Axonal Transport Of Proteins Research Articles

Accumulation of [ 3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co 2+-containing or Ca 2+ -free medium

Previous in vitro studies have established that Co 2+-containing or Ca 2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [ 3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16–17 h in Ca 2+-free medium or in medium containing 0.18 mM Co 2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental-cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca 2+-free medium and the Co 2+-containing medium both reduced by approximately 80% the quantity of [ 3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [ 3H]fucose into glycoproteins. The results indicate that in the presence of Co 2+ or in the absence of Ca 2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca 2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system.

Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neurosecretory system

In vitro biosynthesis of neurosecretory peptides specific to the X-organ sinus gland (XOSG) of the land crabCardisoma carnifex was studied. The neurosecretory somata comprising the X-organ were selectively exposed to saline containing3H-leucine, glucose and antibiotics for 5 h (pulse) followed by 19–24 h of superfusion with nutrient media (chase). Analysis of extracts of somata and terminal regions by high voltage paper electrophoresis demonstrated the uptake and incorporation of3H-leucine into protein by X-organ somata and its axonal transport to terminal regions. Tritiated-leucine incorporation was reduced 90% by 50 μg/ml puromycin, indicating ribosome-mediated synthesis. Axonal transport was blocked by ligation of the axons; this did not affect the rate of3H-leucine incorporation by the somata. Radiolabelled peptide hormones were identified by comigration of radiolabel with RPLC and/or SDS-PAGE determined regions of bioactivity and by comparison of RPLC elution patterns with those of sinus gland peptides purified and analyzed for amino acid composition by Newcomb (1983a). Erythrophore concentrating hormone (ECH) and at least two 6,000 Dalton peptides having crustacean hyperglycemic hormone activity (CHH) were identified among peptides biosynthetically radiolabelled. Comparison of XOSG systems receiving 5 h of incubation with those receiving a pulse-chase (5 h∶19 h) regime demonstrates the existence of polypeptide precursors to the XOSG peptide hormones.

Radioautographic study of the axonal transport of proteins into the sensory nerve endings of avian mechanoreceptors

The axonal transport of proteins to the nerve endings of Herbst and Grandry sensory receptors has been investigated by electron-microscope radioautography. Soon after the injection of [ 3H]leucine into the trigeminal ganglia of young ducks, labeled proteins are conveyed along the suborbital sensory nerves to the sensory nerve endings at rates of at least 200–280 mm/day. Most of these rapidly transported proteins accumulate in areas containing vesicles of various kinds and along the axolemmal region. Later, the bulk of labeled proteins migrate along the axons at rates of about 15 mm/day and are distributed mainly to the mitochondria. A small portion of labeled material is transferred to the adjoining modified Schwann and specialized Grandry receptor cells. It is concluded that the transport of proteins from sensory ganglia to sensory nerve endings of mechanoreceptors is conveyed at fast and intermediate rates and is mainly used for the renewal of vesicles, axolemmal constituents and mitochondria.

Velocity of axonal transport of labeled protein is not dependent on local concentration

The velocity of axonal transport of protein labeled with [ 3H]leucine was determined in rat sciatic nerve sensory axons after reversible cooling of the nerve, which provoked a local accumulation of transported material. The velocity of the wavefront of the labeled protein, as well as the slope of the wavefront, was not affected by the duration of cold-block prior to rewarming. In addition, the velocity and slope did not change as the wavefront moved distally from the site of block. In these axons, therefore, velocity of axonal transport is independent of the local concentration of rapidly transported protein.

Ionic requirements for in vitro retrograde axonal transport of acetylcholinesterase

The ionic requirements for retrograde axonal transport of acetylcholinesterase were studied in vitro in desheathed spinal nerves of the bullfrog; the accumulation of enzyme activity distal to a ligature served to evaluate the status of retrograde transport. After an 18 h incubation in control medium, the 2 mm segment of nerve immediately distal to the ligature contained approximately twice as much acetylcholinesterase activity as other more distal segments. Accumulation of acetylcholinesterase distal to the ligature was reduced during incubation in Ca 2+-free medium plus 1 mM EGTA; retrograde transport was not diminished by the substitution of sucrose for the NaCl of the medium. In comparison with ionic requirements for orthograde fast axonal transport of protein, retrograde transport of acetylcholinesterase thus appears to share a Ca 2+ requirement, but not a requirement for NaCl.

Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study.

Beta,beta'-iminodipropionitrile (IDPN) produces a rearrangement of axoplasmic organelles with displacement of microtubules, smooth endoplasmic reticulum, and mitochondria toward the center and of neurofilaments toward the periphery of the axon, whereas the rate of the fast component of axonal transport is unchanged. Separation of microtubules and neurofilaments makes the IDPN axons an excellent model for study of the role of these two organelles in axonal transport. The cross-sectional distribution of [3H]-labeled proteins moving with the front of the fast transport was analyzed by quantitative electron microscopic autoradiography in sciatic nerves of IDPN-treated and control rats, 6 h after injection of a 1:1 mixture of [3H]-proline and [3H]-lysine into lumbar ventral horns. In IDPN axons most of the transported [3H] proteins were located in the central region with microtubules, smooth endoplasmic reticulum and mitochondria, whereas few or none were in the periphery with neurofilaments. In control axons the [3H]-labeled proteins were uniformly distributed within the axoplasm. It is concluded that in fast axonal transport: (a) neurofilaments play no primary role; (b) the normal architecture of the axonal cytoskeleton and the normal cross-sectional distribution of transported materials are not indispensable for the maintenance of a normal rate of transport. The present findings are consistent with the models of fast transport that envision microtubules as the key organelles in providing directionality and propulsive force to the fast component of axonal transport.

Recovery of fast axonal transport and retinal protein synthesis in the rabbits after intraocular administration of vinblastine

Orthograde fast oxonal transport was almost completely blocked one day after the intraocular injection of 10–100 μg of vinblastine (VLB), then recovered gradually for up to 2 months. Recovery was not complete, however; There was a definite relationship between the dose of VLB administered and the degree of recovery of fast axonal transport. Retinal protein synthesis was reduced by intraocular VLB, but there was no relationship between the dose administered and the amount of reduction of protein synthesis. Cytotoxicity of VLB to the retina is less probable. The participation of retrograde axonal transport in the metabolism of cell bodies is discussed.

Axonal transport of protein in motor fibres of experimentally diabetic rats — Fast anterograde transport

Fast axonal transport of radiolabelled proteins in motor fibres of rat sciatic nerves was studied after 14 days of streptozotocin-induced diabetes. The rate of fast transport as measured at two time intervals after application of [3H]leucine to the motor neurone cell bodies in the spinal cord was reduced by 21% in diabetic rats. There was no significant change in the time between injection of isotope and the start of fast transport. The amount of axonal transport of radiolabelled proteins as measured by accumulation of proteins proximal to a ligation on the sciatic nerve was also unchanged. The reduction in fast transport rate in the diabetic rats was eliminated by maintenance of normal blood glucose levels in twice daily insulin administration. The results are discussed with regard to the known effects of experimental diabetes on axonal transport in sensory fibres and to the role of fast axonal transport in peripheral neuropathies in general.

Association of spermine and 4S RNA during axonal transport in regenerating optic nerves of goldfish

Experiments were designed to determine whether polymines are bound to 4S RNA and then transported axonally along regenerating optic axons of goldfish. In one set of experiments, inhibition of retinal RNA synthesis by intraocular injections of 10 μg of cordycepin, blocked the axonal transport of both [ 3H]RNA and [ 14C]spermidine by about 65%, 6 and 14 days after injection. Intraocular injections of vinblastine, (0.1, 0.5 or 1.0 μg) an agent which interrupts axonal transport of proteins, had no effect on retinal RNA synthesis nor on the amount of [ 14C]spermidine incorporated into the TCA-insoluble fraction of retinal extracts. However, the axonal transport of both [ 3H]RNA and [ 14C]polyamines was affected in a dose-dependent fashion; the inhibition of both was approximately 80% at the higher dose. Further evidence for an association between axonally transported 4S RNA and polyamines came from experiments in which regenerating optic axons were cut and allowed to degenerate 6 days after injection of [ 3H]spermidine into the eye. The loss of optic axons from the tectum 7 days after cutting the nerve resulted in an 86% loss of TCA insoluble polyamines, indicating a largely intra-axonal locus. A similar loss of 4S RNA was found in identical experiments following injections of [ 3H]uridine into the eye. Finally, experiments were performed in which [ 3H]spermidine was injected into both eyes of 12 fish whose optic nerves had been regenerating for 18 days. Six days later, fish were sacrificed and RNA was extracted from tectal homogenates by hot phenol and ethanol precipitation. The major stable RNA species were separated by SDS-polyacrylamide disc gel electrophoresis and radioactivity was determined by extraction of 2.0 mm gel slices. Results showed co-migration of 3H with 4S RNA optical density peaks, and not with 28S and 18S ribosomal RNA peaks, suggesting that some polyamine-associated radioactivity is bound to axonally transported 4S RNA. When the nature of that radioactivity was determined on an amino acid analyzer, it was found to be present primarily as spermine and not as the injected compound spermidine. The data are consistent with the hypothesis that some spermine is bound to 4S RNA and then axonally transported along regenerating axons of the goldfish optic nerve.

Acrylamide neuropathy and changes in the axonal transport and muscular content of the molecular forms of acetylcholinesterase.

Acetylcholinesterase (AChE) is present in nervous and muscular tissues of normal chickens in four main molecular forms (G1, G2, G4, and A12), distinguishable by sedimentation analysis. In the sciatic nerve of acrylamide-poisoned chickens, the anterograde axonal transport of A12 AChE was reduced by 60%, and that of G4 by 21%, compared to control values whereas the slow axoplasmic transport of G1 and G2 was unaffected. Regarding the leg muscles, only the tibialis anterior revealed dramatic alterations in the distribution of it AChE forms coinciding with a large reduction in the number of nerve endings. In acrylamide poisoning, the AChE molecular forms were considered as very sensitive markers of both axonal transport phases and of the innervation state. Our results support the hypothesis that a defect in the fast axonal transport of proteins might be involved in the degeneration process of the disease.

Retrograde axonal transport of transmitter enzymes, fucose-labeled protein, and nerve growth factor in streptozotocin-diabetic rats.

Rapid axonal transport was studied by several methods in rats with serum glucose levels above 300 mg/dl as a result of treatment with streptozotocin (45–50 mg/kg) 3 days, 1 wk, 4 wk, 8 wk, or 4 mo earlier. With untreated age-matched rats as controls, rapid anterograde axonal transport in the sciatic nerves of diabetic rats appeared entirely normal. Statistically significant differences were never observed between experimental and control nerves in the basal content of acetylcholinesterase (AChE) and dopamine-β-hydroxylase (DBH) activity or in the rate of accumulation of these enzymes proximal or distal to a ligature. Therefore, the basic capacity for rapid anterograde and retrograde axonal transport of proteins was probably unimpaired in the diabetic nerves. The accumulation of labeled glycoproteins proximal to ligatures on the contralateral nerves of the same rats after injection of the L5 dorsal root ganglion with 3H-fucose was likewise normal. However, rats with 1, 4, and 8 wk of diabetes did show reduced accumulation of fucose-labeled protein distal to nerve ligations, indicating a long-lasting abnormality of retrograde axonal transport. Furthermore, this abnormality was reversed by daily insulin treatment during the second half of an 8-wk experimental period. It is therefore unlikely that the depression of retrograde transport reflects direct toxic effects of streptozotocin. We conclude that streptozotocin-induced diabetes leads to: (1) abnormal delay in the turnaround of transported proteins in distal nerve regions, perhaps combined with (2) an abnormal metabolism of glycoproteins. A delayed onset of retrograde transport is consistent with present observations of reduced accumulation of 125I-labeled nerve growth factor (NGF) below a midthigh ligature on the sciatic nerve after injection of this protein into the hindfoot of rats with 3–5 wk of diabetes. Further work on the factors controlling rapid retrograde transport of proteins in diabetic nerve is warranted.

Studies on axonal transport in dopaminergic neurons: Effect of neuropharmacologic lesions

Axonal transport of [ 3H]protein to the nucleus accumbens, olfactory tubercle, septal region, caudate nucleus, and hypothalamic region was investigated in rats after unilateral injection of [ 3H]lysine into the substantia nigra. Co-injection of 2 μg of colchicine with the [ 3H]lysine depressed the recovery of [ 3H]protein from forebrain structures by over 70 percent without altering incorporation into midbrain protein, whereas 1 or 2 μg of cycloheximide decreased the incorporation of labelled lysine into both midbrain and forebrain protein by 69 to 76 percent. Partial 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra decreased striatal dopamine levels by 78 percent and reduced axonal protein transport by 47 to 82 percent. Injecting the [ 3H]lysine 2 mm dorsal to the substantia nigra decreased transport by 95 percent. Unilateral kainic acid-induced lesions of the caudate, which decreased striatal glutamic acid decarboxylase activity by 44 percent and spared striatal dopamine content, did not alter the transport of [ 3H]protein. Thus, axonal transport of protein in dopamine-containing systems is dependent upon the site of injection of labelled precursor and upon the integrity of a 6-OHDA sensitive pathway. Further, transport is sensitive to inhibitors of both microtubule assembly and protein synthesis, and insensitive to intrastriatal kainic acid lesions.

‘Acrylamide-induced’ neuropathy and impairment of axonal transport of proteins. I. Multifocal retention of fast transported proteins at the periphery of axons as revealed by light microscope radioautography

The axonal transport of proteins was studied in ciliary ganglia of control and acrylamide-treated chickens. After an intracerebral injection of [ 3H]lysine, the distribution of axonally transported proteins was analyzed in the preterminal segments of preganglionic axons and in their caliciform nerve endings by means of quantitative light microscope radioautography. By 7 days after the injection of [ 3H]lysine, the concentration of labeled proteins in the preganglionic axons was either similar or slightly increased in acrylamide-treated chickens as compared to controls. In contrast, by 3 h, whereas the axons of controls were poorly radioactive, 20–30% of the axons in acrylamide-treated chickens displayed focal and intense labeling at their periphery; simultaneously, in acrylamide treated chickens, 20–30% of the nerve endings contained a decreased amount of radioactive proteins. It is concluded that acrylamide induces multifocal retention of fast axonally transported proteins in preterminal segments of certain axons.

Importance of monovalent ions for the fast axonal transport of proteins.

Proteins labeled with [35S]methionine or [3H]leucine were generated in vitro in bullfrog dorsal root ganglia and their fast axonal transport in the spinal nerves was followed during a subsequent incubation period. Incubation of the ganglia in a medium where sucrose, choline chloride, or sodium isethionate replaced NaCl caused respectively an 88, a 37, or a 76% reduction in the quantity of proteins carried by the fast axonal transport system; no decrease in synthesis of labeled proteins was observed and protein transport followed the usual time course. Incubation of desheathed spinal nerves in a medium where sucrose replaced NaCl reduced by 67% the quantity of labeled proteins which were transported past the desheathed region. Although both the axons and the dorsal root ganglia exhibit the requirement for monovalent ions to maintain fast axonal transport, the possibility that the ionic requirements of the ganglia pertain to the somal portion of the nerve cell is discussed.

‘Acrylamide-induced‘ neuropathy and impairment of axonal transport of proteins. II. Abnormal accumulations of smooth endoplasmic reticulum as sites of focal retention of fast transported proteins. Electron microscope radioautographic study

The distribution of fast axonally transported proteins was studied by electron microscope radioautography in ciliary ganglia of chickens treated or not treated with acrylamide. At 3 h after the intracerebral injection of [ 3H]lysine, the preganglionic axons of the untreated chickens displayed few silver grains, mainly associated with smooth endoplasmic reticulum (SER) profiles. In most axons of acrylamide-treated chickens, a similar pattern was observed, except in axons which exhibited focal and intense labeling underneath the axolemma: clusters of silver grains indeed overlayed peripheral accumulations of tubulovesicular profiles of SER, dense core vesicles and mitochondria. After impregnation with heavy metals, electron microscope observation of 1 μm thick sections showed a locally disorganized SER forming a complex network of tubules intermingled with vesicles and mitochondria. Such a local disorganization of the peripheral SER in the distal part of the axons, could be responsible for the focal stasis of fast transported proteins; it seems to be one of the earliest changes detectable in axons damaged by acrylamide treatment.

Axonal transport: a quantitative study of retained and transported protein fraction in the cat.

The fast axonal transport of proteins was studied in the cat sciatic nerve after injection of [3H]leucine into the spinal ganglion or the ventral horn of the seventh lumbar segment. The amount of transported proteins after ganglion injection was linearly related to the amount of label present at the ganglion. At variable intervals after ganglion or spinal cord injection, the sciatic nerves were sectioned in some experiments. The transport of proteins continued in the peripheral nerve stump in a wavelike manner, but the advancing wave leaves a labeled trail behind. A fraction of this trail corresponds to proteins moving at slower velocities than the velocity of proteins in the wave front. Another fraction of the trail corresponds to molecules retained by the axons. Each nerve segment of 5 mm in length retains 1.5% of the transported proteins, and the profile of retained proteins along the sciatic nerves follows a single exponential function. From the proportion of retained proteins, the concentration of transported proteins at the terminals of branching axons as a function of the branching ratio was estimated. In the case of motor axons innervating the soleus muscle of the cat, the concentration of recently transported proteins at the nerve terminals would be approximately 0.83% of the proteins leaving the spinal cord. This low concentration of transported proteins at the nerve terminals may explain the lability of neuromuscular synapses when axonal transport is decreased or interrupted.

Intracellular transport in neurons.

Intracellular transport in neurons.

Axonal transport of proteins and glycoproteins in the rat nigro-striatal pathway and the effects of 6-hydroxydopamine

Following stereotaxic injection of [35S]methionine into the substantia nigra of adult rats, there was rapid local incorporation of radioactivity into acid-insoluble material. Incorporation peaked by 4 h and then decreased. In contrast, acid-precipitable radioactivity in the corpus striatum (the major projection site of the substantia nigra) rose markedly between 1 and 8 h followed by a plateau period and another even more marked increase between 24 h and 6 days. Experiments involving injection of [3H]fucose gave similar results except that most of the acid-precipitable radioactivity in the striatum appeared in an early wave. In each case radioactivity in the contralateral striatum was less than 11% of that on the ipsilateral side. Stereotaxic injection of colchicine (20 microgram) into the nigrostriatal pathway (within the median forebrain bundle) blocked transport of [35S]protein and [3H]glycoprotein by 90% and 50%, respectively. In animals treated with 6-hydroxydopamine (6-OHDA; treated neonatally or as adults) the accumulation of striatal [35S]protein was reduced to 7 to 26% of control levels; striatal [3H]glycoprotein was also reduced, but not as much (29% to 73% of control). In control experiments, [3H]DOPA wa injected into the substantia nigra, and [3H]dopamine was measured in corpus striatum; 6-OHDA treatment reduced the amounts of striatal [3H]dopamine recovered to 3% of control values. The failure of colchicine or 6-OHDA to block transport of incorporated fucose as effectively as the transport of incorporated methionine is possible due to greater diffusion of fucose away from the injection site to non-dopaminergic nuclei projecting to the striatum. The molecular weight distribution of radioactive proteins at the substantia nigra and corpus striatum was analyzed by polyacrylamide gel electrophoresis. For both [35S]methionine and [3H]fucose, the gel electrophoretic pattern of radioactive proteins in the injection site (substantia nigra) was complex and did not change greatly between 2 h and 6 days. At the projection site (striatum) the electrophoretic distribution pattern was initially different from that of the substantia nigra, and changed markedly over the course of several days. In 6-OHDA-treated animals (treated neonatally or as adults), the bulk of proteins transported in nigro-striatal non-dopaminergic neurons appears to be very similar to that transported in the intact pathway in control rats. However, in striata of 6-OHDA-treated animals, a consistent reduction in striatal 35S- and 3H-radioactivitiy was observed in proteins with molecular weight from about 67,000 to 77,000. Assuming that the 6-OHDA treatment did not substantially affect the non-dopaminergic neurons, we interpret this to mean that some of the proteins in this molecular weight range are transported primarily by dopaminergic neurons.

Impaired retrograde axonal transport from a nerve crush in streptozotocin diabetic rats.

The axonal transport of proteins in crushed nerves of streptozotocin (40 mg/kg) diabetic rats was investigated 4 weeks after induction of diabetes. 35S-methionine was used as a marker for protein and 3H-fucose as a marker for glycoprotein. The precursors were injected into the fifth lumbar spinal ganglion and the accumulation of TCA-insoluble activity proximal and distal to a sciatic nerve ligature was measured at different time intervals after application of a crush. The start of accumulation distal to the ligature was delayed by 1 hour for proteins as well as for glycoproteins. Furthermore, the total amount of accumulated protein after 19 h was decreased by 18% while the decrease was 21% for glycoprotein. By insulin treatment the differences could both be prevented and reversed after 3 days of normoglycaemia. These findings demonstrate an impaired response to a nerve crush and might be the explanation for the regenerative abnormalities of peripheral nerves in diabetes.

Relation of somal lipid synthesis to the fast axonal transport of protein and lipid

The role of somal lipid synthesis in the fast axonal transport of protein and lipid was examined in vitro utilizing spinal/sciatic nerve preparations of bullfrog. Inhibition of phospholipid synthesis in dorsal root ganglia by the amphiphilic cation, fenfluramine (0.1–2.0 mM) was monitored as decreased incorporation of [ 3H]choline into phosphatidyl choline. This inhibition was directly proportional to a decrease in the amount of [ 3H]protein undergoing fast axonal transport, the two variables being related by a slope close to unity. [ 3H]Choline-labeled lipid undergoing fast transport in the axon was unaffected by inhibition of somal phospholipid synthesis. Levels of fenfluramine up to 1.0 mM had no effect on uptake or incorporation of [ 3H]leucine. Selective exposure of desheathed nerve trunks to 1.0 mM fenfluramine had no effect on [ 3H]protein translocation, indicating that local phospholipid synthesis is not required to maintain ongoing transport in the axon. Inhibition of cholesterol synthesis in the ganglia with the analog 20,25-diazacholesterol also resulted in depression of [ 3H]-protein transport. Since synthesis of both phospholipid and cholesterol are required at the level of the ganglion, it is suggested that the initiation of fast axonal transport of protein is dependent on the assembly of lipoprotein structures in the soma.