Nicholas A. Ingoglia

Transfer RNA may be axonally transported during regeneration of goldfish optic nerves.

If [3H]uridine is injected into the eyes of goldfish during optic nerve regeneration, then the return of fibers to the optic tectum is accompanied by the appearance of [3H]RNA in the tectum. The amount of [3H]RNA arriving in the tectum is consistently greater than in non-regenerating controls and reaches maximum levels (more than 10 times controls) 24 days after optic nerve crush. When [14C]uridine is injected subarachnoidally 1 day prior to sacrificing, the amount of [14C]RNA in the tectum is approximately doubled throughout the regeneration period. In order to characterize the radioactive tectal RNA in these experiments, we have crushed the optic nerves of 15 fish, and 18 days later injected [3H]uridine into both eyes. Five days later [14C]uridine was injected subarachnoidally and all fish were sacrificed a day later. RNA was extracted and fractionated in 2.0% polyacrylamide gels. The amounts of 3H- and 14C-labeled ribosomal as well as small molecular weight RNAs were increased during regeneration. Analysis of the area under the 28S, 18S and 4-7S RNA peaks indicated a small increase in 14C radioactivity in each peak (1.2, 1.5, and 1.5 times control, respectively). On the other hand, 3H radioactivity showed the greatest increase in the 4-7S fraction (8.0 times control) whereas large molecular weight ribosomal fractions were approximately 3 times control. Electrophoresis of the RNA on 10% polyacrylamide gels demonstrated that all of the small molecular weight RNA was confined to the 4S (tRNA) peak. These results suggest that when optic nerves of goldfish regenerate, they may enter the tectum carrying 4S (transfer) RNA.

Axonal transport of putrescine, spermidine and spermine in normal and regenerating goldfish optic nerves

Radopactove putrescine, spermidine or spermine was injected into the right eye of normal goldfish and fish in which both optic nerves had been crushed 18 days earlier. Fish were sacrificed 0.25-21 days after injection. Trichloroacetic acid-soluble and -insoluble material was extracted from the right retina and both tecta and assayed for radioactivity (significant differences between left and right tecta suggesting axonal transport). The nature of the radioactivity in the TCA-soluble fraction was determined on an amino acid analyzer. Results indicate that putrescine is not axonally transported in intact goldfish optic nerves, but that during regeneration of the optic nerve large amounts of putrescine are axonally transported at rates similar to the fast component of protein transport. Spermidine appears to be axonally transported both in intact optic nerves and in regenerating optic nerves, and at an intermediate rate of transport; the amount of spermidine transported is significantly increased during regeneration. Spermine is also axonally transported in intact and regenerating nerves, at a rate similar to the rapid rate of protein transport. The amount of spermine transported appears to be slightly less in regenerating than in intact nerves during early stages of regeneration, but increases during later stages of nerve regeneration. The results suggest that putrescine and spermidine may be preferentially transported during nerve regeneration, while spermine and spermidine are transported extensively in intact nerves.

Interconversion of putrescine, spermidine and spermine in goldfish and rat retina.

Abstract Labelled putrescine is converted to spermidine and spermine in the retina of both the goldfish and of the rat, but the bulk remains as putrescine and spermidine in the goldfish retina whereas the bulk is present as spermine in the rat retina. Labelled spermidine is converted to spermine and to putrescine in the retina of both species, most remaining as spermidine in the goldfish retina whereas most is converted to spermine in the rat retina. Labelled spermine is converted to both spermidine and putrescine in the retina of both species with a greater conversion in the goldfish retina than in the rat retina. These results provide direct evidence of the interconversion of putrescine, spermidine and spermine in neural tissue from both fish and mammals and suggest that spermine should not be regarded solely as an end-product of putrescine metabolism but also as a source of spermidine and putrescine. The pattern of distribution of putrescine and the polyamines, spermidine and spermine, in goldfish retina is the reverse of that in rat retina: Putrescine is the most abundant in goldfish retina whereas spermine is most abundant in rat retina suggesting that the individual polyamines are of different importance in the two species.

Posttranslational Protein Modification by Amino Acid Addition in Intact and Regenerating Axons of the Rat Sciatic Nerve

Abstract: Experiments were performed to determine whether ppsttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 ·g, and analyzed for the ability to incorporate 3H‐amino acids into proteins. No incorporation of amino acids into proteins was found in the high‐speed supernatant, but when the supernatant was passed through a Sephacryl S‐200 chromatography column (removing molecules less than 20 kD), [3H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15–150 times that in intact nerves. The results indicate that the components of this reaction are transported axonally in rat sciatic nerves and that the reaction is increased dramatically in growing axons during nerve regeneration.

Axonal Transport of Polyamines in Intact and Regenerating Axons of the Rat Sciatic Nerve

The axonal transport of putrescine or its polyamine derivatives spermidine or spermine is a subject of some debate. We investigated this question by injecting [3H]putrescine into the lumbar spinal cord of the rat and measuring the accumulation of radioactivity central to ligatures placed on intact and regenerating sciatic nerves. In normal nerves, approximately twice as much radio activity built up proximal to these ligatures 2 or 3 days after injection than at more distal ligatures used to control for accumulation of radioactivity which might be due to tissue damage alone. In regenerating nerves the amount of radioactivity accumulating at the ligature was approx imately five times that at the distal ligature and two to three times greater than in intact nerves. The identity of the radioactivity in regenerating nerves, determined on an amino acid analyzer, was found to be primarily spermidine and an unknown compound that migrated as a frontal elution peak. Autoradiographic analysis showed that the radioactivity was largely confined to axons, but a significant amount of the silver grains was associated with Schwann cells and myelin sheaths surrounding labeled axons in both intact and regenerating nerves. The data indicate that polyamine derivatives of putrescine are transported axonally in rat sciatic nerves, and some of this transported material accumulates in Schwann cells surrounding the labeled axons. These processes are apparently augmented during regeneration of the injured axons.

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [3H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light‐microscope autoradiographs, a class of large‐caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build‐up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.

Comparison of posttranslational protein modification by amino acid addition after crush injury to sciatic and optic nerves of rats

Posttranslational protein modifications by the addition of amino acids are reactions which occur in intact sciatic and optic nerves of rats. The nerves differ, however, in that 2 h after crush injury these reactions are activated in sciatic but not in optic nerves. As sciatic nerves will eventually regenerate, whereas optic nerves will not, we have proposed that the activation of these reactions is correlated with the ability of a nerve to regenerate. The current experiments examined the posttranslational addition of amino acids to proteins at times greater than 2 h after nerve crush, during sciatic nerve regeneration and optic nerve degeneration. We also examined the optic nerve for morphologic correlates to changes in protein modification and partially characterized the proteins modified by [3H]Lys in the regenerating sciatic nerve using two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In a segment of sciatic nerve taken from a region just proximal to the site of crush, protein modification by covalent addition of [3H]Arg, [3H]Lys and [3H]Leu increased during both posttraumatic (2 h postcrush) and regenerative (6 days and 14 days postcrush) stages. Two-dimensional PAGE of [3H]Lys modified sciatic nerve proteins 6 days after crush injury showed labeling of proteins having molecular masses in the 18,000- to 20,000-, 30,000- to 40,000-, and 80,000- to 100,000-Da ranges, with neutral or basic isoelectric points (pI 7.1 to 8.0). In the retinal portion of the crushed optic nerve, incorporation of the same amino acids was unchanged or depressed to 21 days postcrush, except at 6 days postcrush when the incorporation of all three amino acids into proteins was increased threefold. These increases correlated with the appearance of terminal end bulbs in the portion of nerve analyzed. Histological examination of each nerve 2 h postcrush showed marked edema in the optic but not the sciatic nerve, a condition which may be related to the ability of sciatic and inability of optic nerves to activate protein modification reactions.

Axonal migration of taurine in the goldfish visual system

Taurine is an ubiquitous amino sulfonic acid present in high concentrations in neural and muscle tissue. Except for its role in the conjugation of bile acids, its biological significance is uncertain 5,7. However, there is considerable recent evidence which suggests that it may function as a neurotransmitter or neuromodulator in nervous tissue and in retina 1, and that it might be involved in brain development 1~-16. The present experiments were performed in order to test the possibility that taurine might be axonally transported in the optic nerve of the goldfish, a system which has been used extensively to study the axonal transport of macromolecules 4,6,1°. We also have used this system to investigate the axonal migration of cysteine, a precursor of taurine, and of?-aminobutyric acid (GABA), an amino acid analog of taurine and also a putative neurotransmitter. [85S]Taurine (64 mCi/mmole, obtained from Amersham/Searle Corp.), [~5S] cysteine (22 mCi/mmole, obtained from Amersham/Searle Corp.), or [14C]GABA (49 mCi/mmole, obtained from New England Nuclear Corp.) were dissolved in 0.9 saline. Four microliters of each solution (containing 0.5-1.0/~Ci) were injected in separate experiments into the vitreous of the right eye of goldfish (4-5 in. in length, obtained from Ozark Fisheries, Stoutland, Mo.). Fish were killed at various times after injection and their right retinas and both optic tecta were removed and homogenized in deionized H20. Trichloroacetic acid (TCA), 30 ~o w/v, was added to the homogenate to give a final concentration of 1 0 ~ TCA. Determination of TCAinsoluble and TCA-soluble radioactivity was performed as described previously 6. Since visual fibers of the goldfish cross completely at the optic chiasm, radioactivity in the tectum ipsilateral to the injected eye (right tectum in this case) represents material which has arrived by general circulation, whereas radioactivity in the left tectum represents material which has arrived by axonal transport or axonal diffusion

Oxidative stress increases ubiquitin--protein conjugates in synaptosomes.

Synaptosomes were incubated in the presence of FeSO4 to test the hypothesis that iron-catalyzed oxidative damage causes an increase in the ubiquitination of synaptosomal proteins. Incubation with 10 or 50 microM FeSO4 caused concentration-dependent increases in carbonyl groups (an indication of protein oxidation) and ubiquitinated proteins (determined by probing Western blots with a monoclonal antibody to ubiquitin). Differences in protein ubiquitination occurred within 5 min of incubation, indicating a rapid response to oxidative stress. Results of experiments with MG-132, an inhibitor of the degradation of ubiquitinated proteins, suggested that oxidative damage stimulated ubiquitination rather than inhibited degradation of ubiquitinated proteins. The data are consistent with the hypothesis that synaptic terminals utilize the ubiquitin/proteasome proteolytic pathway to degrade oxidatively damaged proteins.

A comparison of the axonal transport of taurine and proteins in the goldfish visual system.

Abstract— Radioactive cystathionine, a metabolic precursor of taurine, was injected into the right eye of goldfish. At various times after injection the retina and both optic tecta were extracted with trichloroacetic acid (TCA) and the amount and nature of the radioactivity was determined. Radioactive taurine and inorganic sulfate were present in the TCA‐soluble extract of retina and radioactive taurine and a small amount of inorganic sulfate was found in the contralateral optic tectum. That taurine is migrating intraaxonally and is not diffusing in extraaxonal spaces is suggested from experiments in which the migration of taurine was compared with that of [14C]mannitol, used here as a marker of extracellular diffusion. In the time studied (up to 15 h) mannitol did not migrate to the tectum, whereas taurine was detectable in the tectum as early as 8 h after injection. Since intra‐axonal diffusion of amino acids and other small molecules in this system has been ruled out, it is likely that taurine is being transported axonally.