152
Brain Research. 102 (1976} 152 155 i' Elsevier Scientific Publishing (~ompany, Amsterdam Printed in The Nethcrland~
Short Communications
Factors affecting retrograde axonal transport of horseradish peroxidase in the visual system ANN H. BUNT, RICHARD H. HASCHKE, RAYMOND D. LUND AND DIANNE F. CALKINS Departments of Ophthalmology, Anesthesiology and Biological Structure, University of Washington, Seattle, Wash. 98195 (U.S.A.)
(Accepted October 8th, 1975)
Horseradish peroxidase (HRP) has proven valuable for demonstrating uptake and retrograde axonal transport of exogenous protein by neurons, and this property has been used recently as an experimental tool in hodological neuronography~L Other exogenous proteins have also been demonstrated to move retrogradely in axons ~].t6,22 and the transport of nerve growth factor (NGF) is of particular interest because it has enabled study of the specificity and biological significance o f this process. Thus, whereas 125I-labeled N G F was taken up and transported retrogradely by sympathetic axons, several other proteins and aged or oxidized N G F which had lost biological activity were transported poorly 22. Retrograde transport of the glycoprotein HRP, while demonstrated in a number of neuronal systems, also shows specificity. Nauta et al. 14 found that neurons of the neocortex did not transport H R P as expected after its injection into caudoputamen, and St6ckel et al. 2~ also found lack of transport of HRP in sympathetic axons (but see refs. 5 and 8). Preliminary experimentsZ have indicated a lack of retrograde transport of a number of other proteins (including bovine serum albumin, alkaline phosphatase, hemocyanin, and lactoperoxidase) in the axons of rat optic nerve, which do transport HRP. This suggested a specificity of retrograde transport in axons of the visual system, but since these proteins differ considerably in physical and chemical properties, little could be deduced about the factors responsible for specificity of uptake and/or transport in these axons. It appeared more appropriate to test which features of the HRP molecule might be related to this specificity, so that this information might provide a general understanding of the characteristics of proteins normally transported retrogradely. The visual cortex or superior colliculus of a total of 18 adult albino rats was injected with 1--5 ,ul of a 1 0 - 3 0 ~ saline solution of the test protein. After 24 h, the animals were perfused with 4 ~ paraformaldehyde in 0.1 M phosphate buffer and the retinae and brains processed cytochemically6 with appropriate controts a. The test proteins were as follows.
153 (1) HRP (Sigma, type VI). It was determined that greater than 8 0 ~ of the enzymatic activity of this product was due to isoenzyme C (ref. 9). (2) Periodate-oxidized-borohydride-reduced HRP isoenzyme C (ref. 21). (3) HRP isoenzymes A, B and C. HRP was purified from fresh horseradish roots 2° through ion-exchange columns (CM-52 for the B and C isoenzymes and DE52 for the A isoenzyme). Analytical polyacrylamide gel electrophoresis at pH 4.5 and pH 8.9 (see refs. 4 and 17) indicated that the A, B and C isoenzymes were not intercontaminated. The reaction mixture for the spectrophotometric assay contained 2.5 ml of 20 m M sodium phosphate buffer pH 6.0; 0.2 ml 150 m M H202; 0.3 ml 5 ~ pyrogallol and appropriately diluted enzyme (1 /~g). The oxidation of pyrogallol to purpurogallin was followed at 420 nm (activity unit: micromoles purpurogallin formed per minute). The specific activities of the isoenzymes used in the injections into the brain were: A, 1372; B, 1409 and C, 2423 U/rag. Following periodate oxidation and borohydride reduction 21 the specific activity of the C isoenzyme was 635 U/rag. Following injection of HRP type VI or isoenzymes B or C into the visual cortex, peroxidase granules were readily demonstrated in neurons of the ipsilateral dorsal lateral geniculate nucleus (dLGN) and also cortical neurons adjacent to the injection. Similarly, after injections into the superior colliculus, ganglion cell somata of the contralateral retina contained the tracer granules. When the injected HRP was restricted to one side of the visual cortex or tectum, labeled neurons were not found in that d L G N or retina projecting to the uninjected side, indicating that the HRP-positive granules did not result from blood-borne label 3. No retrograde transport of HRP isoenzyme C which had been periodate-oxidized and borohydride-reduced or ofisoenzyme A was found in neurons of the d L G N or retina. Filled profiles of axons and cells were visible within the injection site, and the cytoplasm of some of these neurons appeared somewhat granulated, but no neurons could be found in the tissue beyond the zone of diffusion of HRP which contained peroxidase granules. The extent of diffusion from the injection sites of the periodateborohydride-treated isoenzyme C and the A and B isoenzymes was noticeably diminished, as compared with injections of equal volumes of isoenzyme C. This may reflect the lower specific activities of these proteins (see above), although the centralmost parts of the injection sites were equally well stained and the isoenzyme B did give rise to well-labeled neurons. The results suggest a specificity of retrograde transport, involving the uptake and/or actual transport process, in axons of the rat visual system. Whereas transport of HRP type VI and isoenzymes B and C was readily detected, the periodate-borohydride-treated isozyme C and isozyme A either did not enter cells or were not transported, as detected histochemically. The preliminary findings suggest that the carbohydrate moiety, which is altered by the periodate-borohydride treatment, may be involved in the specificity. The specific activity of the enzyme was reduced after this procedure, suggesting that other groups may have also been altered13, 23, so that the direct involvement of the carbohydrate remains to be demonstrated. In attempting to determine the biochemical basis for the difference in retrograde transport between the A compared with the B and C isoenzymes, it should be noted
154 that these differ from each other significantly in at least two aspects. A substanlially different amino acid composition is found for the A isoenzyme (isoelectric point pH 4.5) as compared with the B and C isoenzymes (isoelectric point pH 9.0)15,2L Each isoenzyme contains approximately 19 ;",i carbohydrate by weight, but the A isoenzyme differs tYom B and C in carbohydrate composition, containing galactose, arabinose and mannose as neutral sugars and the amino sugar mannosamine; the B and ( i s o enzymes contain xylose, fucose and mannose as neutral sugars and the amino sugars mannosamine and galactosamine t'j. These differences suggest that the specificity of transport may be related to differences in the carbohydrate composition, as shown in a number of other recognition systems 1. Alternatively, it may be relevant that proteins with net positive charges (such as the B and C isoenzymes) are more readily phagocytosed by a variety of cell types than are proteins with net negative charges (sttch as the A isoenzyme) I~. These findings are relevant to the problem of iontophoresis of HRP. Since over 80 ~ of the enzyme activity of type VI H R P is due to the basic isoenzyme C, we suggest that its iontophoretic migration will be maximized by use of a buffer with pH lower than 8.6 (ref. 7). Also, the differences in charge of the A vs. B and C isoenzymes, and the lack of retrograde transport of the A, must be considered when attempting to inject H R P iontophoretically, and experiments are underway to examine this problem further 10. This work was supported by U SPH S Research Grants EY01311, GM 15991 and EY01086, and by a grant from Fight For Sight, Inc., New York City (to A.H.Bj. The authors are grateful to Ms. I. Klock and Mr. T. Ahern for technical assistance and to Ms. E. Patton for secretarial help.
1 ASHWELL, G., AND MORELL, A. G., The role of surface carbohydrates in the hepatic recognition and transport of circulatory glycoproteins. In A. MASTER(Ed.), Advances in Enzymology, VoL 41,
Wiley, New York, 1974, pp. 99-128. 2 BUNT,A. H., ANDLUND,R. D., unpublished observations. 3 BUNT,A. H., LUND,R. D., ANDLUND,J. S., Retrograde axonal transport of horseradish peroxidase by ganglion cells of the albino rat retina, Brain Research, 73 (1974) 215-228. 4 DAVIS,B. J., Disc electrophoresis - - 1I. Method and application to human serum proteins, Ann. N. Y. Acad. Sci., 121 (1964) 404-427. 5 ELLtSON,J. P., AND CLARK,G. M., Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves, J. comp. NeuroL, 161 (1975) 103-114. 6 GRAHAM,R. J., JR., ANDKARNOVSKY,M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 7 GRAYBIEL,A. M., ANDDEVOR,M., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 8 HANSSON,H. A., Uptake and intracellular bidirectional transport of horseradish peroxidase in retinal ganglion cells, Exp. Eye Res., 16 (1973) 377-388. 9 HASCHKE,R. H., unpublished observations. 10 HENRY, G., AND ANDERSON, M., personal communication. 11 KRISTENSSON,K., OLSSON,Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 12 LAVAIL, J. H., The retrograde transport method, Fed. Proc., 34 (1975) 1618-1624.
155 13 LEE, Y. C., AND MONTGOMERY, R., The carbohydrate of ovalbumin, Arch. Biochem., 95 (1961) 263-270. 14 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 15 PAUL, K. G., AND STIGBAND,T., Four isoperoxidases from horseradish root, Acta chem. stand., 24 (1970) 3607-3617. 16 PRICE, O. L., GRIFFINS,J., YOUNG, A., PECK, K., AND STOCKS, A., Tetanus toxin: direct evidence for retrograde intraaxonal transport, Science, 188 (1975) 945-947. 17 REISFELD,R. A., LEWIS, V. J., AND WILLIAMS, O. E., Disk electrophoresis of basic proteins and peptides on polyacrylamide gels, Nature (Lond.), 195 (1962) 281-283. 18 RYSER, H. J.-P., Uptake of protein by mammalian cells. An underdeveloped area, Science, 159 (1968) 390-396. 19 SHANNON, L. M., KaY, E., AND LEW, J. Y., Peroxidase isozymes from horseradish roots. I. Isolation and physical properties, J. biol. Chem., 241 (1966) 2166-2172. 20 SHIH, J. H. C., SHANNON, L. M., KAY, E., AND LEW, J. Y., Peroxidase isoenzymes from horseradish roots. IV. Structural relationships, J. biol. Chem., 246 (1971) 4546-4551. 21 SPIRO, R. G., Characterization of carbohydrate units of glycoproteins. In E. F. NEUFELD AND V. GINSBERG (Eds.), Methods in Enzymology, Academic Press, New York, 1966, pp. 4447. 22 STGCKEL,K., PARAVICINI,U., AND THOENEN, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413421. 23 WEINRVB, J., The oxidation of horseradish peroxidase by periodate, Biochem. biophys. Res. Commun., 31 (1968) 110-112. 24 WELINDER,K. G., SMILLIE,L. B., AND SCHONBAUM,G. R., Amino acid sequence studies of horseradish peroxidase. I. Tryptic peptides, Canad. J. Biochem., 50 (1971) 44-62.
Brain Research. 102 (1976} 152 155 i' Elsevier Scientific Publishing (~ompany, Amsterdam Printed in The Nethcrland~
Short Communications
Factors affecting retrograde axonal transport of horseradish peroxidase in the visual system ANN H. BUNT, RICHARD H. HASCHKE, RAYMOND D. LUND AND DIANNE F. CALKINS Departments of Ophthalmology, Anesthesiology and Biological Structure, University of Washington, Seattle, Wash. 98195 (U.S.A.)
(Accepted October 8th, 1975)
Horseradish peroxidase (HRP) has proven valuable for demonstrating uptake and retrograde axonal transport of exogenous protein by neurons, and this property has been used recently as an experimental tool in hodological neuronography~L Other exogenous proteins have also been demonstrated to move retrogradely in axons ~].t6,22 and the transport of nerve growth factor (NGF) is of particular interest because it has enabled study of the specificity and biological significance o f this process. Thus, whereas 125I-labeled N G F was taken up and transported retrogradely by sympathetic axons, several other proteins and aged or oxidized N G F which had lost biological activity were transported poorly 22. Retrograde transport of the glycoprotein HRP, while demonstrated in a number of neuronal systems, also shows specificity. Nauta et al. 14 found that neurons of the neocortex did not transport H R P as expected after its injection into caudoputamen, and St6ckel et al. 2~ also found lack of transport of HRP in sympathetic axons (but see refs. 5 and 8). Preliminary experimentsZ have indicated a lack of retrograde transport of a number of other proteins (including bovine serum albumin, alkaline phosphatase, hemocyanin, and lactoperoxidase) in the axons of rat optic nerve, which do transport HRP. This suggested a specificity of retrograde transport in axons of the visual system, but since these proteins differ considerably in physical and chemical properties, little could be deduced about the factors responsible for specificity of uptake and/or transport in these axons. It appeared more appropriate to test which features of the HRP molecule might be related to this specificity, so that this information might provide a general understanding of the characteristics of proteins normally transported retrogradely. The visual cortex or superior colliculus of a total of 18 adult albino rats was injected with 1--5 ,ul of a 1 0 - 3 0 ~ saline solution of the test protein. After 24 h, the animals were perfused with 4 ~ paraformaldehyde in 0.1 M phosphate buffer and the retinae and brains processed cytochemically6 with appropriate controts a. The test proteins were as follows.
153 (1) HRP (Sigma, type VI). It was determined that greater than 8 0 ~ of the enzymatic activity of this product was due to isoenzyme C (ref. 9). (2) Periodate-oxidized-borohydride-reduced HRP isoenzyme C (ref. 21). (3) HRP isoenzymes A, B and C. HRP was purified from fresh horseradish roots 2° through ion-exchange columns (CM-52 for the B and C isoenzymes and DE52 for the A isoenzyme). Analytical polyacrylamide gel electrophoresis at pH 4.5 and pH 8.9 (see refs. 4 and 17) indicated that the A, B and C isoenzymes were not intercontaminated. The reaction mixture for the spectrophotometric assay contained 2.5 ml of 20 m M sodium phosphate buffer pH 6.0; 0.2 ml 150 m M H202; 0.3 ml 5 ~ pyrogallol and appropriately diluted enzyme (1 /~g). The oxidation of pyrogallol to purpurogallin was followed at 420 nm (activity unit: micromoles purpurogallin formed per minute). The specific activities of the isoenzymes used in the injections into the brain were: A, 1372; B, 1409 and C, 2423 U/rag. Following periodate oxidation and borohydride reduction 21 the specific activity of the C isoenzyme was 635 U/rag. Following injection of HRP type VI or isoenzymes B or C into the visual cortex, peroxidase granules were readily demonstrated in neurons of the ipsilateral dorsal lateral geniculate nucleus (dLGN) and also cortical neurons adjacent to the injection. Similarly, after injections into the superior colliculus, ganglion cell somata of the contralateral retina contained the tracer granules. When the injected HRP was restricted to one side of the visual cortex or tectum, labeled neurons were not found in that d L G N or retina projecting to the uninjected side, indicating that the HRP-positive granules did not result from blood-borne label 3. No retrograde transport of HRP isoenzyme C which had been periodate-oxidized and borohydride-reduced or ofisoenzyme A was found in neurons of the d L G N or retina. Filled profiles of axons and cells were visible within the injection site, and the cytoplasm of some of these neurons appeared somewhat granulated, but no neurons could be found in the tissue beyond the zone of diffusion of HRP which contained peroxidase granules. The extent of diffusion from the injection sites of the periodateborohydride-treated isoenzyme C and the A and B isoenzymes was noticeably diminished, as compared with injections of equal volumes of isoenzyme C. This may reflect the lower specific activities of these proteins (see above), although the centralmost parts of the injection sites were equally well stained and the isoenzyme B did give rise to well-labeled neurons. The results suggest a specificity of retrograde transport, involving the uptake and/or actual transport process, in axons of the rat visual system. Whereas transport of HRP type VI and isoenzymes B and C was readily detected, the periodate-borohydride-treated isozyme C and isozyme A either did not enter cells or were not transported, as detected histochemically. The preliminary findings suggest that the carbohydrate moiety, which is altered by the periodate-borohydride treatment, may be involved in the specificity. The specific activity of the enzyme was reduced after this procedure, suggesting that other groups may have also been altered13, 23, so that the direct involvement of the carbohydrate remains to be demonstrated. In attempting to determine the biochemical basis for the difference in retrograde transport between the A compared with the B and C isoenzymes, it should be noted
154 that these differ from each other significantly in at least two aspects. A substanlially different amino acid composition is found for the A isoenzyme (isoelectric point pH 4.5) as compared with the B and C isoenzymes (isoelectric point pH 9.0)15,2L Each isoenzyme contains approximately 19 ;",i carbohydrate by weight, but the A isoenzyme differs tYom B and C in carbohydrate composition, containing galactose, arabinose and mannose as neutral sugars and the amino sugar mannosamine; the B and ( i s o enzymes contain xylose, fucose and mannose as neutral sugars and the amino sugars mannosamine and galactosamine t'j. These differences suggest that the specificity of transport may be related to differences in the carbohydrate composition, as shown in a number of other recognition systems 1. Alternatively, it may be relevant that proteins with net positive charges (such as the B and C isoenzymes) are more readily phagocytosed by a variety of cell types than are proteins with net negative charges (sttch as the A isoenzyme) I~. These findings are relevant to the problem of iontophoresis of HRP. Since over 80 ~ of the enzyme activity of type VI H R P is due to the basic isoenzyme C, we suggest that its iontophoretic migration will be maximized by use of a buffer with pH lower than 8.6 (ref. 7). Also, the differences in charge of the A vs. B and C isoenzymes, and the lack of retrograde transport of the A, must be considered when attempting to inject H R P iontophoretically, and experiments are underway to examine this problem further 10. This work was supported by U SPH S Research Grants EY01311, GM 15991 and EY01086, and by a grant from Fight For Sight, Inc., New York City (to A.H.Bj. The authors are grateful to Ms. I. Klock and Mr. T. Ahern for technical assistance and to Ms. E. Patton for secretarial help.
1 ASHWELL, G., AND MORELL, A. G., The role of surface carbohydrates in the hepatic recognition and transport of circulatory glycoproteins. In A. MASTER(Ed.), Advances in Enzymology, VoL 41,
Wiley, New York, 1974, pp. 99-128. 2 BUNT,A. H., ANDLUND,R. D., unpublished observations. 3 BUNT,A. H., LUND,R. D., ANDLUND,J. S., Retrograde axonal transport of horseradish peroxidase by ganglion cells of the albino rat retina, Brain Research, 73 (1974) 215-228. 4 DAVIS,B. J., Disc electrophoresis - - 1I. Method and application to human serum proteins, Ann. N. Y. Acad. Sci., 121 (1964) 404-427. 5 ELLtSON,J. P., AND CLARK,G. M., Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves, J. comp. NeuroL, 161 (1975) 103-114. 6 GRAHAM,R. J., JR., ANDKARNOVSKY,M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 7 GRAYBIEL,A. M., ANDDEVOR,M., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 8 HANSSON,H. A., Uptake and intracellular bidirectional transport of horseradish peroxidase in retinal ganglion cells, Exp. Eye Res., 16 (1973) 377-388. 9 HASCHKE,R. H., unpublished observations. 10 HENRY, G., AND ANDERSON, M., personal communication. 11 KRISTENSSON,K., OLSSON,Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 12 LAVAIL, J. H., The retrograde transport method, Fed. Proc., 34 (1975) 1618-1624.
155 13 LEE, Y. C., AND MONTGOMERY, R., The carbohydrate of ovalbumin, Arch. Biochem., 95 (1961) 263-270. 14 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 15 PAUL, K. G., AND STIGBAND,T., Four isoperoxidases from horseradish root, Acta chem. stand., 24 (1970) 3607-3617. 16 PRICE, O. L., GRIFFINS,J., YOUNG, A., PECK, K., AND STOCKS, A., Tetanus toxin: direct evidence for retrograde intraaxonal transport, Science, 188 (1975) 945-947. 17 REISFELD,R. A., LEWIS, V. J., AND WILLIAMS, O. E., Disk electrophoresis of basic proteins and peptides on polyacrylamide gels, Nature (Lond.), 195 (1962) 281-283. 18 RYSER, H. J.-P., Uptake of protein by mammalian cells. An underdeveloped area, Science, 159 (1968) 390-396. 19 SHANNON, L. M., KaY, E., AND LEW, J. Y., Peroxidase isozymes from horseradish roots. I. Isolation and physical properties, J. biol. Chem., 241 (1966) 2166-2172. 20 SHIH, J. H. C., SHANNON, L. M., KAY, E., AND LEW, J. Y., Peroxidase isoenzymes from horseradish roots. IV. Structural relationships, J. biol. Chem., 246 (1971) 4546-4551. 21 SPIRO, R. G., Characterization of carbohydrate units of glycoproteins. In E. F. NEUFELD AND V. GINSBERG (Eds.), Methods in Enzymology, Academic Press, New York, 1966, pp. 4447. 22 STGCKEL,K., PARAVICINI,U., AND THOENEN, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413421. 23 WEINRVB, J., The oxidation of horseradish peroxidase by periodate, Biochem. biophys. Res. Commun., 31 (1968) 110-112. 24 WELINDER,K. G., SMILLIE,L. B., AND SCHONBAUM,G. R., Amino acid sequence studies of horseradish peroxidase. I. Tryptic peptides, Canad. J. Biochem., 50 (1971) 44-62.
