Axonal Transport Of Proteins Research Articles

Increased synthesis of membrane macromolecules is an early response of retinal neurons to trimethyltin intoxication

We studied the synthesis and axonal transport of proteins and glycoproteins in the visual system of adult Long-Evans rats that had received 4 weekly doses of trimethyltin hydroxide (TMT, 4 mg/kg b. wt.) by gastric intubation. One week following the last dose, an in vitro assay was used to study the rate of incorporation of radioactive precursors into various macromolecules of isolated retinas. Retinas from TMT-treated rats showed increased apparent rates of synthesis, relative to retinas from control rats, for proteins [( 35S]methionine precursor) and glycoproteins [( 3H]fucose precursor). Gel electrophoretic analysis of newly synthesized proteins indicated that the increased synthesis was a generalized effect, i.e. it was not restricted to a select subset of proteins. The axonal transport of these macromolecules by retinal ganglion cells to axons (optic tract) and nerve endings (superior colliculus) was examined in vivo following intraocular precursor injection. The amount of material transported, relative to that synthesized in the retina, was not appreciably altered in TMT-treated rats, indicating that TMT did not selectively impair axonal transport. The biochemical changes were accompanied by minimal ultrastructural alterations and little neuronal necrosis in the retina. We suggest that TMT induces increased synthesis of membrane macromolecules in retinal neurons; this may reflect an early reactive (compensatory) response rather than a regressive (degenerative) response of retinal neurons to TMT. Our data do not support the hypothesis that TMT induces a functional impairment of neuronal endoplasmic reticulum or Golgi apparatus.

The relationship between axonal transport of protein and demyelination in the optic nerves of mice infected with Semliki Forest Virus

Fast and slow axonal transport of protein have been studied in the optic nerves of mice infected with Semliki Forest Virus (SFV) that causes patchy demyelination throughout the CNS. Intravitreal injections of [ 3H]proline were given at regular intervals after virus inoculation, the labelled protein in the superior colliculi was then measured after survival periods of 18 h or 10 days, for fast and slow axonal transport studies, respectively. Fast transport studies showed an enhanced amount of protein arriving at the optic nerve terminals (superior colliculus) of the SFV-infected mice prior to the onset of demyelination. In contrast, the slow transport studies showed an enhanced amount of protein at the superior colliculus of the SFV-infected mice during the demyelination period. There was no concomitant increase in labelled protein in the retina at any time after the SFV infection. It is proposed that alteration in the transport of the protein constituents other than major myelin specific components may cause disruption of myelin maintenance in SFV infection.

Effects of Systemic Methyl Mercury‐Adulterated Water Consumption on Fast Axonal Transport in the Rat Visual System

The present study was designed in an effort to determine whether changes in fast axonal transport in the mature rat visual system can be directly correlated with the onset of neurological dysfunction. Methyl mercury was administered in the drinking water at a concentration of 54 micrograms Hg/ml. Fast axonal transport of proteins in the optic nerve and tract was quantified by scintillation spectrometry of protein-bound radioactivity along the visual pathway after an intraocular injection of 3H-proline. At 8 hours after injection the labeled protein had reached the lateral geniculate body both in controls and treated animals. However, two-way analysis of variance revealed a significant decrease in the volume of transported protein-bound radioactivity along the visual pathway. Thus, while the rate of fast axonal transport does not seem to be correlated with the onset of motor dysfunction, the onset of neurological symptoms may be associated with abnormal transport capacity. Treatment lowered body weight to the same extent in males and females. Hind limb cross-over occurred after 25.6 +/- 0.8 days and was followed quickly by hind limb paralysis (32 +/- 0.6 days). The cerebellum revealed pyknotic nuclei throughout the internal granular layer. Purkinje cells appeared normal. No pathological changes were noted in the kidneys.

Reduction of axonal transport in the rat optic system after direct application of methylmercury

Fast axonal transport of proteins in the optic nerve and tract was quantified by scintillation counts of protein-bound radioactivity along the visual pathway after an intraocular injection of [ 3H]proline. In control rats the label traveled at a rate of about 60 mm/day, reaching the optic chiasm at 4 h and the lateral geniculate body at 8 h postinjection. When methylmercury was injected simultaneously with [ 3H]proline, the label traveled at a rate of about 30 mm/day. At 8 h postinjection, the labeled protein had reached the optic chiasm, but the more distal pathway was unlabeled. The same pattern was observed histologically by emulsion autoradiography of the pathway. Some label was detected in the lateral geniculate of methylmercury-treated animals at 8 h, but this may have resulted from local incorporation, as judged by a similar level of labeling in the contralateral visual pathway. Alternatively, it may be the case that a small fraction of the axons in the treated pathway continued to transport proteins in a normal fashion. The very heavy label observed throughout the pathway in controls was present only in the proximal half of the pathway in methylmercury-treated rats. Methylmercury signficantly reduced incorporation of [ 3H]proline in the rat retina, but this reduction was not as great as the effect in the optic nerve. In contrast, cycloheximide, a potent protein synthesis inhibitor, reduced labeled protein in the optic nerve only to the same extent as it reduced incorporation. These results suggest that methylmercury's effect on transport is not dependent solely on its effects on protein synthesis, but represents a separate mechanism of neurotoxicity.

Slow axonal protein transport and axoplasmic organization

A recently formulated structural theory of axonal transport suggests that each group of transported material is a distinct class of functionally related structures and that the proteins making up those structures, with few exceptions, are found in only one rate component, thus maintaining a close noncovalent association during transport. If such a relationship exists, one would postulate that the labelled proteins at the leading edge and trailing edge of a rate component following a pulse of radioactive amino acid would be present in similar proportions. After retinal ganglion cell proteins were labelled by intraocular injection of radioactive amino acid in the guinea pig eye, the optic nerve and tract were analyzed at several post-injection intervals. The data thus derived support the existence of a close relationship among the “soluble” proteins of slow component b and are consistent with the structural concept of axonal transport. If diffusion were the mechanism of transport, the larger proteins would be expected to move slower than the smaller proteins. Such insights into the process of axonal transport should help to identify the variables critical to survival of neurons following acute trauma and degenerative diseases.

Inhibition of the retrograde axonal transport of dopamine-β-hydroxylase antibodies by the calcium ionophore A23187

High levels of calcium, as well as calcium ionophores, have been reported to inhibit the anterograde transport of proteins. The effect of the calcium ionophore. A23187, on the retrograde axonal transport of proteins was therefore investigated. The uptake of antibodies to dopamine-β-hydroxylase (anti-DβH) by sympathetic nerve terminals in the iris and their subsequent accumulation in the superior cervical ganglion was inhibited by up to 65% by A23187 (6 nmol, i.o.). At this dose, catecholamine fluorescence in the iris was reduced, indicating a high rate of exocytosis, but tyrosine hydroxylase levels and the capacity of the treated irides to take up noradrenaline were unaffected. Higher amounts of A23187 (28 nmol, i.o.) did not cause a greater degree of inhibition of retrograde transport. However, this dose was toxic to the neurons, as shown by a 68% decrease in the ability of the nerve terminals in the iris to take up [ 3H]noradrenaline. This loss of function occurred gradually over a 12-h period. On the other hand, tyrosine hydroxylase levels were unaffected by 28 nmol A23187. The toxicity of A23187 may be a consequence of a build up in intracellular calcium, but such toxicity did not lead to any apparent loss of nerve terminals within a 3-day period.

Blockage at two points of axonal transport in glaucomatous eyes

Blockage of axonal protein transport in intraocular hypertensive primates (Macaca irus) was studied autoradiographically and quantitatively and the findings compared with our previous work on rabbits. Fast axonal transport was blocked at two points, at the lamina scleralis and at the edge of posterior scleral foramen, and reduced by 25% when intraocular pressure of 50 mmHg continued for 6 h. The importance of the blockage at the lamina scleralis and at the edge of scleral foramen for the explanation of paracentral scotomas and the peripheral nasal step (Rønne) is discussed.

Comparison of the effects of sciatic nerve crush or resection on the proteins of fast axonal transport in rat dorsal root ganglion cell axons

Proteins of fast axonal transport in rat sciatic nerve axons were separated and characterized by SDS-polyacrylamide gel electrophoresis and fluorography, after injection of l-[ 35S]methionine into the dorsal root ganglion. The effects of crushing or resecting the sciatic nerve on the relative labeling of specific polypeptide bands were compared. Initially, both types of axon injury produced the same response, but after 3 weeks there was a partial return to normal composition in crushed nerves. In resected nerves, the changes characteristic of axon injury persisted beyond 7 weeks. Behavioral testing showed that crushed nerves reinnervated foot skin, whereas no reinnervation was detected after resection. We conclude that in sensory neurons, as in several other neuronal types, the restoration in normal composition of fast-transported protein after axon injury depends on reinnervation of target tissues. This aspect of the cell body reaction to injury seems to be regulated by a retrograde trophic interaction with the target.

Proteins in fast axonal transport are differentially transported in branches of sensory nerves

Radioactively labeled, fast-transported proteins were collected at ligatures placed on peripheral and central branches of spinal sensory nerves of the bullfrog. In agreement with previous studies, we found that the same species of proteins were transported down each branch. For each protein species analyzed we have measured the amount of radioactivity reaching each ligature, and calculated the ratio of radioactivity reaching the peripheral ligature to that reaching the central ligature. Not all protein species have the same ratio. This suggests that there may be differential transport of fast-transported proteins in axonal branches.

Synthesis and fast axonal transport of proteins in the isolated Aplysia nervous system

Fast axonal transport and neuronal protein synthesis was studied in the isolated nervous system of Aplysia californica. The abdominal ganglion with attached pleural-abdominal connectives (PAC) was removed and the ganglion pulse-labelled with [ 35S]methionine for 30 min in vitro. The axon containing connectives were ligated 24–28 mm from the ganglia and the system was perfused with chase media for 6–72 h to allow labelled rapidly transported proteins to accumulate at the ligature. One-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography was used to analyze the distribution of rapidly transported proteins along the right PAC. By 12 h, a significant accumulation of labelled proteins at the ligature was present but the build up was not complete until 48 h when almost no trailing of rapidly transported proteins was observed. Quantitation of the transport profiles of several rapidly transported protein ssuggested a discontinuous release of proteins from the cell body. Analysis using two-dimensional PAGE revealed 10 major groups of rapidly transported proteins. These proteins were all identified among the total complement of newly synthesized proteins in cell R2. Not all rapidly transported proteins are cleared from the cell body at the same rate. Several of the major groups were no longer present in the neuron cell body 24 h after labelling, indicating that these species are selectively exported; others were still present after 3 days, suggesting that these proteins with a longer residence time have functions in both somatic and axonal regions of the neuron.

Removal of optic tectum prolongs the cell body reaction to axotomy in goldfish retinal ganglion cells

Injury to the optic axons of goldfish elicits dramatic changes in the cell bodies of the neurons from which these axons arise, the retinal ganglion cells. The changes include a large increase in cell size and in synthesis and axonal transport of protein. The cells begin to return to normal about 3 weeks after the injury, when the axons invade the contralateral (homotopic) lobe of the optic tectum, and recovery is essentially complete by 8–10 weeks after the lesion. However, if the homotopic lobe of the tectum was removed at the time of nerve crush, we found that the cell body reaction was greatly prolonged. The cells remained enlarged, and [ 3H]proline incorporation and fast axonal transport of protein remained elevated, until at least 10–12 weeks after nerve crush, although by this time most of the regenerating axons had probably regained their normal length and many had entered the remaining ipsilateral (heterotopic) lobe of the tectum. The cells showed partial recovery by the latest time tested, 26 weeks after nerve crush, when the projections from the two eyes had segregated into separate bands in the heterotopic tectal lobe.

Ca 2+-activated protease activity in frog sciatic nerve: Characterization and effect on rapidly transported axonal proteins

Protease activity was studied in the frog sciatic nerve. The activity was measured as the release of TCA-soluble radioactivity from either 3H-labelled proteins transported by rapid axonal transport (AXT) or 3H-labelled ganglionic proteins. In nerve homogenates containing transported substrates, protease activity exhibited two peaks, one around pH 5 and one around pH 8. Ca 2+ at 100 μM or higher concentrations only stimulated the latter, which was inhibited by 1 mM parachloromercuric benzoate, a sulphydryl reagent, but unaffected by ATP (1 mM). The proteolytic activity was recovered in the 10 5 g supernatant of the homogenate. In desheathed nerves containing 3H-labelled transported proteins, the protease activity could be activated by exposing the nerve to a Ca 2+-ionophore, X-537 A, or to an elevated Ca 2+-concentration (50 mM). These conditions were also shown to increase the influx and efflux of 45Ca 2+ in the nerves. The results indicate the presence within axons of a Ca 2+-activated soluble protease, which degrades rapidly transported proteins. The finding that the protease degraded ganglionic soluble proteins to about the same extent suggests a broad substrate specificity. The present system should be useful for further characterization of protease activity during various physiological conditions.

The blocking of axonal transport of protein by local cooling of the axon in Aplysia

The blocking of axonal transport of protein by local cooling of the axon in Aplysia

The blocking of axonal transport of protein by local cooling of the axon in aplysia

The blocking of axonal transport of protein by local cooling of the axon in aplysia

Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat.

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.

Is protease activity involved in fast axonal transport?

N-alpha-p-Tosyl-L-Lysine Chloromethyl Ketone (TLCK), a protease inhibitor, was found to irreversibly inhibit rapid axonal transport of protein in vitro in the frog sciatic nerve. TLCK exerted its action at the axonal level and seemed to depress the rate rather than the amount of transported protein. The efficiency of TLCK as a protease inhibitor was demonstrated by polyacrylamide gel electrophoresis, which showed that degradation of high molecular weight proteins (presumably neurofilament subunits) into a 25000 dalton protein could be induced by exposing the frog nerves to triton-X and prevented by the presence of TLCK. Findings that TLCK, at a transport inhibiting concentration (0.1 mM), had little or no effects on either protein synthesis or ATP levels, suggest that TLCK did not affect transport due to general cytotoxic properties. The effects of TLCK is discussed in relation to possible roles of protease activity in axonal transport.

Axonal transport of [ 35S]Methionine labeled proteins in Xenopus optic nerve: Phases of transport and the effects of nerve crush on protein patterns

Axonal transport of proteins in the Xenopus optic nerve was examined by labeling proteins in the eye with [ 35S]methionine injected intraocularly and then analyzing the labeled proteins in the eye, nerve, and tectum on linear gradient SDS polyacrylamide gels at different times after the injection. Because the optic nerve in Xenopus is short, in order to distinguish transported proteins from locally synthesized proteins, the optic nerve on one side of the animal was crushed at the orbit (to stop axonal transport) 5–30 min prior to injection and the crushed and normal nerve segments were compared. Proteins in the intact nerve which were absent in the crushed nerve were identified as axonally transported proteins. By such criteria several waves corresponding to transported material moving at⩾ 6mm/day, 1.6–2.8 mm/day, and approximately 0.2 mm/day were detected in the nerve. The most rapid phases of transport could be further resolved in the optic tectum into 3 additional components at 60–96 mm/day, 30–48 mm/day, and 6–11 mm/day. Analysis of labeled proteins in the crushed nerves distal to the crush, near the injury site, revealed several locally synthesized proteins (mol. wt. 54,000, 48,000, 43,000 daltons) which were not present in normal, uninjured nerves. Such proteins are probably synthesized by glia in response to injury.

Increase in axonal transport in demyelinating optic nerve fibres in the mouse infected with Semliki Forest virus.

Since the integrity of myelin and axon is closely linked with the transport of substances along the axon, changes in the fast and slow axonal transport in the demyelinating optic nerves of mice infected with Semliki Forest virus were studied. Radioactive analysis of the superior colliculi and the optic nerves was thus made following different survival times after an intravitreal injection of tritiated proline by autoradiographic and liquid scintillation counting techniques. The amounts of both the fast and slow axonal transport of proteins were significantly higher in the optic nerves of mice infected with the virus than those found in the optic nerves of control mice. These results suggest that demyelinating fibres send a signal to the perikarya which then responds by synthesizing more proteins.

Antibodies to gangliosides inhibit goldfish optic nerve regeneration in vivo.

Intraocular injection of antiserum to mixed ganglioside or to GM1 inhibited the regeneration of goldfish optic axons following an optic nerve crush. For example, injections of antiserum on 5 consecutive days beginning the day before the crush resulted in a decrease of about 40% in the axonal outgrowth distance measured at 10 days after the crush. The inhibition was observed even when the treatment was begun a few days after the lesion, and greater degrees of inhibition were observed when the treatment was given later in regeneration. This indicates that the antiganglioside serum interfered with axonal elongation more than with the initial sprout formation. The antiganglioside treatment did not impair the enlargement of the cell bodies and nucleoli that accompanies regeneration, nor did it affect fast axonal transport of protein, glycoprotein, or glycolipid in regenerating nerves. Thus inhibition of outgrowth by antiganglioside treatment was not mediated by a gross change in the metabolism of the regenerating neurons. Treatment of normal neurons with the antiserum produced a 20-30% increase in the amount of 3H-glucosamine-labeled glycoproteins and glycolipids conveyed by fast axonal transport. These results suggest that membrane gangliosides may normally influence the supply of axonally transported glycosylated macromolecules. However, the effect of antiganglioside on axonal transport of glycosylated molecules and on axonal outgrowth are not necessarily related to each other.

Retrograde and orthograde axon transport by brief-exposure to nickel chloride: Methodology and parameters for success in the brain-retrocerebral complex of the cockroach Diploptera punctata

Exposure of the intact brain-retrocerebral complex of the cockroach Diploptera punctata to 500 mM NiCl 2 in 0.1% bovine serum albumen for 10 min resulted in the filling of neurones connecting the components of the brain-retrocerebral complex. Following severance of one corpus cardiacum from the brain prior to exposure to NiCl 2, a larger number of neurones filled in the contralateral side of the pars intercerebralis. Reducing exposure time to NiCl 2 to 30s did not result in backfilling of intact preparations. However, if the nervi corporis allati I (NCA 1) were sectioned, then exposed to NiCl 2 for 30s and incubated in culture medium at 4°C for 24 h, neurones of the severed NCA I backfilled to somata originating in the pars intercerebralis and pars lateralis. In addition, the axons distal to the cut also filled with the tracer in the orthograde direction, revealing a complex network of axon terminals investing the corpora allata. Sectioned nervi corporis allati I exposed to the tracer solution, then incubated in culture medium for 0.5 h, rapidly backfilled at a rate of 30–100 mm/day. Rapid backfilling did not occur in the absence of BSA. One per cent fluorescein isothiocyanide-labelled BSA backfilled as rapidly as the NiCl 2 BSA solution. 2,4-Dinitrophenol and cytochalasin B inhibited brief-exposure backfilling whereas colchicine had no effect in short term incubations but partially inhibited backfilling in long term incubations. Backfilling is thus a metabolically mediated event and the rate of transport of the tracer is similar to that of retrograde fast axonal transport of proteins.