Fast Axonal Transport Of Proteins Research Articles

Human amyloid-β1–42 applied in vivo inhibits the fast axonal transport of proteins in the sciatic nerve of rat

Human amyloid-β1–42 has been suggested to be a pathogenetic factor in Alzheimer's disease. The precise mechanism by which this peptide causes the degeneration of neurons in the affected brain is not yet fully understood. By using immunohistochemistry we explored the inhibitory effects of human amyloid-β1–42 applied in vivo on the fast axonal transport of acetylcholinesterase, the amyloid precursor protein, the vesicular acetylcholine transporter and synaptophysin in the sciatic nerve of rat. Our findings provide evidence for the in vivo neurotoxic effect of human amyloid-β peptide.

Optic neuritis in chronic relapsing experimental allergic encephalomyelitis in Biozzi ABH mice: Demyelination and fast axonal transport changes in disease

The encephalitogenicity of optic nerve tissue was demonstrated in Biozzi ABH (H-2 dq1) mice. Acute experimental allergic encephalomyelitis (EAE) occurred in 11/14 animals and 4/5 exhibited relapse. The involvement of the optic nerve in spinal cord homogenate induced chronic relapsing EAE (CREAE) was demonstrated by mononuclear cell infiltration and myelin degradation in the optic nerve prior to and during clinical disease. During the relapse phase gross pathological assessment revealed swollen and translucent plaques on the optic nerves. Advanced lesions showed widespread demyelination, astrocytic gliosis and fibrotic changes of the blood vessels. Physiologically, the fast axonal transport of proteins from the retina to the optic nerve and superior colliculus was significantly decreased during relapse. The association of inflammation and demyelination with physiological deficit in the optic nerve highlights the usefulness of this model in the study of multiple sclerosis in which acute monosymptomatic unilateral optic neuritis is a common manifestation. Furthermore, the novel induction of CREAE with optic nerve homogenate suggests that optic nerve antigens play a significant role in the pathophysiology and progression of neurological disease in CREAE which may be relevant to studies of optic neuritis in multiple sclerosis.

Tumour necrosis factor causes an increase in axonal transport of protein and demyelination in the mouse optic nerve

An increase in fast axonal transport of protein by the optic nerve was found in mice following a single combined injection of human recombinant tumour necrosis factor alpha (rTNF) and [ 3H]proline into the vitreous chamber. Demyelination was observed in optic nerve fibres arising from the eyes of mice which received a single rTNF injection. No such changes were detected when heat-inactivated rTNF was injected with the label. The effects of intravitreal injection of rTNF on the pathophysiology of mouse optic nerve resembled those found in mice infected with Semliki Forest virus (SFV), an animal model of multiple sclerosis. We suggest that TNF could mediate at lease some of the pathophysiological changes found in SFV-infected mice and may provide a clue concerning the disease mechanism in multiple sclerosis.

Evidence for a T-cell related factor as the cause of demyelination in mice following Semliki Forest virus infection

Patchy demyelination throughout the central nervous system, including the optic nerve, is known to occur following infection with Semliki Forest virus (SFV) in Swiss/A 2G mice. An increase in the fast axonal transport of protein in optic nerves occurred before they showed signs of demyelination in Swiss/A 2G mice, heterozygous nude mice and nude mice reconstituted with T-cells. SFV infection, however, caused neither an increase in the fast axonal transport of protein, nor optic nerve demyelination in T-cell-deficient nude mice. Thus, the increase in both axonal transport and demyelination following SFV infection appear to be T-cell-mediated events, rather than direct effects of the virus.

Pressure-induced inhibition of fast axonal transport of proteins in the rabbit vagus nerve in galactose neuropathy: prevention by an aldose reductase inhibitor

Fast and slow anterograde axonal transport and retrograde axonal transport of proteins were studied in the mainly non-myelinated sensory fibres of the vagus nerve of rabbits fed a diet of 50% galactose over a period of 29 days. Galactose feeding had no effect on the rate or protein composition of slow transport nor on the amount of retrogradely transported proteins. There was a slight retardation of fast transported proteins although their composition was unchanged. The galactose feeding led to a significant increase (p less than 0.005) in nerve water content and nerve galactitol but no significant change in myo-inositol. When 20 mm Hg pressure was applied locally to the cervical vagus nerve, fast transported proteins accumulated proximal to the compression zone in the galactose-fed but not in control rabbits. Administration of the aldose reductase inhibitor Statil (ICI 128436) throughout the experiment prevented the increased susceptibility to pressure and the increase in nerve galactitol and water content. The effects of pressure are similar to those found in the streptozotocin-diabetic rat although the underlying mechanisms may differ.

The disturbance of the fast axonal transport of protein by passive stretching of an axon in Aplysia.

1. Radioactive amino acid, either [3H]leucine or [3H]proline, was injected into neurone R2 in the abdominal ganglion of Aplysia kurodai to investigate the intra-axonal transport of protein in a single axon. 2. Some of the injected amino acid which was not utilized for protein synthesis diffused intra-axonally with a diffusion constant of 6.0 X 10(-6) cm2/s (25 degrees C), which is the value expected from the known diffusion constants of acetylcholine and gamma-aminobutyric acid in the axoplasm (Koike & Nagata, 1979). The true length of the zigzagging axon in the nerve bundle was measured by the diffusion distances. 3. The radioactive proteins newly synthesized in the cell body from either of the injected amino acid were transported axonally in a single axon of R2. 4. Elongation of the axon resulted in a suppression of the fast axonal transport of the proteins and the amount of protein transported. This contrasts with the observation that action potential propagation along the elongated axon never failed. 5. A possible site for the suppression of the axonal transport would be axonal microtubules whose structure is not likely to be resistant to the distortion caused by the elongation.

Fast axonally transported proteins in regenerating goldfish optic axons

Fast axonal transport of protein was examined in regenerating goldfish optic axons after a lesion of either the optic tract or optic nerve, which revealed changes in the original intact optic axon segments or in the newly regenerated axon segments, respectively. In animals killed either 6 or 24 hr after injection of 3H-proline into the eye, labeling of total fast-transported protein in the original axon segments was increased by 2 d after the lesion, reached a peak of nearly 20 X normal at 2 weeks, and then declined to a level somewhat above normal at 12 weeks. When the labeling of individual transported proteins was examined by 2-dimensional gel electrophoresis, it was found that no new labeled proteins appeared during regeneration, but all proteins examined showed an increase in labeling. Among the various proteins, there was great variation in the magnitude and time course of the labeling increase. The largest increase, to nearly 200 X normal with 6 hr labeling, was seen in a protein with a molecular weight of 45 kDa and a pl of about 4.5, resembling a protein that has previously been designated a "growth-associated protein" (GAP-43; Skene and Willard, 1981a). The proteins showing increased labeling included a small fraction of cytoskeletal proteins (alpha-tubulin, beta-tubulin, and actin) that was apparently transported at a much faster rate than is usually expected of these constituents. In the new axon segments, the total protein labeling was increased by 1 week after the lesion, remained elevated at a nearly constant level of about 7 X normal from about 2 to 5 weeks, and then declined to levels somewhat above normal by 12 weeks. The 45 kDa protein again showed the largest increase, and became the single most prominently labeled constituent in the new axons. On the basis of the time course of labeling in both original and new axon segments during regeneration, the fast-transported proteins were tentatively separated into 5 classes that may represent groups of proteins that are coregulated during regeneration. They may conceivably correspond to different functional or structural entities within the neuron.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.