RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING PROTEINS IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

—The redistribution of rapidly migrating [³H]leucine-labelled proteins was studied using double ligatures applied to the vagus nerve and single ligatures, applied to the hypoglossal nerves. Rapidly migrating proteins accumulating for 16 h proximal to a distal ligature of the cervical vagus redistributed to give a retrograde accumulation distal to a second ligature. Within 6 h a substantial redistribution occurred indicating a rapid retrograde transport. After 21 h there was a further accumulation with 70 per cent of the labelled material accumulating at the distal end of the isolated nerve segment and 16 per cent accumulating at the proximal end. It was shown that about a half of the retrograde accumulation was dependent on the distal accumulation zone. Rapidly migrating proteins accumulated distal to a ligature applied to the hypoglossal nerve 16 h after labelling of the nerve cell bodies indicating that a rapid retrograde transport of labelled macromolecules occurs from the peripheral parts of the nerve in the tongue. Labelled proteins accumulated proximal to ligatures and transections of both the hypoglossal and vagus nerve when applied 16 h after labelling of the nerve cell bodies, indicating the presence of axonal proteins, migrating at a rate of transport intermediate to that of rapidly and slowly migrating proteins.     

Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons

The accumulation of [³H]leucine- and [³H]fucose-labelled axonal proteins, acetyl-CoA : choline O-acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was studied proximal to a ligature applied to the hypoglossal nerve of the rabbit at different phases of nerve regeneration. After 1 week of regeneration, the accumulation of rapidly migrating [³H]leucine-labelled proteins, ChAc and AChE was reduced as compared to that of the contralateral nerve. In contrast, the accumulation of [³H]fucose-labelled glycoproteins was markedly increased. After a regeneration period of 4-6 weeks, the accumulation of proteins and glycoproteins in the regenerating nerve was increased whereas the accumulation of ChAc and AChE was almost normal. The results indicate an initial depression of the synthesis and axonal transport of the bulk of rapidly migrating proteins, ChAc and AChE in the chromatolytic hypoglossal neurons whereas the synthesis and transport of rapidly migrating glycoproteins is increased. These initial changes are less pronounced during the subsequent regeneration period.     

DECREASED AXONAL FLUX OF RETROGRADELY TRANSPORTED GLYCOPROTEINS IN EARLY EXPERIMENTAL DIABETES

Abstract— Anterograde and retrograde flux of axonal transported glycoproteins were examined in streptozotocin diabetic rats with 4 weeks'duration of the metabolic derangement.
[³H]Fucose and [¹⁴C]NeuNAc were injected into the fifth lumbar root ganglion and the accumulation of TCA-PTA insoluble activity proximal and distal to a sciatic nerve ligature was measured.
Accumulation of glycoproteins during 2 h collection periods was decreased distal to a ligature in diabetic animals whereas no abnormality of proximal accumulation was observed. These findings demonstrate an abnormality of the retrograde transport of glycoproteins in early experimental diabetes.     

TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

Axoplasmic transport and possible recycling of opiate receptors labelled with 3H-lofentanil

3H-Lofentanil, an extremely potent opiate drug with a very long duration of action was injected intravenously into rats immediately after a ligature had been tied around the vagus nerve. Radioactivity accumulated on both sides of the ligature 24 hours and, to a larger extent, 48 hours after the injection. In contrast, there was no accumulation in animals pretreated with naloxone, neither in ligated sciatic nerves nor between two ligatures in the vagus nerve. An accumulation of stereospecific 3H-lofentanil binding sites measured in vitro was only detected above the ligature, thus in the proximal part of the nerve. When 3H-lofentanil was injected at different time intervals after ligation, we observed a tremendous drop of labelling in the distal and also but more slowly in the proximal part of the nerve. This could be due to a possible recycling or re-utilization of 3H-lofentanil binding sites. The present data are compatible with an axoplasmic flow and a possible recycling of opiate receptors labelled in vivo after intravenous injection of 3H-lofentanil.     

The intra-axonal transport of acetylcholine and cholinergic enzymes in rat sciatic nerve during regeneration after various types of axonal trauma

The proximo-distal intra-axonal transport of acetylcholine (ACh) and cholinergic enzymes (choline acetyltransferase, CAT, and ACh-esterase, AChE) in rat regenerating sciatic nerve was studied by accumulation technique. Four types of axonal trauma were performed: freezing with solid CO₂, crushing, ligating the nerve with remaining tight silk ligature, and cutting the nerve. Normal and sham-operated rats were used as controls. One to twenty-nine days later, the nerves were crushed about 15 mm proximal to the trauma. The nerve segment proximal to this crush was dissected out 12 hr later and assayed for ACh-content and enzyme activities. The increase in this segment 12 hr after crushing was taken as an indication of proximo-distal transport in the regenerating nerves. ACh transport did not seem to vary during regeneration as compared to controls. In contrast, the transport of both CAT and AChE was initially markedly depressed. Towards the end of the observation period (29 days), a recovery of CAT-transport occurred in all groups. Recovery of AChE-transport was marked in the freeze and crush groups. In the cut group no recovery was seen and in the ligated group only a small recovery occurred. Thus, in the nerves where regeneration was facilitated by the presence of intact connective tissue sheaths (freezing and crushing) recovery of transport occurred earlier than in cut or ligated nerves.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Axonal Transport of Synaptic Vesicles and Muscarinic Receptors: Effect of Protein Synthesis Inhibitors

The effect of cycloheximide, a protein synthesis inhibitor, was studied on the axonal transport of noradrenergic synaptic vesicles and presynaptic muscarinic receptors, identified by in vitro binding of [3H]dihydrotetrabenazine and [3H]quinuclidinylbenzilate, respectively, in rat sciatic nerve. Cycloheximide (1.5 mg/kg) administered subcutaneously 2 h before ligation decreased by approximately 50% the accumulation of vesicles and receptors in the proximal segment above the ligature placed on the nerve; its action was detectable after a lag period of 10 h and disappeared 96 h after administration. Double ligatures were placed on the nerve at various time intervals between the first (distal) and the second (proximal) ligature, and the accumulation of vesicles and receptors proximal to the second ligature was measured; the first ligature diminished the accumulation above the second ligature. At an interval of 96 h between the first and the second ligature, cycloheximide completely prevented the accumulation of vesicles and receptors proximal to the second ligature. The effects of double ligatures and the response to cycloheximide treatment can best be explained on the assumption that an important proportion of synaptic vesicles and presynaptic receptors is being recycled in the nerve cell bodies after retrograde transport.     

Posttranslational Protein Modification by Amino Acid Addition in Intact and Regenerating Axons of the Rat Sciatic Nerve

Abstract: Experiments were performed to determine whether ppsttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 · g , and analyzed for the ability to incorporate ³H-amino acids into proteins. No incorporation of amino acids into proteins was found in the high-speed supernatant, but when the supernatant was passed through a Sephacryl S-200 chromatography column (removing molecules less than 20 kD), [³H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15–150 times that in intact nerves. The results indicate that the components of this reaction are transported axonally in rat sciatic nerves and that the reaction is increased dramatically in growing axons during nerve regeneration.     

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.     

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.     

EFFECTS OF COLCHICINE ON AXONAL TRANSPORT IN PERIPHERAL NERVES

—Colchicine injected intracisternally markedly inhibited the rapid migration (300-400 mm/day) of labelled proteins in the hypoglossal and vagus nerve of the rabbit. The transport of acetylcholinesterase (EC 3.1.1.7) and choline acetyltransferase (EC 2.3.1.6) previously shown to move with the slow (5-26 mm/day) phase of axoplasmic transport in these nerves, was only partially blocked. In view of this differential effect on axonal flow, we suggest that the neurotubules, on which colchicine acts preferentially, are primarily involved in the rapid (300-400 mm/day) axoplasmic flow. After local injection of colchicine into the nerves both the rapidly migrating labelled proteins and the enzymes (AChE and ChAc) accumulated above the site of injection to the same degree as they accumulate above a nerve ligation. Since this blockage of enzyme transport occurred after concentrations of colchicine much higher than those used for intracisternal injections these findings after local injection may represent more severe effects on axonal transport systems.     

Protein Synthesis and Axonal Transport During Nerve Regeneration

Abstract— Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [³⁵S]-methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two-dimensional polyacryl-amide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.     

Axonal growth during regeneration: a quantitative autoradiographic study

The intraaxonal distribution of labeled glycoproteins in the regenerating hypoglossal nerve of the rabbit was studied by use of quantitative electron microscope autoradiography. 9 d after nerve crush, glycoproteins were labeled by the administration of [3H]fucose to the medulla. The distribution of transported 3H-labeled glycoproteins was determined 18 h later in segments of the regenerating nerve and in the contralateral, intact nerve. At the regenerating tip, the distribution was determined both in growth cones and in non-growth cone axons, 6 and 18 h after labeling. The distribution within the non-growth cone axons of the tips was quite different at 6 and 18 h. At 6 h, the axolemma region contained < 10% of the radioactivity; at 18 h, it contained virtually all the radioactivity. In contrast, the distribution within the growth cones was similar at both time intervals, with 30% of the radioactivity over the axolemmal region. Additional segments of the regenerating nerve also showed a preferential labeling of the axolemmal region. In the intact nerve, 3H-labeled glycoproteins were uniformly distributed. These results suggest that: (a) in this system the labeled glycoproteins reaching the tip of the regenerating axons are inserted into the axolemma between 6 and 18 h after leaving the neuronal perikaryon; (b) at the times studied, there is a fairly constant ratio between glycoproteins reaching the growth cone through axoplasmic transport and glycoproteins inserted into the growth cone axolemma; (c) the axolemma elongates by continuous insertion of membrane precursors at the growth cone; the growth cone then advances, leaving behind an immature axon with a newly formed axolemma; and (d) glycoproteins are preferentially inserted into the axolemma along the entire regenerating axon.     

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.     

Different Polypeptides Are Rapidly Transported in Auditory and Optic Neurons

Abstract: Rapidly transported proteins and glycoproteins in the auditory and optic nerves of the guinea pig were analyzed by electrophoresis and two-dimensional electrofocusing/electrophoresis. Proteins transported in the auditory nerve were analyzed in the cochlear nucleus 3 h after cochlear injection of radioactive precursor, and proteins transported in the optic nerve were analyzed in the superior colliculus 6 h after intraocular injection of radioactive precursor. Two-dimensional analysis showed that several rapidly transported polypeptides were present in one system, but not in the other. By use of [³H]fucose as a precursor or by separating [³⁵S]methionine-labeled polypeptides on immobilized concanavalin A or wheat germ agglutinin, it was shown that most of the proteins transported in only one system are glycoproteins. As previously reported a polypeptide of molecular weight 140,000 was a major labeled species in the auditory nerve. This polypeptide was also found in the optic nerve, but only as a minor species. Two other polypeptides with molecular weights and isoelectric points similar to those of the 140,000 molecular weight polypeptide were present in both systems, but were much more abundant in the optic nerve. The major labeled polypeptide in both systems had a molecular weight of 25,000.     

Insulin-like growth factor binding protein-1 is pre-synaptic at mouse neuromuscular synapses and is transported in nerve

In a previous study, we localized insulin-like growth factor binding protein 1 (IGFBP-1) to mouse neuromuscular junctions, and intramuscular nerves. To determine if pre-synaptic accumulation of IGFBP-1 occurred, we used double ligation of sciatic nerve in adult mice at different time points. IGFBPs were deteced by Western ligand blot (WLB) with¹²⁵I-IGF-I. WLB and Western immunoblot (WIB) analysis of extracts from double-ligated nerves showed a delayed (6 days) increase of IGFBP-1 in the soluble fraction between the ligatures and distal to the distal ligature. For comparison we evaluated transport of neurofilament components, using WIB and confirmed the primarily anterograde transport of these intraaxonal proteins. These data suggest that expression of IGFBP-1 is both by activated Schwann cells as well as retrograde axonal transport with likely entry into the axon at the synapse.Special issue dedicated to Dr. Sidney Ochs.     

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.     

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.