A and B proteins from the same male newborn were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [³H]pargyline-labeled peptides were obtained. These findings indicate that distinct enzyme molecules are associated with the A and B types of human MAO activity.     

Reversal of Axonal Transport: Similarity of Proteins Transported in Anterograde and Retrograde Directions

Reversal of axonal transport of endogenous labeled protein was studied in intact and injured nerve axons. Nerve crushes were used to collect labeled protein transported in anterograde and retrograde directions in rat sciatic nerve motoneuron axons after administration of L-[35S]methionine to the vicinity of the cell bodies. The collected proteins were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent fluorography. In injured nerves, where the nerves were ligated distally at the time of precursor injection, the polypeptide composition of proteins moving in anterograde and retrograde directions, 9-11 h after precursor injection, was identical, indicating that reversal at a ligature is a nonselective process. In intact nerves, protein moving in the anterograde direction 22-24 h after injection was different from that found 9-11 h after injection, and was also different from protein moving in the retrograde direction 22-24 h after injection. However, protein moving in the retrograde direction 22-24 h after injection was similar to protein moving in the anterograde direction 9-11 h after injection. Thus it appears that the same group of proteins originally transported into the axon are later returned toward the cell body. In intact axons, also, reversal was nonselective, except that one major labeled polypeptide was reduced in amount in the protein moving in the retrograde direction.     

Proteins of Yaba Monkey Tumor Virus I. Structural Proteins

Yaba virus proteins were characterized by polyacrylamide gel electrophoresis. Electrophoresis of Yaba virion (proteins) dissociated by sodium dodecyl sulfate and 2-mercaptoethanol in continuous and discontinuous buffer systems yielded 37 polypeptide species by staining and by counting bands of radioactively labeled polypeptides. The molecular weights of the viral polypeptide species were found to range from 10,000 to 220,000 by comparing the relative distance of migration of viral proteins with proteins of known molecular weights. Two polypeptides were removed from purified virions by nonionic detergent nonidet P -40 treatment, and the amount of one polypeptide was reduced. Purified cores yielded 21 polypeptide species, none of which was labeled with radioactive glucosamine.     

Synthesis of Cytoskeletal Proteins in Bulk-Isolated Neuronal Perikarya Takashi Nakayama

Abstract: Neuronal perikarya were isolated from young rat brain by sucrose density gradient centrifugation of the tissue, dissociated with a low concentration of trypsin. The isolated cells retained their endogenous proteins, and were capable of active protein synthesis. After incubation with L-[³⁵S]methionine, perikarya were homogenised and separated into soluble and particulate fractions by centrifugation at 70,000 g. Newly synthesised polypeptides in each fraction were resolved by SDS-gel and two-dimensional gel electrophoresis coupled with fluorography. Neuronal perikarya synthesised predominantly actin, and α¹-, α² and β-tubulin. In addition, polypeptides with molecular weights of 35,000, 68,000 and 85,000 were heavily labelled. On two-dimensional electrophoresis, microheterogeneities were seen in soluble actin as well as in soluble tubulins, indicating that heterogeneities reported for brain actin and tubulins are inherent in neuronal actin and tubulins, but not owing to the heterogeneity of cells in the brain tissue. Structural differences between soluble tubulins and those associated with the particulate fraction were indicated by two-dimensional gel electrophoresis and also by one-dimensional peptide maps. The 68,000 molecular weight polypeptide synthesised in neuronal perikarya in vitro yielded a peptide map virtually identical with that generated from the major component of the neurofilament triplet polypeptides that were synthesised in situ. The 160,000 and 200,000 components of the neurofilament triplet were also synthesised in perikarya in vitro , but to disproportionately weaker extents compared with the 68,000 component.