Fast axonal transport of tyrosine sulfate-containing proteins: Preferential routing of sulfoproteins toward nerve terminals

The presence of a subset of fast-transported proteins containing sulfate while lacking carbohydrate residues [Stone et al. (1983). J. Neurochem. 41:1085-1089] was confirmed by two-dimensional gel electrophoretic analysis of individual fast-transported proteins double-labeled with 35SO4 and [3H]mannose. Analysis by high-pressure liquid chromatography revealed that the sulfate moieties of these "sulfoproteins" are linked to tyrosine residues. Separation of fast-transported 35SO4-labeled proteins delivered to local regions of axon from proteins en route toward terminal regions demonstrated, on the basis of acid lability of tyrosine-bound sulfate, that the sulfoproteins were localized preferentially in the wavefront of fast-transported proteins. Analysis of individual sulfoproteins confirmed differential transport in that sulfoproteins were present at threefold greater amount in the wavefront than in material off-loaded to local regions of the axon. By contrast, nonsulfated species of molecular weights similar to those of the sulfoproteins were detected in nearly equal amounts in both regions of the transport profile. Treatment of nerve segments containing total 35SO4-labeled fast-transported proteins with sodium carbonate led to solubilization of half the protein-bound sulfate. Exposure of the solubilized proteins to mild acid resulted in the release of approximately 80% of the 35SO4 associated with this fraction. Two-dimensional gel patterns displaying carbonate releasable or nonreleasable fractions are consistent with the most abundantly labeled sulfoproteins being transported within membrane-bound organelles. In terms of apparent destination and subcellular compartmentalization, the sulfoproteins meet critical requirements for consideration as secretable fast-transported proteins.     

Subsets of Axonally Transported and Periaxonal Polypeptides Are Released from Regenerating Nerve

Using two-dimensional polyacrylamide gel electrophoresis to analyze proteins, we have found subsets of periaxonal and fast-transported axoplasmic proteins that are released in vitro from regenerating sciatic nerve into a surrounding bath. Of the fast-transported proteins that are released from nerve, there is a subset of at least five polypeptides that appears in greater relative abundance in the bath than in the nerve. Some of these released, fast-transported proteins are glycosylated. Several periaxonally synthesized polypeptides are released in significantly greater amounts from regenerating nerve, and of these polypeptides, two are released in greater amounts from nerve only at regions of regeneration or distal to regeneration. These released polypeptides do not represent the most abundant of the locally synthesized proteins. The released, fast-transported and periaxonal proteins may play a role in intercellular signaling or in modulation of the extracellular environment during nerve regeneration.     

Similar polypeptide composition of fast-transported proteins in rat motor and sensory axons.

SDS-polyacrylamide gel electrophoresis was used to characterize labeled proteins transported in rat motor and sensory axons after application of 3H-leucine to the neuron cell bodies. Two types of experiments were performed: first, transported protein accumulating proximal to a ligature placed on the sciatic nerve was analyzed; second, the segment of sciatic nerve nearest to the "wavecrest" of transported protein travelling down the nerve was analyzed. In both cases, no significant differences in peak position or amplitude were found in gels containing labeled proteins from motor or sensory axons. This may mean that the majority of fast-transported protein is involved in an axonal function common to the two types of neuron.     

Functions of retrograde axonal transport

Retrograde axonal transport conveys materials from axon to cell body. One function of this process is recycling of materials originally transported from cell body to axon. In motoneurons, 50% of fast-transported protein is returned. Reversal probably occurs mainly at nerve terminals and, for labeled proteins, is nonselective. Proteolysis is not required, although changes in tertiary protein structure may occur with a repackaging of molecules in organelles different from those in which they were anterograde-transported. A second function is transfer of information about axonal status and terminal environment. Premature reversal of transport adjacent to an axon injury may be a component of a signal that initiates cell body chromatolysis. Transport of target cell-derived molecules with trophic effects on the cell body is exemplified by nerve growth factor transport in neurons dependent on it, and is probably a widespread phenomenon in the developing nervous system. Disorders in retrograde transport or reversal occur in some experimental neuropathies, and certain viruses, as well as tetanus toxin, may gain access to the central nervous system by this route.     

Similar polypeptide composition of fast-transported proteins in rat motor and sensory axons

SDS-polyacrylamide gel electrophoresis was used to characterize labeled proteins transported in rat motor and sensory axons after application of 3H-leucine to the neuron cell bodies. Two types of experiments were performed: first, transported protein accumulating proximal to a ligature placed on the sciatic nerve was analyzed; second, the segment of sciatic nerve nearest to the “lwavecrest” of transported protein travelling down the nerve was analyzed. In both cases, no significant differences in peak position or amplitude were found in gels containing labeled proteins from motor or sensory axons. This may mean that the majority of fast-transported protein is involved in an axonal function common to the two types of neuron.     

Proteins of axonal transport: investigation of solubility characteristics and behaviour in gel filtration

Rapid axonal transport of proteins in retinal ganglion cells of the rabbit was studied following intraocular injections of labelled amino acids. Approximately 10% of the transported radioactivity was found in the supernatant following homogenization and high-speed centrifugation of the nerve terminal region. Relatively simple manipulations with ionic strength, pH and the presence of a chelating agent could solubilize an equivalent amount of radioactivity from the pellet. Lithium diiodosalicylate solubilized most rapidly transported membrane proteins. Gel filtration of readily soluble rapidly transported radioactivity gave a main macromolecular radioactive peak with an approximate mol. wt. of 500,000 dalton as determined on Sephadex G-200. However, gel filtration on Sepharose CL-6B gave a mol. wt. of about 160,000 for the same radioactive peak. SDS polyacrylamide gel electrophoresis of rapidly transported soluble proteins and fractions derived from these proteins via gel filtration and ion exchange chromatography revealed in all cases a very complex picture of labelled polypeptides. Thus rapid axonal transport of soluble proteins in this system seems to involve many different macromolecules.     

Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats.

Proteins synthesized by soma located in L4 dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [3H] leucine into the L4 dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.     

Glycosylation as a Criterion for Defining Subpopulations of Fast-Transported Proteins

The role carbohydrate residues may play in the sorting of newly synthesized fast-transported proteins during the initiation of fast axonal transport has been examined by identifying individual fast-transported glycoproteins that contain either or both fucose and galactose. [3H]Fucose or [3H]galactose was incorporated together with [35S]methionine in vitro in bullfrog dorsal root ganglia. Fast-transported proteins that accumulated proximal to a ligature on the spinal nerve were separated via two-dimensional gel electrophoresis, and 92 gel spots were analyzed quantitatively for the presence of 35S and 3H. Of these spots, 56 (61%) contained either or both fucose and galactose. Glycomoieties were generally associated with families of charged spots whose isoelectric points could be altered with neuraminidase treatment. Single spots tended to be unglycosylated and were unaffected by neuraminidase. The prevalence of glycoproteins was considerably greater in the higher-molecular weight range. Of the 55 spots analyzed with molecular weight greater than approximately 35,000 daltons, 89% were glycosylated, whereas only 19% of the 37 spots with lower molecular weight contained sugar moieties. When considered in light of previous studies in which similar subpopulations have been described, the current findings suggest that the presence or absence of glycomoieties may represent another criterion by which proteins are sorted during the initiation of fast axonal transport.     

Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats

Proteins synthesized by soma located in L₄ dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [³H] leucine into the L₄ dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.     

INFLUENCE OF TEMPERATURE ON THE VELOCITY AND ON THE ISOTOPE PROFILE OF SLOWLY TRANSPORTED LABELED PROTEINS

Abstract— Slow intra-axonal flow of [³H]leucine labeled proteins has been studied in the garfish olfactory nerve. Because of the homogeneity of the nerve a very well defined peak of slowly transported radioactivity is observed. The velocity of slow flow increases linearly with temperature. Between 14 and 28°C, the rate of the peak apex increases from 0.26 to 1.57 mm/day and the rate of the leading edge of the wavefront from 0.54 to 2.75 mm/day. Extrapolation of the rate-temperature function indicates that slow flow should stop at 11°C. However, a velocity of 0.1 mm/day was determined for experiments conducted at 10°C. Between 15 and 25°C a Q ₁₀ of 3.7 was determined for the peak apex and of 3.3 for the leading edge of the wavefront. The Q₁₀'s are significantly larger than the value of 2.2 found for fast transport (G ross & B eidler , 1975) and support the possibility of at least partial differences between the mechanisms of fast and slow transport. A very small peak was found to migrate in front of the main peak. The positioning of this peak seems to be similar to one found by L asek & H offman (1976) in rat ventral motor neurons.
A temperature dependent exponential decrease of the slow moving peak height was measured and it can be estimated that only 1% of the slowly transported radioactivity reaches the synapses. Most of the slow radioactivity appears to remain in the axon behind the peak. The plateau height was also found to decrease exponentially with time. The rate of disappearance greatly affects the profile determined by the slowly transported labeled proteins along the nerve.     

Isolation and Characterization of Rapid Transport Vesicle Subtypes from Rabbit Optic Nerve

Subcellular fractionation of rabbit optic nerve resolves three populations of membranes that are rapidly labelled in the axon. The lightest membranes are greater than 200 nm and are relatively immobile. The intermediate density membranes consist of 84 nm vesicles which disappear from the nerve with kinetics identical to those of the rapid component. A third population of membranes, displaying a distinct protein profile, is present in the most dense region of the gradient. Immunological characterization of these membranes suggests the following. (1) The lightest peak contains rapidly transported glucose transporter and most of the total glucose transporters present in the nerve; this peak is therefore enriched in axolemma. (2) The intermediate peak contains rapidly transported glucose transporters and synaptophysin, an integral synaptic vesicle protein, and about half of the total synaptophysin; this peak therefore contains transport vesicles bound for both the axolemma and the nerve terminal, and these subpopulations can be separated by immunoadsorption with specific antibodies against the aforementioned proteins. (3) The heaviest peak contains rapidly transported synaptophysin and tachykinin neuromodulators and about half of the total synaptophysin, and 80% of the total tachykinins present in the nerve; this peak appears to represent a class of synaptic vesicle precursor bound for the nerve terminal exclusively. (4) Synaptophysin is present in the membranes of vesicles carrying tachykinins. (5) Both the intermediate and the heaviest peaks are enriched in kinesin heavy chain, suggesting that both vesicle classes may be transported by the same mechanism.     

Differential Axonal Transport of Soluble and Insoluble τ in the Rat Sciatic Nerve

Abstract: Axonal transport of microtubule-associated protein τ was studied in the motor fibers of the rat sciatic nerve 1–4 weeks after labeling of the spinal cord with [³⁵S]methionine. As 60–70% of low molecular weight τ in this system was found to be insoluble in 1% Triton-containing buffer, labeled proteins in 6-mm consecutive nerve segments were first separated into Triton-soluble and insoluble fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-tau antibody confirmed the presence of τ among labeled, transported proteins in both fractions. Isoform composition of labeled τ was similar to that of bulk axonal τ, the most acidic species with apparent molecular mass of 66 kDa being the major component. Transport profiles obtained by measuring radioactivities associated with this major isoform showed that soluble and insoluble τ were transported at different rates. Insoluble τ, which contained the majority of τ-associated radioactivity, was transported at 1.7 mm/day in slow component a (SCa), whereas soluble τ was transported faster, at 3 mm/day, corresponding to the rate of slow component b (SCb). Cotransport of insoluble τ with insoluble tubulin in SCa suggests its association with stable microtubules.     

Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post- translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.     

IS THERE BIDIRECTIONAL TRANSPORT OF NORADRENALINE IN SYMPATHETIC NERVES?

The experiments were designed to detect somatopetal transport of [¹⁴C]noradrenaline in the postganglionic sympathetic nerves supplying the cat spleen and sheep eye. The animals were treated with nialamide to protect the radioactive noradrenaline, after uptake into the nerve terminals, from monoamine oxidase. In the spleen, the transmitter stores were labelled by infusion of [¹⁴C]noradrenaline into a branch of the splenic artery. The branches of the nerves to the infused and non-infused sides of the spleen were ligated in an attempt to arrest, distal to the constriction, any noradrenaline transported somatopetally in the axons from their terminals. After 24 hr, however, there was less radioactivity in the nerves distal compared to proximal to the constriction, despite heavier labelling of the terminal transmitter stores in the infused portion of the spleen. The proximal accumulation of radioactivity could be attributed to a somatofugal transport of [¹⁴C]noradrenaline. Experiments were also done on the intact sympathetic nerve supply of the sheep eye. The sympathetic nerve terminals in the smooth muscle of the left eye were heavily labelled 5 days after the injection of [¹⁴C]noradrenaline into the left vitreous humour. However, both superior cervical ganglia were only lightly labelled, and there was no significant difference in the radioactivity present in the two ganglia. The results provide no support for a bidirectional transport of noradrenaline in sympathetic nerves but are consistent with a somatofugal transport of the amine storage vesicles from their site of synthesis in the soma to the axon terminals.     

Metalloendoprotease Inhibitors Block Fast Axonal Transport

Metalloendoprotease activity that was sensitive to the metal chelator 1,10-phenanthroline and to synthetic dipeptide substrates of the enzyme was detected in homogenates of dorsal root ganglia (DRG) and spinal nerve from the bullfrog. Exposure of an intact in vitro preparation of DRG and spinal nerves to 1,10-phenanthroline led to a dose-dependent depression in the accumulation of fast-transported 3H-labeled protein proximal to a nerve ligature. In nonligated preparations, the chelator treatment reduced the amount of transported protein entering the nerve; no marked effect on the transport rate was observed. Exposure of a desheathed region of spinal nerve to 1,10-phenanthroline, while DRG were maintained in control medium, resulted in a slight depression of fast transport. This effect was not dose dependent over the range that produced a dose response when both DRG and spinal nerve were exposed to the drug. Treatment of DRG and spinal nerve with the metalloendoprotease substrate analogues carbobenzoxy (CBZ)-Ser-Leu-amide or CBZ-Gly-Leu-amide inhibited fast axonal transport, whereas treatment with CBZ-Gly-Gly-amide, which is not a substrate, had no detectable effect on transport. Selective exposure of desheathed nerve trunk to CBZ-Ser-Leu-amide inhibited fast transport, but the effect was less marked than when DRG and nerve trunk were treated. Although previous studies have focused on the role of metalloendoprotease activity in exocytosis, the present data suggest that the enzyme may also be involved in earlier stages of intracellular transport.     

Regenerating Peripheral Axons Transport and Release Low-Molecular-Mass Materials In Vitro

Abstract: The release of radiolabeled material from regenerating frog sciatic nerves was studied using a multicom- partment chamber, in which the ganglia and the outgrowth region, respectively, were separated from the rest of the nerve. The nerves were incubated with radioactive amino acids in the ganglionic compartment, and the material transported to and released at the outgrowth region was collected and analyzed. Approximately 10% of the transported radioactivity was released over a 24-h incubation period. Of the released materials, 84% had a molecular mass of < 1,000 daltons [the low-molecular-mass (LM) fraction] as determined by exclusion chromatography. The presence of LM material could not be explained by leakage, nor was it due to intracellular or extracellular degradation of radiolabeled, transported proteins. It was reduced by cold and was shown by the use of vinblastine to be dependent on axonal transport. According to TLC, both the original precursor and metabolites thereof could be detected among the released LM material. The present results demonstrate the existence of a transport system for LM material in peripheral axons. The preferential release of LM over high-molecular-mass material at the outgrowth region suggests that it could serve specific functions during regeneration.     

Anatomical consideration of reverse-flow island flap transfers from the midpalm for finger reconstruction.

Primary soft-tissue coverage for large palmar defects of the fingers is a difficult problem for cases in which homodigital or heterodigital flaps cannot be used. The aim of this study was to explore the vascular and neural anatomy of the midpalmar area to assess the possibility of reverse island flaps from this area. In 24 cadaver hands perfused with a silicone compound, the arterial pattern of the superficial palmar arch and common palmar digital artery was examined. The cutaneous perforating arteries and nerve branches supplying the midpalmar area were dissected, and the number, location, and arterial diameter of these branches were measured. In six other specimens, the common palmar digital artery was injected to determine the skin territory supplied by the artery. The superficial palmar arch contained the three common palmar digital arteries and its terminal branch coursed along the radial margin of the index metacarpus. This terminal branch had three to six cutaneous perforators (diameter range, 0.1 to 0.5 mm) and supplied the radial aspect of the midpalmar area located over the ulnar half of the adductor pollicis muscles. The midpalmar area was divided into two regions-the proximal and distal-according to the vascular distributions. The proximal region contained dense aponeurosis and thin subcutaneous tissue, and the cutaneous perforators were rather sparse (between three and nine) and had a small diameter (0.1 to 0.3 mm). The distal region, which had loose aponeurosis and abundant subcutaneous tissue, had a rich vascular supply from the common and proper digital artery. Perforating arteries of this region coursed frequently in an oblique fashion and the number of perforators (between eight and 15) and their arterial diameters (diameter range, 0.1 to 0.5 mm) were higher than those of the proximal region. The area of skin perfused by the common palmar digital artery was 5 x 3 cm at the distal midpalmar region. There were three to five cutaneous nerve branches from the palmar digital nerve supplying the midpalmar area. From this study, two different reverse flaps were proposed. First, a 5 x 2 cm flap from the distal midpalmar region was elevated on the basis of the common and proper palmar digital artery. Measurement of the rotation arc revealed that the pivot point of this flap was located at the proximal interphalangeal joint level and could cover the finger pulp of the digits. The second flap candidate was that from the radial aspect of the midpalm, which was supplied by the terminal branch of the superficial palmar arch. In studies with cadaver hands, connection of this artery with the deep arterial system enabled this flap to reach the thumb pulp. These flaps may be a useful reconstruction option for significant palmar soft-tissue loss of the fingers.     

Differential effects of cobalt on the initiation of fast axonal transport

Effects of Co²⁺ on the fast axonal transport of individual proteins were examined in vitro in bullfrog spinal/sciatic nerves.³⁵S-methionine-labeled proteins, fast-transported in control and Co²⁺-treated preparations were separated via two-dimensional gel electrophoresis. While the overall amount of protein transported was reduced, no qualitative differences could be seen when gel fluorographic patterns were compared. Quantitative analyses of the 48 most abundantly transported species revealed two significantly different populations (p < 0.01) differentially sensitive to Co²⁺ and distinguishable to a large extent by molecular weight. Those proteins less sensitive to Co²⁺ ranged from ~20,000 to 35,000 daltons while those more sensitive to Co²⁺ were >~35,000 daltons. The finding that all proteins are affected by Co²⁺ supports the proposal that fast-transported proteins are subject to a common Co²⁺-sensitive, Ca²⁺-requiring step. The observed differential effects are consistent with more than one Ca²⁺-dependent step occurring during the initiation phase of fast transport.This research was supported by a Muscular Dystrophy Association postdoctoral fellowship to G.C.S., and by research grants from NSF (BNS 79-24125) and the National Multiple Sclerosis Society (RG 1296-A-1) to R.H.     

A Comparison of the Effects of Acrylamide and Experimental Diabetes on the Retrograde Axonal Transport of Proteins in the Rat Sciatic Nerve: Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis

Retrogradely transported proteins that accumulated for 18 h distal to a ligature on the sciatic nerve of rats were separated by two-dimensional polyacrylamide gel electrophoresis and visualised with silver stain. A total of 14 proteins were detectable in this way as being retrogradely transported. In rats rendered diabetic 14 days previously by a single intravenous dose of streptozotocin, the accumulation of four of those proteins was noticeably decreased. Administration of acrylamide to a cumulative dose of 150 mg.kg-1 or 350 mg.kg-1 decreased the accumulation of four and eight proteins, respectively. Three of the four protein changes were common to the diabetic and acrylamide-treated animals, and were present before signs of neuropathy could be detected. The results suggest that those alterations may play a causal role in the development of neuropathy.     

TRANSPORT AND TURNOVER OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Axonal transport and turnover rate of proteins in the supraoptico-neurohypo-physial tract were studied after injection of ³⁵S cysteine into the region of the supraoptic nucleus. The proximo-distal migration of labelled proteins from the nerve cell bodies to the axon terminals in the neurohypophysis was followed by measuring the radioactivity of neurohypophysial proteins at various time intervals (4 h to 30 days) after isotope injection. A rapidly transported phase of proteins with a minimal transport rate of approximately 60 mm/day was demonstrated. An accumulation of protein-bound radioactivity was also observed in the neural lobe at 9 days after isotope injection, representing slowly transported proteins (0-5 mm/day). In addition, an intermediate phase of axonal transport (1-5 mm/day) was found. Fractionation of neurohypophysial proteins by polyacrylamide gel disc electrophoresis revealed that a predominating portion of the radioactivity was recovered in a single protein component (fraction A) at 4 h as well as at 30 days after isotope injection. This protein component was shown to be a constituent both of the rapid and the slow phase of axonal transport. With time an increasing amount of radioactivity was found in another protein component (fraction B), which reached a maximum at 14 days after injection and then remained fairly constant up to 30 days. When the turnover rates of neurohypophysial proteins were estimated, a half-life of 1-2 days and 8 days was calculated for the rapidly and slowly transported proteins, respectively.