A COMPARISON OF THE AXONAL TRANSPORT OF TAURINE AND PROTEINS IN THE GOLDFISH VISUAL SYSTEM N. A. INGOGLIA, J. A. STURMAN, D. K. RASIN a n d T. D. LINJJQUIST Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, NJ 07103, U.S.A. and Department of Human Development and Genetics, Institute for Basic Research in Mental Retardation, Staten Island, NY 10314. U.S.A. (Received 4 November 1977. Accepted 30 January 1978)

Abstract-Radioactive cystathionine, a metabolic precursor of taurine, was injected into the right eye of goldfish. A t 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 intra-axonally and is not diffusing in extra-axonal 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 I5 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. The axonal transport of taurine was found to be similar to the fast component of protein transport because: ( I ) their rates of transport are similar, (2) the transport of both is blocked by the protein synthesis inhibitor cycloheximide, (3) vinblastine, which disrupts neurotubules, appears to have similar effects on both protein and taurine transport, and (4) both rapidly transported proteins and taurine remain mostly intra-axonal once they have been transported to the tectum. Taurine and proteins differ in that rapidly transported proteins are primarily particulate in nature and localized to a large extent in nerve endings, while taurine is primarily in a soluble fraction and is present in nerve endings only in trace amounts. We suggest that taurine may be loosely linked to a newly synthesized protein in the soma and is then transported along with that protein on a similar conveying mechanism in the axoplasm

OVERthe past several years a great deal of attention has been focused o n the sulfonic amino acid, taurine. Taurine is a ubiquitous amino acid in eukaryotes and has been shown to be present in high concentration in brain (JACOBSEN & SMITH,1968). The concentration of taurine in developing brain is several-fold higher than in adult brain and it has been suggested that taurine may play a special role in developing neural tissue (STURMAN et a/., 1977). It has also been shown to possess a strong inhibitory action o n spinal cord neurons (CURTIS& WATKINS,1960). and more recently has been proposed as an inhibitory neurotransmitter (DAVISON & KACZMAREK, 1971 ; PASANTES-MORELES et al., 1973; OJA& LAHDESMAKI, 1974). Other studies have suggested that the role of taurine in neural tissue may be as a n intracellular membrane stabilizer and that the inhibitory effects attributed to taurine might be explained by this action and not by postulating taurine as an inhibitory neurotranset a/., 1976). mitter (GRUENEK When [3SS]taurine is injected intravitreally into goldfish, it migrates along tne optic axons of the retinal ganglion cells t o the termination of those axons in the contralateral optic tectum (INGOGLIA et

nl., 1976). The present experiments were designed to

examine some of the characteristics of axonally migrating taurine in an attempt t o understand better the role of this molecule in nervous tissue. MATERIALS AND METHODS Goldfish (Carrasius aurafus) approx 1&12 cm in length were obtained from Ozark Fisheries, Stoutland, MO. Radioisotopes ["S]cystathionine (9.4 mCi/rnmol), ["Sltaurine (64mCi/mmol), [3H]taurine (18 Ci/mrnol). [3H]leucine (55 Ci/mmol) and C3HJproIine (29 Ci/mmol) were obtained from Amersham/Searle Corp. (Chicago, 11.). and [14C]mannitol (50.6 mCi/mmol) was obtained from New England Nuclear Corp. (Boston, MA.). The metabolic inhibitors used in this study, cycloheximide and vinblastine sulfate, were obtained from Sigma Chemical Co. (St. Louis. MO.). In general, the experiments were performed by injecting labelled materials into the right eye of groups of goldfish, killing the fish at various times after injection and assaying radioactivity present in the right retina and both optic tecta. In some experiments the right retina and both tecta were rapidly removed, homogenized in distilled H,O. and then extracted with 10: trichloroacetic acid (TCA) according to techniques already described (INGOGLIA er al.. 1973).

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This procedure gives a TCA-soluble fraction containing small molecules (e.g.amino acids) and an insoluble fraction containing macromolecules (eg. proteins). These fractions were dissolved in Hydromix (Yorktown Industries, Hackensack. NJ) and counted in a liquid scintillation spectrometer. In other experiments where it was not necessary to separate small molecules and proteins, retinas and tecta were digested in a commercial tissue solubilizer (Soluene 350, Packard Instruments, Downers Grove, IL.). dissolved in a Toluene/Liquiflour (New England Nuclear Corp.) scintillation cocktail and counted in a liquid scintillation spectrometer. Counts per minute were converted to disintegrations per minute using appropriate quench correction curves and simultaneous equations for analysis of dual "S or and 'H labelled experiments. All calculations of radioactivity measurements allowed for decay of "S and 'H and were referred to the day of measurement of the specific radioactivity. Subcellular fractions of goldfish tecta were prepared 24 h after intraocular injection of ["'Sltaurine (1 pCi) and ['Hlproline (5.3 pCi) or after intraocular injection of ["Sltaurine (8 pCi) and intracranial injection of C3H]taurh e (2pCi). In some experiments [35S]taurine (1 pCi) and [3H]taurine (5 pCi) were added to ice-cold homogenates prepared from brains of fish which had not had an isotopically labelled compound administered in uiuo. The latter experiment was designed to ensure that the subcellular distribution of taurine was not an artifact of the procedure used to prepare the fractions. In each experiment the tecta from 20 goldfish injected with isotope were pooled and the brains from 15 goldfish which had not been injected with an isotope added as unlabelled carrier for the subcellular fractions The brain material was homogenized in 0.32 M sucrose (10 ml/g) in a motor-driven Teflon-pestled glass homogenizer and fractionated using the method de& WHITTAKER (1962).The following fracveloped by GRAY tions were prepared and identified as described previously (RASIN& GAULL,1975):crude nuclie, PI (sedimented by centrifugation at IOOOg for 10 min); crude mitochondria and synaptosomes, P2 (sedimented by Qntrifugation at 12,OOO g for 20 min); microsomes, P, (sedimented by centrifugation at 50,OOOg for 2 h); and soluble fraction, S3. Fraction P2 was further fractioned by means of discontinuous sucrose gradients consisting of 6ml each of 0.4, 0.6.0.8, 1.0 and 1.2 M-SUCTOS~and centrifugation at 50,OOOg for 2 h. After P, was resuspended in water the following fractions were obtained: synaptosomal cytoplasm, 0; synaptic vesicles, D; membrane and myelin fragments, E, F, G; partially disrupted synaptosomes H; and mitochondria, I. After P, was resuspended in iso-osmotic sucrose the following fractions were obtained: myelin, A; synaptosomes, B; and mitochondria, C. Samples of the homogenate and of the various fractions were analyzed for [35S]taurine, ['Hltaurine and ['HJproline as follows: 1 vol of each fraction was added to 3 vol of cold 10% TCA and centrifuged, the supernatant solution was collected added to liquid scintillation fluid (RASSIN el a/., 1977)and counted in a liquid scintillation spectrometer (Packard model 3320) set for double label counting The TCA precipitate was washed twice with cold 10% TCA. 1 ml ether was added and allowed to dry overnight in a fume hood. The dried pellet was dissolved in 1 ml soluene and counted in a toluene based scintillation counting cocktail. In autoradiographic experiments 4 pCi of C3H]taurine was injected into both eyes of a group of fish and 24 h

a/.

later the fish were anaesthetized by immersion into ice water. Retinas and tecta were fixed by perfusion of the tissue with 4% glutaraldehyde in cacodylate buffer (0.1M, pH 7.4). In the case of the retinas, two puncture holes were made in the sclera and fixative was injected into one and allowed to drain out of the other. After perfusion retinas were removed from the fish and placed in fixative. In the case of the tecta, skulls were removed and tecta were fixed by flooding the dorsal surface with fixative and then injecting additional fixative into the ventricle, thus flooding the ventral surface. Retinas and tecta were then passed through four 30 min washes of fixative, placed in fresh fixative overnight, post-fixed for 2 h in 1% osmium tetroxide and embedded in epon. One micron sections were cut, slides were dipped in Kodak NTB emulsion, exposed for 2 or 4 weeks and developed in Kodak D19 developer.

RESULTS Taurine is synthesized directly from the sulfur amino acid cysteine by a series of reactions one of which involves the enzyme cysteinesulfinic acid decarboxylase. This enzyme has been shown to be present in the goldfish retina (Sturman, unpublished data). In an earlier study, we found that [35S]cysteine, when injected into the eye of goldfish, was rapidly metabolized in the retina to taurine which then migrated along optic axons to the contralateral tectum. Radioactivity in the TCA-soluble fraction of the tecta was found t o be present only as taurine and inorganic sulfate but not in cysteine (or cystine) (INGOGLIAet al., 1976). This suggested a rather specific migration of taurine in the goldfish visual system. In the present experiments, we have investigated this finding further by studying axonal migration following intraocular injection of cystathionine, a metabolic precursor of cysteine. [35S]Cystathionine was dissolved in phosphate buffer, (PH7.4.0.16 M) and 4 p1 (0.1 yCi) was injected into the right eye of 16 fish. Fish were killed I,3,6 or 14 days later and TCA-soluble and -insoluble radioactivity was extracted from the right retina and both tecta. TCA-insoluble radioactivity migrated to the contralateral tectum by 1 day after injection (Fig. I). It is likely that this material represents radioactivity in [3sS]cysteine formed by enzymatic cleavage of [?S]cystathionine and incorporated into proteins in the retina: these proteins are then axonally transported in the fast component of protein transport. It is possible that some of this TCA-insoluble radioactivity in retina may be present as sulfated lipids, although this would not affect the results obtained from the tectum, since sulfated lipids are not transported axonally (ELAMet a/., 1970; ELAM et a!., 1971). The peak of TCA-soluble radioactivity appears slightly later (3 days after injection), but it is clear that this component too has migrated along the optic axons (significant left vs right tectal differences in radioactivity). In separate experiments 15 fish were injected in both eyes with 2.0@ [35S]cystathionine and 3 days later the 30 retina and 30 tecta were pooled and

Axonal transport of taurine

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are taken up by cells such as amino acids (ELAM& AGRANOFF,1971), the diamine putrescine (INCOGLIA 0 et al., 1977) and inorganic sulfate (ELAMet al., 1970). have been shown not to diffuse to the tectum within the time period we are studying. In order to determine whether taurine is diffusing extra-axonally or rather is being transported axonally, experiments were performed in which fish were injected into the right eye with [14qmannitol, as a marker of extracellular diffusion, or with a solution I of [3H]leucine and ["S]taurine. All fish were sacrificed at times ranging from 2 to 15 h after injection I and the retinas and tecta were removed and dissolved in Soluene. Radioactivity was then determined in the right retina and both tecta. Results showed no differiL I ences in left vs. right tecta radioactivity following inw 1 0 ' I I traocular injections of [i4C]mannitol (Fig. 2). DifferI 3 6 14 DAYS AFTER INJECTION OF ["S] CYSTATHONINE ences were noted, however, beginning at 8 and persisting at 1 5 h in fish injected with [3HJleucine and FIG.I. [3JS]Cystathioninewas injected into the right eye of a group of fish and TCA-soluble (0)and insoluble ( 0 ) ["Sltaurine. These results suggest that taurine is not radioactivity was extracted from both tecta at various reaching the tectum by extracellular diffusion but is, times after injection. Values are the mean S.E.M. of 6 fish in all probability, being transported axonally. and show transported radioactivity (left tectum-right tec- Further, these experiments show a similarity in the tum) plotted against time after injection. The radioactivity time of arrival of the fast component of protein transin the TCA-soluble fraction was found to be composed port and taurine transport primarily of taurine. In order to study this association more closely, we next investigated the effect of cycloheximide, an inextracted with cold 10% TCA. The extract was then hibitor of protein synthesis, and vinblastine an inhibianalyzed using an automatic amino acid analyzer tor of protein transport on the axonal transport of (Beckman 12OC) in conjunction with a flow cell scin- taurine. tillation spectrometer (Packard model 3142) and the Cycloheximide, in doses of 0, 1, 5, or 10 pg (in 5 pI nature of the radioactivity was determined (STLIRMAN of distilled H20), was injected into the right eye of & GAULL,1974). [35S]Taurine and ['5S]inorganic 24 fish. One hour later a solution of [35S]taurine sulfate were present in the TCA-soluble extract of (0.20 pCi) and [3H]proline (1.54 pCi), in a vol of 5 pi, retina, and radioactivity was present in the TCA-ins* luble fraction of retina, presumably as [%]cysteine 0 35S-TAURINE formed from [35SJcystathionine and incorporated 0 3H-LEUCINE into protein. ['%]Sulfate comprised approx 40% of the TCA-soluble radioactivity and its identity was further confirmed by precipitation with BaCI,. After this step, there was no radioactivity eluted with the solvent front. [35S]Taurine and small amounts of [35S]inorganic sulfate were present in the tectum, with no radioactive cystathionine or cysteine present. This data suggests that the TCA-soluble material migrating along optic axons to the tectum contains only taurine and not its metabolic precursors and demonstrates further the uniqueness of taurine migration among molecules of similar molecular weight and structure. The small amount of labelled inorganic sulfate in the tectum (