Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells

In an effort to understand the regulation of the transition of a mature neuron to the growth, or regenerating, state we have analyzed the composition of the axonally transported proteins in the retinal ganglion cells of the toad Bufo marinus after inducing axon regeneration by crushing the optic nerve. At increasing intervals after axotomy, we labeled the retinal ganglion cells with [35S]methionine and subsequently analyzed the labeled transported polypeptides in the crushed optic nerve by means of one- and two-dimensional electrophoretic techniques. The most significant conclusion from these experiments is that, while the transition from the mature to the regenerating state does not require a gross qualitative alteration in the composition of axonally transported proteins, the relative labeling of a small subset of rapidly transported proteins is altered dramatically (changes of more than 20-fold) and reproducibly (more than 30 animals) by axotomy. One of these growth-associated proteins (GAPs) was soluble in an aqueous buffer, while three were associated with a crude membrane fraction. The labeling of all three of the membrane-associated GAPs increased during the first 8 d after axotomy, and they continued to be labeled for at least 4 wk. The modulation of these proteins after axotomy is consistent with the possibility that they are involve in growth-specific functions and that the altered expression of a small number of genes is a crucial regulatory event in the transition of a mature neuron to a growth state. In addition to these selective changes in rapidly transported proteins, we observed the following more general metabolic correlates of the regeneration process: The total radioactive label associated with the most rapidly transported proteins (groups I and II) increased three to fourfold during the first 8 d after the nerve was crushed, while the total label associated with more slowly moving proteins (group IV) increased about 10-fold during this same period. Among these more slowly transported polypeptides, five were observed whose labeling increased much more than the average. Three of these five polypeptides resemble actin and alpha- and beta-tubulin in their electrophoretic properties.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Protein synthesis and transport in the regenerating goldfish visual system

The nature of the proteins synthesized in the goldfish retina and axonally transported to the tectum during optic nerve regeneration has been examined. Electrophoretic analysis of labeled soluble retinal proteins by fluorography verified our previous observation of a greatly enhanced synthesis of the microtubule subunits. In addition, labeling of a tubulin-like protein in the retinal particulate fraction was also increased during regeneration. Like soluble tubulin, the particulate material had an apparent MW of 53–55K and could be tyrosylated in the presence of cycloheximide and [³H]tyrosine. Comparison of post-crush and normal retinal proteins by two-dimensional gel electrophoresis also revealed a marked enhancement in the labeling of two acidic 68–70K proteins. Analysis of proteins slowly transported to the optic tectum revealed changes following nerve crush similar to those observed in the retina, with enhanced labeling of both soluble and particulate tubulin and of 68–70K polypeptides. The most striking change in the profile of rapidly transported protein was the appearance of a labeled 45K protein which was barely detectable in control fish.     

Fodrin: axonally transported polypeptides associated with the internal periphery of many cells

Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d--slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins. In this communication, we report the intracellular distribution of fodrin. Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence. Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e.g., skeletal muscle, uterus, intestinal epithelium). Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining). In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro. We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities: (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm; and (c) that fodrin could serve to link other proteins and organelles to a submembrane force-generating system.     

Slow components of axonal transport: two cytoskeletal networks

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.     

Electrophoretic Analysis of Axonally Transported Proteins in Toad Retinal Ganglion Cells

As a preliminary step to studying changes in axonal transport in regenerating neurons, we have analyzed the composition and organization of polypeptides normally axonally transported in a neuronal system capable of regeneration, i.e., the retinal ganglion cells of the toad, Bufo marinus. We labeled proteins synthesized in the retina with 35S-methionine and subsequently used one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to analyze labeled, transported proteins in tissues containing segments of the axons (the optic nerve, optic tract, and optic tecta) of the retinal ganglion cells. The transported polypeptides could be divided into five groups according to their apparent transport velocities. Many of the polypeptides of each group were electrophoretically similar to polypeptides of corresponding groups previously described in rabbit and guinea pig retinal ganglion cells, and in some cases, additional properties of the polypeptides indicated that the transported materials of the two vertebrate classes were homologous. These results serve two purposes. First they establish the retinal ganglion cells of the toad Bufo marinus as a model system in which changes in gene expression related to regeneration may be studied. Second they show that the organization and many aspects of the composition of axonal transport in retinal ganglion cells have been conserved in animals as unrelated as amphibians, and mammals.     

Axonal transport of lipid in goldfish optic axons

After injection of labeled glycerol, choline, or serine into the eye of goldfish, labeled lipids were axonally transported along the optic nerve to the optic tectum. although the different precursors were presumably incorporated into somewhat different lipid populations, all three were approximately equally effective in labeling the lipids transported to the tectum, but the amount of transported material remaining in the nerve was different, being highest with choline and lowest with serine. The labeled lipids appeared in the tectum within 6 hr of the injection, indicating a fast rate of transport, but continued to accumulate over a period of 1–2 weeks, which presumably reflects the time course of their release from the cell body. Since there was a gradual increase in the proportion of labeled lipid in the tectum during this period, some other process in addition to fast axonal transport may have affected the distribution of the lipids along the optic axons. When [³H]choline was used as precursor, the transported material included a small amount of TCA-soluble material, which was probably mainly phosphorylcholine, with labeled acetylcholine appearing in only insignificant amounts. With serine, which gave rise to a large amount of axonally transported protein in addition to lipid, a late increase in the amount of labeled lipid in the tectum was seen, accompanied by a decrease in labeling of the protein fraction.     

AXONAL TRANSPORT AND TURNOVER OF PROLINE- AND LEUCINE-LABELED PROTEIN IN THE GOLDFISH VISUAL SYSTEM

Abstract— The suitability of radioactively labeled proline as a marker of axonally transported protein in the goldfish visual system is further investigated and compared with another amino acid, leucine, in double-label experiments. Intraocularly injected proline is incorporated into protein in the eye S times more efficiently than is leucine, while local labeling of brain protein from precursor which has left the eye and entered the blood, (observed in the ipsilateral optic tectum) is five- to eight-fold less from proline than from leucine. The difference is attributed to the superior transport of leucine, an essential amino acid, into the brain from the blood. Once in the brain, the apparent rates of incorporation of the two amino acids are similar. Proline- or leucine-labeled, axonally transported proteins have a longer apparent half-life in the brain than do proteins labeled from intracranial injection of the precursors. By either route, proline-labeled proteins have a longer apparent half-life than leucine-labeled proteins. It is proposed that proline, released from protein breakdown is reutilized to a greater extent than is leucine.     

Taurine in the developing rabbit visual system: changes in concentration and axonal transport including a comparison with axonally transported proteins.

[35S]Taurine injected intravitreally into rabbits was transported axonally to the optic nerve terminals. Considerably more [35S]taurine was transported in young rabbits than in mature rabbits. The time course of taurine transport did not parallel that of proteins labeled with [3H]proline in the same system. The concentration of taurine in all components of the visual system, except retina, was greater in young animals than in mature animals, and was especially high in optic nerve. The possible functions of the high concentrations of taurine and the greater amount of axonally transported taurine in developing mammalian CNS are discussed.     

Investigation of the Axonal Transport of Three Acidic, Soluble Proteins (14-3-2, 14-3-3, and S-100) in the Rabbit Visual System

Abstract: The question of whether three acidic, water-soluble proteins (14-3-2, 14-3-3, and S-100, the first and last known to be brain-specific) are axonally transported was investigated in the rabbit visual system. The water-soluble proteins were obtained from individual optic nerves, combined optic tracts and lateral geniculate bodies, superior colliculi, and, in some instances, retinas at various times (1–56 days) after monocular injections of [³H]leucine. These proteins were separated by a two-step polyacrylamide gel electrophore-sis procedure that isolated 14-3-2, 14-3-3, and S-100 almost uncontaminated by other radioactivity. The isolated 14-3-2 and S-100 were demonstrated to be approx. 90% pure by a new method based on retarding the migration of these proteins by immunoadsorption during the first step of electrophoresis. An analysis of the radioactive labeling of the total soluble proteins (TSP) and the isolated acidic proteins revealed that: (1) S-100 was not axonally transported; (2) both 14-3-2 and 14-3-3 were part of one of the slow components of axonal transport (2-4 mm/day); (3) the radioactivity of 14-3-2 and 14-3-3 represented about 2.7% and 3.2%, respectively, of the radioactivity incorporated into the axonally transported TSP; (4) the ultimate distributions of the radioactively labeled 14-3-2 and 14-3-3 were the same (about 70% of each destined for the superior colliculus) and differed from that of the TSP; and (5) the rates of catabolism of the axonally transported 14-3-2 and 14-3-3 were slightly greater than that of the TSP, with half-lives for 14-3-2 and 14-3-3 estimated to be 11 and 10 days, respectively.     

Taurine in the developing rabbit visual system: Changes in concentration and axonal transport including a comparison with axonally transported proteins

[³⁵S]Taurine injected intravitreally into rabbits was transported axonally to the optic nerve terminals. Considerably more [³⁵S]taurine was transported in young rabbits than in mature rabbits. The time course of taurine transport did not parallel that of proteins labeled with [³H]proline in the same system. The concentration of taurine in all components of the visual system, except retina, was greater in young animals than in mature animals, and was especially high in optic nerve. The possible functions of the high concentrations of taurine and the greater amount of axonally transported taurine in developing mammalian CNS are discussed.     

Lack of physiological stimulation induces decreased proteolytic activity in nerve terminals

The effect of optic nerve transsection on proteolytic degradation of axonally transported proteins in the superior colliculus of the rabbit was studied. Proteolysis of labeled proteins was determined in vitro in small pieces of the superior colliculus. Within 2 hours after sectioning the optic nerve there was a decreased degradation of slowly transported labeled proteins in the nerve terminals in the superior colliculus.Special Issue dedicated to Prof. Holger Hydén.     

The identification of two intra-axonally transported polypeptides resembling myosin in some respects in the rabbit visual system

Two polypeptides (M1 and M2) which co-sediment with F-actin in an ATP- reversible way have been detected in extracts of tissue from the rabbit visual system. Both polypeptides resemble skeletal muscle myosin in their ATP-sensitive co-sedimentation with actin, while they resemble the heavy chain of myosin and the lighter polypeptide of erythrocyte spectrin in their electrophoretic mobilities. (The estimated molecular weights are: MI congruent to 195,000; myosin congruent 200,000; M2 and spectrin congruent to 220,000). M1 and M2 were labeled in the cell bodies of the retinal ganglion cells with a radioactive amino acid and subsequently recovered in tissues (optic nerve, optic tract, lateral geniculate nucleus, and superior colliculus) containing segments of the retinal ganglion cell axons. The temporal sequence of labeling M1 and M2 in these tissues indicated that both polypeptides were synthesized in the cell bodies of retinal ganglion cells and subsequently transported down their axons at different maximum velocities. The estimated velocities were: M1, 4-8 mm per day; and M2, 2-4 mm per day.     

Acrylamide impairs fast and slow axonal transport in rat optic system

Effects of single and repeated doses of acrylamide on fast and slow axonal transport of radio labeled proteins following the injection of L-[4,5-³H] leucine have been studied in the optic system of male Sprague-Dawley rats. A single dose of acrylamide (100 mg/kg) had no effect, but higher concentrations (200–300 mg/kg) altered the distribution of fast axonally transported materials in optic nerves and optic tracts. Repeated doses of acrylamide (30 mg/kg/day, 5 days per week for 4 weeks) produced degeneration of tibial nerves but spared optic nerves and optic tracts. Fast axonal transport rate in optic axons was reduced by 50% (reduced to 4 mm/h from 8 mm/h) in acrylamide treated animals. Acrylamide also slowed the velocity of slow axonal transport of labeled proteins in optic axons to 1.0 mm per day from 1.3 mm per day. Since acrylamide impaired the rate of both fast and slow axonal transport in the absence of overt morphological damage, it can be concluded that deficit in axonal transport is an important factor in the pathogenesis of axonal degeneration in acrylamide neuropathy.     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

Cholesterol Synthesis and Nerve Regeneration

Abstract: In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [³H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25-diazacholes-terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia-zacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled des-mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [³H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro , diazacholesterol did not inhibit optic nerve regeneration in vivo , as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.     

TRANSPORT OF CHOLESTEROL IN THE CHICK OPTIC SYSTEM

After injection of radioactive cholesterol into the vitreous humour of chicks, cholesterol is axonally transported along the optic nerve to the contralateral optic tectum. In the optic nerve of young chicks cholesterol is only transported at the slow rate whereas in the sciatic nerve of adult chicks it is transported at both the fast and slow rates. This difference is not due to age or a peculiarity of avian species as the same difference exists in mice. Estimates of the cholesterol content of tissues and the amount of cholesterol transported suggests the existence of small rapidly turning over pools of cholesterol in nerves. Due to its great metabolic stability, cholesterol is a useful and specific marker for slow transport in the optic nerve and thus may prove suitable for the mapping of neuronal projections.     

Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo

The phosphorylation and dephosphorylation of specific proteins was demonstrated directly in the intact vertebrate nervous system in vivo. By exploiting the neurons' ability to segregate a select group of cytoskeletal proteins from most other phosphorylated constituents of the cell by axoplasmic transport, we were able to examine the dynamics of phosphate turnover on neurofilament proteins in mouse retinal ganglion cell neurons simultaneously labeled with [32P]orthophosphate and [3H]proline in vivo. Three [3H]proline-labeled neurofilament protein (NFP) subunits, designated H (160-200 kDa), M (135-145 kDa), and L (68-70 kDa), entered optic axons in a mole:mole ratio similar to that of isolated axonal neurofilaments, supporting the notion that newly synthesized NFPs are transported along axons as assembled neurofilaments. NFP subunits incorporated high levels of 32P before reaching axonal sites at the level of the optic nerve. As neurofilaments were transported along axons, however, many initially incorporated [32P]phosphate groups were removed. Loss of these phosphate groups occurred to a different extent on each subunit. A minimum of 50-60 and 35-40% of the labeled phosphate groups was removed in a 5-day period from the L and M subunits, respectively. By contrast, the H subunit exhibited relatively little or no phosphate turnover during the same period. Dephosphorylation of L in axons is accompanied by a decrease in its net state of phosphorylation; changes in the phosphorylation state of H and M, however, also reflect ongoing addition of phosphates to these polypeptides during axonal transport (Nixon, R.A., Lewis, S.E., and Marotta, C.A. (1986) J. Neurosci., in press). The possibility is raised that dynamic rearrangements of phosphate topography on NFPs represent a mechanism to coordinate interactions of neurofilaments with other proteins as these elements are transported and incorporated into the stationary cytoskeleton along retinal ganglion cell axons.     

Cotransport of Glyceraldehyde-3-Phosphate Dehydrogenase and Actin in Axons of Chicken Motoneurons

1.To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [³⁵S]methionine and, 1–20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I–Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography.2.In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).3.The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods.4.Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3–5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity.5.Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.     

Effect of light on the labeling of optic tectum gangliosides after an intraocular injection of N-[3H]acetylmannosamine.

Axonally transported gangliosides from retina were more labeled in the optic tectum of chickens exposed to light compared to those maintained in the dark. No differences were observed between the labeling of retinal gangliosides from the two groups. These results indicate that light modifies either the labeling of ganglion cell gangliosides or their axonal transport.