Fate of axonally transported taurine and proteins in the developing rabbit visual system following optic nerve section

These experiments were performed to characterize the axonally transported taurine in the visual system of developing rabbits. [³⁵S]Taurine, transported axonally after intravitreal injection, disappeared from the components of the visual system more rapidly after nerve section than it did with intact nerves. The decrease was most rapid in the youngest animals, and tended to be most pronounced in the elements nearest to the section (optic nerve, optic tract).³H-labeled proteins present in the visual system changed less markedly than [³⁵S]taurine after nerve section; only in the youngest rabbits was there a marked decrease. These results suggest that a greater proportion of the intraaxonal taurine is labile in young rabbits than in mature rabbits.     

Structural analysis of glycosaminoglycans derived from axonally transported proteoglycans in regenerating goldfish optic nerve

Structural characteristics of glycosaminoglycans (GAGs) derived from axonally transported proteoglycans (PGs) were compared in 21 day regenerating and intact goldfish optic tracts. Twenty one days following unilateral optic nerve crushes, fish received intraocular injections of³⁵SO₄. Eight hours post injection, tracts were removed and the³⁵SO₄-labeled GAGs, chondroitin sulfate (CS) and heparan sulfate (HS), isolated. The HS from regenerating optic tracts had a DEAE elution profile indicative of decreased charge density, while heparitinase treatment of HS followed by Sephadex G50 analysis of the resulting fragments showed a change in the elution pattern, suggesting reduced overall sulfation. HPLC analysis of HS disaccharides revealed a difference in the sulfation pattern of regenerating tract HS, characterized by the reduced presence of tri-sulfated disaccharides. Other structural features, such as the sizes of CS and HS, and the sulfation of CS, showed no changes during regeneration. These results indicate that changes in the structure of axonally transported HS accompany regeneration of goldfish optic axons.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Evidence for multiple somatic pools of individual axonally transported proteins

The idea that individual axonally transported proteins can exist in several kinetically distinct pools within the cell body was studied using the presumptive neurosecretory low molecular weight (LMW) proteins of Aplysia neurons L11 and R15. Pulse-chase experiments revealed that the loss of labeled LMW proteins from the soma by axonal transport does not follow single exponential kinetics as it should if they are being removed from single pools. Rather, decay of label occurs in at least two phases having half-lives of approximately 1 and 40 h. The LMW proteins are homogeneous by sequential SDS gel electrophoresis and isoelectric focusing, indicating that individual protein species exhibit multiphasic decay kinetics. Two types of evidence imply that the bulk of cellular LMW protein turns over at the slower rate: the LMW pool does not reach constant specific activity at the rapid rate during continuous exposure to labeled precursor, and long-term blockade of axonal transport does not produce an appreciable accumulation of these species in the cell body. These results suggest that some of the newly synthesized LMW protein enters a small somatic pool from which it is rapidly subjected to axonal transport, while the remainder enters a larger pool where it can mix with previously synthesized protein before transport. A cellular mechanism that would yield this behavior is suggested.     

Differential turnover of axonally transported glycoproteins

Metabolic turnover of axonally transported glycoproteins has been examined in membranous and soluble subfractions of goldfish optic tectum following intraocular injection of [³H]fucose. Radioactivity in total transported glycoproteins reached a maximum in the tectum after 24–30 hr, then declined with a half-life of approximately 20 days. Radioactivity in the total membranous subfraction declined with a similar half-life of 20–21 days while radioactivity in the soluble fraction showed a significantly shorter half-life of approximately seven days. Various sized glycopeptides derived from the membranous subfraction showed differential rates of loss of radioactivity with the lower molecular weight nondialyzable molecules displaying the most rapid turnover. In contrast, the glycopeptides derived from the soluble fraction showed relatively uniform rates of turnover. The results are discussed in the context of metabolic compartmentalization between membranous and soluble glycoproteins and among the carbohydrate chains of the membranous molecules.Supported by NIH grant NS 11456.     

Multiple-rate components of axonally transported proteins in the hypothalamo-neurohypophysial system of the rat

The transport of labeled proteins from the hypothalamus to the neurohypophysis following 35S-methionine injection into the rat supraoptic nucleus was studied using a unique approach adapted for the study of short-axon systems. Multiple-rate components to those found in other neuronal systems were demonstrated. Neurosecretory vesicle-containing proteins (e.g., neurophysins) were transported at fast rates (greater than 120 mm/day), whereas the cytoskeletal protein, actin, moved principally in the slow component of transport. Two-dimensional gel electrophoresis was used to analyze the diverse patterns of labeled proteins found in the various rate components of axonal transport in this system.     

Calpain-mediated degradation of rapidly and slowly axonally transported proteins in retinal ganglion cells

Labelled axonally transported proteins belonging to four different phases of transport in the retinal ganglion cells of the rabbit were used as substrates in order to study proteolytic degradation in axons and nerve terminals.Proteins of both rapidly and slowly transported phases of axonal transport were easily degraded in small intact pieces of the superior colliculus.Addition of the Ca-dependent neutral protease, calpain, to isolated soluble and membrane fractions from the superior colliculus resulted in an increased rate of degradation of axonally transported components. The effects of calpain was most marked toward components in phases II and V of axonal transport in this system (Karlsson and Sjöstrand, 1971; Willard and Hulebak, 1977). The latter phase contains slowly transported neurofilament and microtubular protein while the former one contains rapidly transported membrane proteins.     

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 extraction of axonally transported proteoglycans

Axonally transported proteoglycans were differentially solubilized by a sequence of extractions designed to infer their relationship to nerve terminal membranes. Groups of goldfish were injected unilaterally with³⁵SO₄ and contralateral optic tecta containing axonally transported molecules were removed 16 h later. Tecta were homogenized in isotonic buffer and centrifuged at 100,000g for 60 min to create a total supernatant fraction. Subsequent homogenizations followed by recentrifugation were with hypotonic buffer (lysis extract), 1 M NaCl, Triton X-100 or alternatively Triton-1 M NaCl. Populations of proteoglycans in each extract were isolated on DEAE ion exchange columns and evaluated for content of glycosaminoglycans (GAGs). Results show the distribution of transported proteoglycans to be 26.3% total soluble, 13.7% lysis extract, 13.8% NaCl extract, 12.2% Triton extract, and 46.2% Triton-NaCl extract. Proteoglycans from all fractions contained heparan sulfate as the predominant GAG, with lesser amounts of chondroitin (4 or 6) sulfate. The possible localizations of transported proteoglycans suggested by the extraction results are discussed.     

Further characterization of axonally transported proteoglycans

We report further analysis of axonally transported proteoglycans in soluble and membranous subfractions of goldfish optic tectum. Distribution of transported³⁵SO₄ radioactivity was 35.2% soluble, 63.4% Triton-NaCl extractable and 1.4% unextracted. Proteoglycans isolated on DEAE cellulose were treated with chondroitinase AC or nitrous acid and remaining heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) were sized on Sepharose CL-6B. Kav values and estimated molecular weights were: Soluble CSPG-0.36 (160 kDa), Triton-NaCl extracted CSPG-.031 (200 kDa), Soluble HSPG-0.37 (150 kDa), Triton-NaCl extracted HSPG-0.37 (150 kDa). For constituent CS and HS chains the Kav values and estimated molecular weights on CL-6B were: Soluble CS-0.55 (15 kDa), Triton-NaCl extracted CS-0.55 (15 kDa), Soluble HS-0.59 (13 kDa) and Triton-NaCl extracted HS-0.65 (9 kDa). CS was shown to be sulfated exclusively at carbon 4 for both soluble and Triton NaCl extracted fractions.     

The release of axonally transported material from an in vitro amphibian sciatic nerve preparation

The rapid axonal transport of a pulse of [35S]methionine-labelled material was used to study the release of transported material from amphibian nerve maintained in vitro. Following creation of a moving pulse of activity in a dorsal root ganglion-sciatic nerve preparation, the ganglion was removed and the nerve placed in a three-compartment tray, the section of nerve in the middle compartment containing no truncated branches (unbranched section). All three compartments were filled with a saline solution that in some studies contained nonradioactive methionine (1.0 mmol/L). Analysis of studies in which nonradioactive methionine was absent revealed that labelled material appeared in the bathing solution of the end compartments that contained truncated branches, but not in the solution of the middle (unbranched) compartment. The quantity of label released in the branched compartments was approximately 6% of that remaining in the corresponding section of nerve following an 18-20 h incubation period. However, when nonradioactive methionine was present, all compartments showed an additional activity in the bathing solution of approximately 10% of that remaining in the nerve. In another study in which a position-sensitive detector of ionizing radiation was used to monitor progress of the pulse, it was found that activity did not enter the bathing solution of a compartment prior to the pulse of activity. It is concluded that in the absence of methionine from the bathing solution, axonally transported material is released only from regions of nerve that contain severed axons; however, the presence of methionine allows transported material to be released from nerve containing intact axons. Ultrafiltration studies and thin-layer chromatography revealed the majority of material released to be of low-molecular weight (less than 30,000 daltons) and not free [35S]methionine.     

Transfer of axonally transported phospholipids into myelin isolated from the rabbit optic pathway

The contribution of the axonal transport to the biosynthesis of myelin phospholipids was investigated in the rabbit optic pathway. A double labeling technique was used. The same animals were injected with one isotope intravitreally and the other intraventricularly. This procedure allows double labeling of the optic nerves, optic tracts, lateral geniculate bodies (LGB), and superior colliculus (SC). The precursors simultaneously injected were: [1-¹⁴C]palmitate (15 Ci intravitreally in both eyes or 50 Ci intraventricularly) and [2-³H]glycerol (50 Ci intravitreally in both eyes or 100 Ci intraventricularly). Twenty four hours and 10 days after the injections, myelin was purified from pooled optic nerves and optic tracts as well as from pooled LGBs or SCs. The phospholipids were extracted and then separated by thin-layer chromatography; the specific radioactivity of the various classes of phospholipids was determined. Using both administration routes of¹⁴C-or³H-precursors, the distribution of label and specific radioactivity of myelin phospholipids in the retina and in all other optic structures were very similar. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphoinositol were preferentially labeled with both precursors. These results suggest that, in the rabbit optic pathway the phospholipids synthesized in the retinal ganglion cells and transported along the axons, could undergo transaxonal transfer into myelin.     

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.     

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.     

Postnatal changes in [3H]fucosyl glycoconjugates axonally transported into hamster optic nerve endings

The subsynaptosomal distribution of [³H]fucosyl glycoproteins axonally transported into the optic nerve endings of neonatal and adult hamsters changed dramatically at eye-opening. In 12 day-old previsual hamsters, the highest concentration of incorporated fucose was in the axoplasmic reticulum/synaptic vesicle fraction (51%), with only 6% in the dense synaptic membrane fraction. By the end of the eye-opening period four days later proportional labeling of the dense synaptic membrane fraction had increased four-fold to 23% of total sub-synaptosomal radioactivity. Labeling of the synaptic membrane doubled again in adults (41%). Total synaptosomal radioactivity was greatest in 16 day-olds. These results imply that utilization of [³H]fucose by the retinal ganglion cells, as well as composition of the synaptic membrane, change in association with the onset of functional visual activity.     

Subcellular distribution of axonally transported adenylate cyclase: effect of nerve constriction and comparison with acetylcholinesterase

Abstract: Despite several studies indicating that cyclic nucleotides and their associated enzymes are present in peripheral nerves, their role in neuronal function remains unknown. One possible role is that of a modulating influence in the processes associated with axonal growth and maintenance, and in axonal regeneration. This study has used the frog sciatic nerve as a preparation for investigating the subcellular distribution of neuronai adenylate cyclase activity in normal and crush-injured nerves. The experiments have focused primarily on the axonal transport of adenylate cyclase activity and its subcellular redistribution at the site of constriction. The adenylate cyclase activity measurements were also compared with similar measurements of acetylcholinesterase distribution. Adenylate cyclase activity in normal sciatic nerves increased in the segment proximal to a nerve constriction over time, but did not increase distal to the constriction. Subcellular fractionation of the accumulating activity indicated that the majority of axonally transported enzyme was associated with microsomal organelles; however, an additional transported component was found in the nuclear/mitochondrial fraction. The transport velocities of these two components were different. The microsomal activity appeared to be transported with Group I proteins, while the nuclear/mitochondrial activity was transported with Group II. Rapidly transported Group I proteins have been suggested to be destined principally for the axolemma or the agranular reticu-lum, and the more slowly transported Group II proteins to be associated with intracellular organelles, including synaptic structures. Thus, axonally transported adenylate cyclase activity may have more than one functional role in peripheral nerve. The association of both adenylate cyclase and Protein I, an endogenous substrate for cyclic AMP, with Group II transport offers the intriguing possibility of a structural correspondence. Adenylate cyclase activity in Group I, however, did not appear to be transported with organelles which also contained acetylcholinesterase. The two enzymes, in terms of both velocity of transport and susceptibility to retrograde transport, were handled differently by the neuron. The subcellular distribution of adenylate cyclase activity in an isolated nerve segment was also found to change over time. Microsomal activity decreased, while nuclear/mitochondrial activity transiently increased and then also decreased. This may offer some indication of the morphological location of adenylate cyclase and its potential involvement in Wallerian degeneration and nerve regeneration, particularly in view of recent reports concerning the importance of local injury-induced changes to the initiation of nerve regeneration. We have proposed a dynamic association between axonal calcium and cyclic AMP concentration, which provides a method for membrane renewal or degradation in the intact axon and may offer a molecular basis for the structural reorganization occurring in the proximal segment of an injured nerve.