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.     

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.     

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.     

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.     

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.     

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.     

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.     

Xefiltin,a Xenopus laevis neuronal intermediate filament protein,is expressed in actively growing optic axons during development and regeneration

Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as α-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, α-internexin and gefiltin. α-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 811–824, 1997     

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.     

Dynamic properties of axonal transport of proteins and glycoproteins: A study based on the effects of metaphase blocking drugs in the developing optic pathway of chick embryos

Some properties of the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos and newly hatched chicks were studied by labelling retinal ganglion cells with 3H-proline or 3H-fucose. A study of the effects of colchicine (COL) and vinblastine (VLB) on embryonic axonal transport was also carried out. Marked changes in the efficiency of axonal transport were found throughout development. In particular, the fraction of retinal ganglion cell proteins which is rapidly exported toward tectal terminals increases during embryonic life but steadily decreases after hatching. Glycoprotein transport behaves similarly except that its efficiency is relatively higher at stages when critical events of synaptic maturation in the tectum are reported to occur. Embryonic axonal transport is blocked by COL and VLB at very low intravitreal concentrations. Retinal protein synthesis and the morphology of ganglion cells are profoundly altered by the drugs: in general, COL and VLB effects were much more marked in embryonic than in mature neurons. An analysis of the time course of rapid transport along embryonic optic axons was carried out by reducing the efflux of labelled proteins from the eye by giving VLB intravitreally 2 h after the pulse. It revealed some peculiar features in the retino-tectal migration of glycoproteins and confirmed their progressive accumulation within terminals as previously described by radioautography. These results suggest that axonal transport of proteins during embryonic life undergoes changes in parallel with synaptic maturation. It may thus be considered as one of the factors controlling the genesis of neuronal networks.     

Compositional analysis of growing axons from rat sympathetic neurons

We describe culture systems for neurons of an adrenergic autonomic ganglion which: (a) permit cultivation of neurons without supporting cells, (b) permit separate harvest of somal and axonal material, and (c) permit direct access to the neuronal surface. The antimetabolites used to suppress supporting cell growth did not have demonstrable effects on neuronal polypeptide synthesis. Rapid neurite outgrowth, which characterized these cultures, was prevented by colchicine or cycloheximide and resumed promptly after their withdrawal. Axons separated from cell bodies showed no incorporation of label from leucine or fucose, but did exhibit incorporation of glucosamine. The major polypeptides present in this neuron, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, are described. No major differences in polypeptide content were observed when soma and axons were compared. Likewise, there were no differences detected in polypeptides synthesized by neurons in suspension or neurons actively extending processes. Analysis of the polypeptides within the neurites after labeling with amino acids indicated transport at a number of different rates; certain of these polypeptides corresponded in size and transport characteristics to polypeptides observed in the rabbit optic nerve after labeling of retinal ganglion cells. Tubulin and actin have been definitively identified in this cell type (18); we found proteins similar in size and proportionate amounts to be among the rapidly transported soluble polypeptides. The prominent polypeptides observed after several methods of surface labeling are described.     

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.     

Inhibition of Axoplasmic Transport in the Optic System by Kainic Acid

Axoplasmic transport along the optic axons was studied after intraocular injections of kainic acid (KA). Transport of labeled material did not initiate from the eye when KA was injected simultaneously with the protein precursor [3H]proline. When KA was injected after axoplasmic transport of labeled proteins had begun, no additional radioactive material moved out of the retinal ganglion cells. However, the labeled material already present in the optic nerve at the time of KA injection continued to move, and accumulated at the nerve endings. Although KA reduces the incorporation of precursor, this effect of KA on axoplasmic transport appears to be more than a consequence of inhibition on precursor uptake or protein synthesis. Recovery from this KA action began 6 h after exposure to KA and was about 50% recovered by 36 h. The extent of the recovery remained at this level for as long as a week, which suggested a partial recovery of the ganglion cells. A second exposure to KA after the inner plexiform layer had virtually disappeared was as effective as the first exposure in preventing the appearance of transported protein in the optic nerve, suggesting a direct action of KA on the ganglion cells. We interpreted the results to indicate that KA interferes with the initiation phase of axoplasmic transport in ganglion cells and this effect is partially reversible.     

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.     

Axonal Transport in the Garfish Optic Nerve: Comparison with the Olfactory System

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.     

Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one- dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton- insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein phosphorylation: Localization in regenerating optic axons

A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration (Larrivee and Grafstein, 1989). (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of [³H]proline and³²P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the [³H]proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the³²P label, suggesting that phosphorylation was largely independent of synthesis. (2) To deterine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced [³H]proline labeling of total protein by 88% and³²P labeling by 49%. Among the individual proteins [³H]proline labeling was reduced by 90% or more in 18 cases but³²P labeling was reduced only by 50% or less. (3) When³²P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.Abbreviations SDS sodium lauryl sulfate - GAP growth associated protein - TCA trichloracetic acid - kD kilodalton     

Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

Axonal transport of actin in rabbit retinal ganglion cells

We labeled proteins in the cell bodies of rabbit retinal ganglion cells with [35S]methionine and subsequently observed the appearance of radioactive actin in tissues containing the axons and synaptic terminals of these neurons, i.e., the optic nerve (ON), optic tract (OT), lateral geniculate nucleus (LGN) and the superior colliculus (SC). The temporal sequence of appearance of labeled actin (which was identified by its specific binding to DNase I, its electrophoretic mobility, and its peptide map) in these tissues indicated that actin is an axonally transported protein with a maximum transport velocity of 3.4--4.3 mm/d. The kinetics of labeling actin were similar to the kinetics of labeling two proteins (M1 and M2) which resemble myosin; these myosin-like proteins were previously found to be included in the groups of proteins (groups III and IV) transported with the third and fourth most rapid maximum velocities. The similarity in transport between actin and myosin-like proteins supports the idea that a number of proteins in the third and fourth transport groups may be functionally related by virtue of their involvement in a force-generating mechanism and suggests the possibility that these proteins may be axonally transported as a preformed force-generating unit.