4S RNA is present in regenerating optic axons of goldfish.

Regenerating optic axons of goldfish were loaded with [3H]RNA by injecting [3H]uridine into the eye and allowing time for the radioactivity to be delivered to the optic tectum. The axons were subsequently removed from the tecta by cutting the optic nerve and allowing the optic axons in the tectum to degenerate. Analysis of tectal [3H]RNA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a selective loss of tritiated 4S RNA and not ribosomal RNA from the denervated tecta. These results support the hypothesis that regenerating optic axons of goldfish grow back into the tectum carrying 4C but not ribosomal RNA.

Protein modification by amino acid addition is increased in crushed sciatic but not optic nerves.

Rat optic and sciatic nerves were crushed, and 10 minutes to 3 days later nerve segments between the crushed site and the cell body were removed and assayed for posttranslational protein modification by amino acid addition. Protein modification was comparable in intact optic and sciatic nerves, but in sciatic nerves increased to 1.6 times control levels 10 minutes after crushing and reached a maximum of ten times control levels by 2 hours. In optic nerves activity was decreased throughout the time course studied. The results indicate that, in a nerve which is capable of regeneration (sciatic), protein modification by the addition of amino acids increases immediately after injury, but a nerve incapable of regeneration (optic) is incapable of activating the modification reaction. These findings may be important in understanding the reasons for the lack of a regenerative response after injury to central mammalian nerves.

The site of amino acid addition to posttranslationally modified proteins of regenerating rat sciatic nerves.

The posttranslational modification of proteins by amino acids has been described in a variety of biological systems. These reactions occur at low levels in intact sciatic nerves of rats but are increased 10-fold following nerve injury and during subsequent regeneration of the nerve. While it has been shown in brain and liver that the site of addition of Arg is to the N-terminus, there is no information on the location at which the other amino acids add on to targeted proteins nor the site of addition of Arg in regenerating nerves. In the present study, we have used manual micro-Edman degradation combined with HPLC, and digestion with carboxypeptidase A and B to determine the site of addition of various amino acids to targeted proteins. Of the 3H-labelled amino acids incorporated posttranslationally into proteins of regenerating sciatic nerves (Arg, Lys, Leu, Phe, Val, Ala, Pro and Ser), only [3H]Arg was found to be present at the N-terminus. To determine whether amino acid additions were occurring at the C-terminus, proteins modified by two of the amino acids incorporated in greatest amounts (Lys and Leu) were incubated with specific carboxypeptidases. [3H]Leucine was not liberated following incubation with carboxypeptidase, suggesting that Leu is not added at the C-terminus of modified proteins. Under similar conditions, some [3H]Lys was liberated, but in amounts not significantly different from controls incubated without carboxypeptidase, indicating a non-specific degradation of Lys modified proteins rather than a specific release of Lys from the C-terminus. These experiments show that in regenerating sciatic nerves of rats, Arg is the only amino acid added posttranslationally to the amino terminus of target proteins, and that Leu, and probably Lys, are not conjugated to proteins at the C-terminus.

Regulation of the post-translational conjugation of amino acids to rat brain proteins.

Post-translational conjugation of arginine (but not other amino acids) to proteins has been reported to occur in a high speed supernatant fraction of rat brain homogenates from which molecules of less than 5000 mol. wt have been removed. In the present study we report that removal of molecules of less than 1000 mol. wt by dialysis, does not result in incorporation of arginine into protein in amounts significantly different than in the undialysed supernatant. The addition of molecules with molecular weights greater than 1000 and less than 5000 to the active fraction, inhibits the incorporation of arginine into proteins in a concentration dependent manner suggesting that the post-translational incorporation of arginine into brain is regulated by a molecule(s) of greater than 1000 and less than 5000 mol. wt. Incorporation of lysine into proteins did not occur following removal of molecules of less than 5000 mol. wt, but did occur in the void volume fraction of a Sephacryl S-200 column (molecular weight cut-off 125,000), suggesting that the incorporation of lysine into proteins is regulated by molecules retained by the S-200 column but greater than 5000 mol. wt. When experiments were repeated using the void volume of a Sephacryl S-300 column (molecular weight exclusion, approximately 200 k), leucine and proline were incorporated in amounts similar to arginine and lysine and serine, alanine, valine, phenylalanine and histidine were incorporated at lower but measurable levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a peptide that inhibits the posttranslational arginylation of proteins in rat brain.

All eukaryotic cells contain enzymes that are able to catalyze the transfer of Arg from tRNA to the N-terminus of naturally short lived or damaged cytosolic proteins. For certain test proteins, it has been shown that the addition of Arg to the N-terminus leads to their degradation via the ubiquitin proteolytic pathway. The mechanisms used by cells for identifying proteins for arginylation and regulating arginylation are not known. The present study reports the isolation of a peptide from rat brain that is able to inhibit the arginylation of proteins in brain extracts. We suggest that this peptide is the physiological regulator of arginylation in rat brain.

Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification.

Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-translational modification of proteins by arginine and lysine following crush injury and during regeneration of rat sciatic nerves.

Following crush injury to rat sciatic nerves, a crude fraction of the 150,000 g supernatant can post-translationally incorporate [3H]Arg and [3H]Lys into endogenous proteins in amounts approximately 10 times uninjured control nerves. These increases occur in the proximal nerve stump within 2 h of injury and 2 weeks later in a distal segment of nerve containing the tips of the regenerating axons. In the present experiments, the endogenous nerve proteins modified by Arg or Lys in these nerve segments have been identified using two-dimensional polyacrylamide gel electrophoresis. The fraction used to assay for protein modification, the void volume of a Sephacryl S-300 column, was found to contain only a few proteins visible by Coomassie blue staining, one of which is likely to be albumin (68 kDa, pI 6.4). While this protein was modified by both Arg and Lys, the majority of label was found in areas not showing Coomassie blue staining. This indicates that of the many potential targets of post-translational arginylation and lysylation, most are proteins of relatively low abundance. A variety of proteins were modified by Arg or Lys alone while others were modified by both Arg and Lys. A high molecular weight protein (175 kDa, pI 9.0) was modified only by Lys and only at 2 h post crush. Of a variety of modified proteins of approximately 17 kDa one (pI 6.3) was modified by both Arg and Lys and at both time points, while another (pI 9.0) was modified at both time points, but only by Lys. The results show that Arg and Lys can be added post-translationally to a large number of low abundance, soluble sciatic nerve proteins, and that some of those proteins are modified only by Arg or Lys while others are modified by both Arg and Lys. Also, the modification of certain proteins appears to be associated specifically with the immediate response of a nerve to injury (e.g. 88 kDa, pI 7.1) while others are associated with the regenerative period (e.g. 56 kDa, pI 7.4).

Posttranslational modification of nerve proteins by amino acids.

Both axonal and glial components of nerve are capable of carrying out reactions in which Arg, Lys, Leu, Pro, Val, AJa and Ser can be covalently linked to endogenous proteins in reactions which require tRNA but occur in the absence of ribosomes and ribosomal RNA. These posttranslational protein modifications appear to play important roles in nerve regeneration since they are increased more than 10-fold within 2 h of a crush injury in nerves which are capable of regeneration, but are not activated in nerves not capable of regrowth following injury. The regulation of the modification of proteins by Arg and Lys in vivo appears to be the function of separate peptides. The exogenous application of serine protease inhibitors (but not other protease inhibitors) mimics the effect of the endogenous peptides, suggesting that the endogenous regulators have serine protease inhibitory activity. The targets for modification are proteins of low abundance and thus far have been identified only in terms of their molecular weights and isoelectric points. The site of addition of Arg, but not the other amino acids, to target proteins is to the amino terminus. The addition of Arg to an amino terminus is likely to be involved in the ubiquitin mediated proteolysis of the modified protein. One of the most unusual findings in these series of experiments is that in regenerating sciatic nerves, amino acid modified proteins aggregate to form complexes of greater than 2 × 106 Da. The significance of this finding is not known. But we speculate that the aggregate may result from the assembly of an insoluble functional unit of the cell from soluble precursor proteins, and that the trigger for their assembly is amino acid modification.

Ubiquitin is associated with aggregates of arginine modified proteins in injured nerves.

Crush injury to rat sciatic nerves results in a 10-fold increase in the post-translational arginylation of proteins. In other systems, N-terminal arginylation leads to ubiquitination and proteolysis of the arginylated proteins. In the present experiments, proteins obtained from the 150 kg supernatant of crushed sciatic nerves were posttranslationally modified by 3H-arginine. These arginine modified proteins formed aggregates (precipitated at 20 kg) which then partially separated by SDS-PAGE were immunoreactive to a monoclonal antibody to ubiquitin. The results indicate that following injury to sciatic nerves, certain proteins are arginylated and ubiquitinated, probably targeting them for degradation. It is likely that these reactions help to rid cells of proteins damaged by the crush which would otherwise be cytotoxic.

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.

Introduction of macromolecules into synaptosomes using electroporation.

Synaptic terminals are sites of high metabolic activity and thus are particularly vulnerable to oxidative stress. Oxidative damage to proteins can be toxic to neurons and may cause irreversible cell damage and neurodegeneration. A neuroprotective mechanism used by cells to combat oxidative damage is to selectively degrade damaged proteins. Therefore, it is of interest to study the mechanism of degradation of oxidatively damaged proteins in synaptosomes. One way of oxidizing synaptosomal proteins in vitro is by incubating intact synaptosomes in the presence of an oxidizing agent. A problem with this approach is that it may also cause oxidative damage to the machinery required to recognize and degrade oxidized proteins. We have, therefore, introduced a fluorescent macromolecule into synaptosomes to assess the feasibility of using this technique to study how oxidized proteins are degraded and removed from synaptic terminals. Synaptosomes were subjected to electroporation in the presence of FITC labelled-dextran with an average molecular weight of 70000 (FD-70) and non-specific binding was determined by running parallel experiments in lysed synaptosomes. Following extensive washing, synaptosomes were assayed for the presence of intra-synaptosomal FD-70 by measuring fluorescence in a microplate fluorescence reader. Significant differences in fluorescence were found between intact and lysed synaptosomes with maximal uptake at 100 V/ 1500 microF (approx. 36 pmol/mg protein). To determine if membrane transport was compromised by electroporation, uptake of 3H-arginine was compared in control and electroporated synaptosomes. While untreated electroporated synaptosomes showed a loss of 22% in the ability to transport arginine, preincubation in the presence of 1 mM ATP resulted in a complete restoration of arginine transport. These results show that electroporation is a potentially useful technique for introducing a specific oxidized protein, into synaptic terminals so its metabolic fate can be examined.

Axonal transport of taurine along neonatal and young adult rat optic axons.

Studies in this laboratory have indicated that taurine is axonally transported along goldfish optic nerves. In the present experiments the axonal transport of taurine was examined in neonatal and young adult rat optic axons. [35S]taurine was injected into the vitreous humor of right eyes of developing (1--15-day-old) or young adult (40-day-old) rats. At various times after injection ranging from 3 h to 7 days, right retinae and left and right geniculates were removed and assayed for radioactivity, left minus right lateral geniculate (L-RLG) radioactivity being used as an index of axonally transported [35S]taurine. Results indicated that taurine was rapidly transported along both neonatal and young optic axons, in contrast to other amino acids (i.e., leucine and proline) which are not axonally transported in this system. Significant developmental variations were seen in both L-RLG and right retinal [35S]taurine activity 24 h after injection. The amounts of L-RLG [35S]taurine corrected for retinal ganglion cell uptake in animals injected at 1,4,7 and 11 days after birth (prior to and during the major period of synaptogenesis in the geniculates) were 4.5, 3.1, 2.3 and 2.6 times higher, respectively, than those in the young adults. In contrast, the amount of corrected L-RLG [35S]taurine in animals injected at 15 days after birth (after synaptogenesis) were not significantly different from that in the young adult.

Axonal transport of nucleosides, nucleotides and 4S RNA in the neonatal rat visual system.

The axonal migration of RNA, the nucleoside uridine and its nucleotide derivates (NS/NT) were compared in neonatal and young adult rat optic axons. Tritiated uridine was injected into right eyes of developing (1- or 4-day-old) and young adult (40-day-old) rats which were sacrificed at times after injection ranging from 6 h to 20 days. Right and left lateral geniculates were removed and assayed for trichloroacetic acid soluble (NS/NT) and RNA radioactivity. Left minus right geniculate (L-RLG) radioactivity was used as an index of axonally migrating radioactivity. Results showed that uridine and its phosphorylated derivatives were transported along both neonatal and young adult rat optic axons. Greater than 90% of right geniculate (blood-borne) TCA soluble radioactivity was metabolized to volatile substances (probably 3H2O) by three days after injection, leaving approximately 3% of the neonatal and approximately 10% of the adult activity as [3H]NS/NT. In left geniculate fractions (containing transported material) approximately 15% and 40% of total TCA soluble radioactivity was present as [3H]NS/NT in neonates and adults, respectively. Thus, axonal NS/NT appears to be relatively protected from degradation when compared with blood-borne NS/NT. The amount of L-RLG [3H]RNA in the neonates was 10 times higher than in young adults. Peaks of neonatal [3H]RNA occurred at 5 and 10 days after birth, whether injections were made at 1 or 4 days of age indicating that this [3H]RNA may be linked to developmental events. Gel electrophoretic analysis of neonatal geniculate RNA indicated that a small portion of the [3H]RNA in the first peak represented axonally transported 4S RNA. The remainder of the L-RLG [3H]RNA in the neonates was probably due to a rapid and efficient incorporation of axonally transported [3H]NS/NT into extraaxonal geniculate RNA. In contrast, little or no axonal RNA transport could be demonstrated in the young adults.

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.

4S RNA is transported axonally in normal and regenerating axons of the sciatic nerves of rats.

Experiments were designed to determine if following injection of [3H]uridine into the lumbar spinal cord of the rat, [3H]RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant RNA species present in these axons. In one experiment the left sciatic nerve of a rat was crushed. Two days later 170 microCi of [3H]uridine was injected into the vicinity of the lumbar ventral horn cells. Ten days after injection, rats were sacrificed and sciatic nerves were prepared for autoradiography. Photomicrographs were taken of labeled areas of intact and regenerating nerves and grains were counted over Schwann cells, myelin, axons and other unspecified areas. In both intact and regenerating sciatic nerves more than 20% of the silver grains were associated with motor axons and approximately 40% were found over cytoplasm of Schwann cells surrounding these axons. These data indicate an intra-axonal localization of RNA in sciatic nerve axons, as well as an active transfer of RNA precursors from axons to their surrounding Schwann cels. In separate studies, the left sciatic nerve was crushed and 10 days later [3H]uridine was bilaterally injected intraspinally into 6 rats. Four control rats were sacrificed at 14 or 20 days after injection. In the remaining 2 rats the sciatic nerve was cut 14 days after injection and the distal part of the nerve was allowed to degenerate for 6 days before sacrificing the rat. Thus, the distal portion of the nerve contained Schwann cells labeled by axonal transport but lacked intact axons. RNA was isolated from experimental and control nerve segments by hot phenol extraction and ethanol precipitation. RNA species (28S, 18S and 4S) were separated by polyacrylamide gel electrophoresis and radioactivity was measured in a liquid scintillation counter. Control groups had RNA profiles similar to those already described, with greater than 30% of the radioactivity present as 4S RNA. The proximal portions of nerve taken from the group in which nerves were cut, had a similar amount of radioactivity present as 4S RNA. However, in the distal segments of these nerves (in which the axons had degenerated thus creating an 'axon-less' nerve) the amount of radioactivity in the 4S peak decreased to approximately 15% of the total RNA, suggesting that 4S RNA is the predominant if not the only RNA present in these axons. These results strongly indicate that both intact and regenerating sciatic nerves of rats selectively transport 4S RNA along their motor axons.

Association of spermine and 4S RNA during axonal transport in regenerating optic nerves of goldfish.

Experiments were designed to determine whether polyamines are bound to 4S RNA and then transported axonally along regenerating optic axons of goldfish. In one set of experiments, inhibition of retinal RNA synthesis by intraocular injections of 10 microgram of cordycepin, blocked the axonal transport of both [3H]RNA and [14C]spermidine by about 65%, 6 and 14 days after injection. Intraocular injections of vinblastine, (0.1, 0.5 or 1.0 microgram) an agent which interrupts axonal transport of proteins, had no effect on retinal RNA synthesis nor on the amount of [14C]spermidine incorporated into the TCA-insoluble fraction of retinal extracts. However, the axonal transport of both [3H]RNA and [14C]polyamines was affected in a dose-dependent fashion; the inhibition of both was approximately 80% at the higher dose. Further evidence for an association between axonally transported 4S RNA and polyamines came from experiments in which regenerating optic axons were cut and allowed to degenerate 6 days after injection of [3H]spermidine into the eye. The loss of optic axons from the tectum 7 days after cutting the nerve resulted in an 86% loss of TCA insoluble polyamines, indicating a largely intra-axonal locus. A similar loss of 4S RNA was found in identical experiments following injections of [3H]uridine into the eye. Finally, experiments were performed in which [3H]spermidine was injected into both eyes of 12 fish whose optic nerves had been regenerating for 18 days. Six days later, fish were sacrificed and RNA was extracted from tectal homogenates by hot phenol and ethanol precipitation. The major stable RNA species were separated by SDS-polyacrylamide disc gel electrophoresis and radioactivity was determined by extraction of 2.0 mm gel slices. Results showed co-migration of 3H with 4S RNA optical density peaks, and not with 28S and 18S ribosomal RNA peaks, suggesting that some polyamine-associated radioactivity is bound to axonally transported 4S RNA. When the nature of that radioactivity was determined on an amino acid analyzer, it was found to be present primarily as spermine and not as the injected compound spermidine. The data are consistent with the hypothesis that some spermine is bound to 4S RNA and then axonally transported along regenerating axons of the goldfish optic nerve.

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.

N-terminal arginylation and ubiquitin-mediated proteolysis in nerve regeneration.

Damaged sciatic nerves of rats respond to injury within minutes by activating reactions that result in the transfer RNA-mediated posttranslational addition of several amino acids to a variety of cytoplasmic proteins. For the most part, the site of addition of individual amino acids and the identity of the target proteins is not known. However, arginine, one of the amino acids added in greatest amounts, has been shown to be covalently linked to the N-terminus of acceptor proteins. In other simpler eukaryotic cells, N-terminal arginylation results in degradation of the arginylated proteins via the ubiquitin proteolytic pathway. Recent experiments have shown that when proteins, obtained from sciatic nerves 2 h after injury, are arginylated in vitro, they form high molecular weight aggregates. Other experiments have shown that these arginylated proteins are immunoreactive to a monoclonal antibody to ubiquitin. These findings suggest that following injury to the sciatic nerve, proteins which are arginylated are candidates for ubiquitin mediated proteolysis. Injury to a nerve incapable of regeneration without experimental intervention, the rat optic nerve, does not result in activation of the arginylation reactions until 6 days following injury. Based on the temporal differences in response to injury of sciatic and optic nerves (2 h vs. 6 days), we propose that the lack of arginylation following injury to the CNS is related to its inability to mount a regenerative response. The association of Arg modification of damaged proteins with the ubiquitin-mediated degradation of those proteins, suggests that regenerative failure in the CNS may be related, in part, to a failure to degrade intracellular proteins at the site of injury.