- Email: [email protected]
Immunocytochemical localization of nervous system specific protein (NSP-R) in rat brain
184
Brain Research, 105 (1976) 184-187 © Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherlands
Immunocytochemical localization of nervous system specific protein (NSP-R) in rat brain
v. M. PICKEL, D. J. REIS, P. J. MARANGOS AND C. ZOMZELY-NEURATH Laboratory of Neurobiology, Department of Neurology, Cornell University Medical Center, New York, N. Y. and (P.J.M., and C.Z.-N.) Roche Institute for Molecular Biology, Nutley, N.J. (U.S.A.)
(Accepted December 9th, 1975)
A soluble acidic protein, specific to the nervous system, has recently been purified from rat brain 6. This protein, designated nervous system specific protein of rat (NSP-R) is antigenically similar 7 to the 14-3-2 protein isolated from bovine brain by Moore 9. Bennett and Edelman reported the isolation of antigen a (ref. 2) which was subsequently shown to be antigenically similar to the 14-3-2 protein 1. Thus, these three proteins represent closely related if not homologous molecules. The reported molecular weight of 14-3-2 protein 10 differs from that of NSP-R and they can be separated on Tris-glycine gels at pH 8.96. Although the subunit molecular weights of antigen a and NSP-R are similar, native NSP-R is homogeneous as determined by equilibrium centrifugation 6. By contrast, native antigen a appears to be heterogeneous as reported by Bennett and Edelman 2. At the present time, it is not possible tc state unequivocally that the three proteins are identical. Therefore, we are calling the protein isolated by our group as NSP-R instead of 14-3-2 (ref. 6). In the present study we have sought to directly establish the cellular localization of NSP-R in rat brain by immunocytochemistry. The study has been facilitated by the development of specific antibody to NSP-R 7. Evidence for the neuronal localization of this protein will be demonstrated. NSP-R was purified from rat brain by DEAE-cellulose and Sephadex chromatography in conjunction with isoelectric focusing 6. Antiserum to purified NSP-R was produced in New Zealand white rabbits by methods previously described 7. The antibody was adjudged specific for NSP-R by Ouchterlony double diffusion analysis and immunoelectrophoresis which yielded a single precipitin band when the antibody was reacted against purified or crude preparation of NSP-R and no reaction against another brain-specific protein such as S-1009. NSP-R antibody was localized to sections of rat brain by our modification 11 of the peroxidase-antiperoxidase (PAP) method12,13. Briefly, male Sprague-Dawley rats were anesthetized and killed by perfusion with formalin (4°/paraformaldehyde in 0.1 M phosphate buffer, pH 7.2) for 15 min. The brains were removed, fixed for 6 h in picric acid-formalin, washed overnight in phosphate-buffered saline, and embedded in paraffin. Sections 5/tm in thickness were sequentially incubated in 30 min steps with
185
Fig. 1. Immunocytochemical localization of NSP-R in neurons of the reticular formation of the medulla of the rat stained by the peroxidase-antiperoxidase method. A and B: phase contrast photomicrograph of neurons (arrows with darkly stained reaction product in the cytoplasm and processes). Other unlabeled neurons (N) are present in the same brain stem area. x 250. C: phase contrast photomicrograph in which control, blocked antiserum, replaced the NSP-R antiserum. Unstained neuron (N). x 250.
186 rabbit antisera to NSP-R, goat antirabbit IgG (Miels Laboratories) and PAP. Each step was followed by two (8 min) washes in Tris-saline. The bound peroxidase was reacted for l0 rain with 0.05 ~ 3,3'-diaminobenzidine (Sigma Chemical Company) and 0.01 ~ hydrogen peroxide in Tris buffer (pH 7.6) to form a characteristic brown reaction product 12,13. After a 15 min wash in distilled water, the sections were dehydrated and mounted in Permount. As controls for non-specific staining, adjacent tissue sections were stained by the procedures described except for the substitution of antibody-free serum for the NSP-R specific antiserum. The antibody-free serum was either: (i) pre-immune serum, i.e., serum collected from the rabbits before immunization with NSP-R, or (ii) blocked antisera, i.e., antisera in which the specific antibodies to NSP-R were removed by precipitation with amounts of purified protein sufficient to remove reactivity in immunoelectrophoresis and double diffusion experiments. Examination of sections of rat brain stem (pons and medulla) demonstrated the presence of brown reaction product within the cytoplasm of numerous neurons and their processes (Fig. 1). Glia were never stained. The perikarya of the labeled neurons were of various sizes and were not contained in any particular brain stem area. Their distribution did not correspond to that of the monoamine-containing nerve cells3. Not all neurons were stained and often stained cells were situated adjacent to unlabeled ones (Fig. 1A). The peroxidase labeled material extended from the perikarya into the axons and dendrites of labeled neurons. Numerous stained processes were seen through the neuropil (Fig. 1A and B). In sections incubated with control serum no specific staining was observed (Fig. IC). Thus, the present study demonstrates unequivocally that the nervous system specific protein of rat, NSP-R, is exclusively neuronal. The cytoplasmic localization of NSP-R in the perikarya and processes of selective neurons is consistent with the view that this protein, like other macromolecules, is synthesized in the perikarya and transported into axons and dendrites. The axoplasmic transport of NSP-R is supported by transport studies in the optic nerve 8. The immunocytochemical localization of NSP-R within processes of specific neurons and the transport from perikarya into axons are commonly observed for neuronal specific proteins used in the synthesis of neurotransmitters, as for example, the monoamine synthesizing enzymes tyrosine hydroxylase, dopamine-fl-hydroxylase, DOPA decarboxylase and tryptophan hydroxylase4,11,14. Preliminary studies utilizing neurotropic agents (in preparation) suggest a role for NSP-R in neurotransmitter function. However, there is no evidence, at the present time, for the participation of NSP-R in the synthesis of any neurotransmitter. The definitive localization of the NSP-R protein in the perikarya and processes of specific brain stem neurons should aid in the interpretation of various biochemical, neurophysiological and pharmacological studies being carried out at the present time to further elucidate the function of NSP-R. We thank Ms. Nancy Shieh for excellent technical assistance. This research was supported by NIH Grants MH 24285 and NS 06911.
187 1 BENNETT,G. S., Immunologic and electrophoretic identity between nervous system-specific proteins antigen alpha and 14-3-2, Brain Research, 68 (1974) 365-369. 2 BENNETT, G. S., AND EDELMAN, G. M., Isolation of an acidic protein from rat brain, J. biol. Chem., 243 (1968) 6234-6241. 3 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 64, Suppl. 247 (1964) 37. 4 GOLDSTEIN,i . s FUXE, K., AND HOKFELT,T., Characterization and tissue localization of catecholamine synthesizing enzymes, Pharmacol. Rev., 24 (1972) 293-310. 5 JOH, T. H., SHIKIMI,T., PICKEL,V. M., AND REIS, n . J., Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain, Proe. nat. Acad. Sci. (Wash.), in press. 6 MARANGOS,P. J., ZOMZELY-NEORATH,C., LUK, D. C. i . , AND YORK, C., Isolation and characterization of the nervous system specific protein 14-3-2 from rat brain. Purification, subunit composition and comparison to the beef brain protein, J. biol. Chem., 250 (1975) 1884-1891. 7 MARANGOS, P. J., ZOMZELY-NEURATH,C., AND YORK, C., Immunological studies of a nerve specific protein, Arch. Biochem., 170 (1975) 289-293. 8 MARANGOS,P. J., ZOMZELY-NEURATH,C., YORK, C., AND BONDY, S. C., Axoplasmic transport of a brain specific soluble protein, Biochim. Biophys. Acta (Amst.), 392 (1975) 75-81. 9 MOORE, B. W., Chemistry and biology of two proteins, S-100 and 14-3-2, specific to the nervous system, Int. Rev. Neurobiol., 15 (1972) 215-225. 10 MOORE, B. W., Brain specific proteins. In D. J. SCHNEIDER(Ed.), Raven Press, New York, 1973, pp. 1-12. II PICKEL,V. M., JOH, T. H., FIELD, P. M., BECKER, C. G., AND REIS, n . J., Cellular localization of tyrosine hydroxylase by immunohistochemistry, J. Histochem. Cytochem., 23 (1975) 1-12. 12 STERNBERGER,L. A., In M. A. HAYATT(Ed.), Enzyme Immunocytochemistry, Electron Microscopy of Enzymes: Principles and Methods, Vol. I, Van Nostrand and Reinholt, New York, 1973, pp. 150-191. 13 STERNBERGER,L. A., HARDY, P. H., CUCULIS,J. J., AND MYER, H. G., The unlabeled antibody enzyme method by immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidtse-antip~roxidase) and its use in identification of spirochetes, J. Histoche*n. Cytschern., 18 (1970) 315-333. 14 THOENEN,H., OTTEN, U., AND OE'~CFI,F., Axoplasmic transport of enzymes involved in the synthesis of noradrenalia: relationship b,~tween the rat~ of transport and subcellular distribution, Brain Research, 62 (1973) 471475.
Brain Research, 105 (1976) 184-187 © Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherlands
Immunocytochemical localization of nervous system specific protein (NSP-R) in rat brain
v. M. PICKEL, D. J. REIS, P. J. MARANGOS AND C. ZOMZELY-NEURATH Laboratory of Neurobiology, Department of Neurology, Cornell University Medical Center, New York, N. Y. and (P.J.M., and C.Z.-N.) Roche Institute for Molecular Biology, Nutley, N.J. (U.S.A.)
(Accepted December 9th, 1975)
A soluble acidic protein, specific to the nervous system, has recently been purified from rat brain 6. This protein, designated nervous system specific protein of rat (NSP-R) is antigenically similar 7 to the 14-3-2 protein isolated from bovine brain by Moore 9. Bennett and Edelman reported the isolation of antigen a (ref. 2) which was subsequently shown to be antigenically similar to the 14-3-2 protein 1. Thus, these three proteins represent closely related if not homologous molecules. The reported molecular weight of 14-3-2 protein 10 differs from that of NSP-R and they can be separated on Tris-glycine gels at pH 8.96. Although the subunit molecular weights of antigen a and NSP-R are similar, native NSP-R is homogeneous as determined by equilibrium centrifugation 6. By contrast, native antigen a appears to be heterogeneous as reported by Bennett and Edelman 2. At the present time, it is not possible tc state unequivocally that the three proteins are identical. Therefore, we are calling the protein isolated by our group as NSP-R instead of 14-3-2 (ref. 6). In the present study we have sought to directly establish the cellular localization of NSP-R in rat brain by immunocytochemistry. The study has been facilitated by the development of specific antibody to NSP-R 7. Evidence for the neuronal localization of this protein will be demonstrated. NSP-R was purified from rat brain by DEAE-cellulose and Sephadex chromatography in conjunction with isoelectric focusing 6. Antiserum to purified NSP-R was produced in New Zealand white rabbits by methods previously described 7. The antibody was adjudged specific for NSP-R by Ouchterlony double diffusion analysis and immunoelectrophoresis which yielded a single precipitin band when the antibody was reacted against purified or crude preparation of NSP-R and no reaction against another brain-specific protein such as S-1009. NSP-R antibody was localized to sections of rat brain by our modification 11 of the peroxidase-antiperoxidase (PAP) method12,13. Briefly, male Sprague-Dawley rats were anesthetized and killed by perfusion with formalin (4°/paraformaldehyde in 0.1 M phosphate buffer, pH 7.2) for 15 min. The brains were removed, fixed for 6 h in picric acid-formalin, washed overnight in phosphate-buffered saline, and embedded in paraffin. Sections 5/tm in thickness were sequentially incubated in 30 min steps with
185
Fig. 1. Immunocytochemical localization of NSP-R in neurons of the reticular formation of the medulla of the rat stained by the peroxidase-antiperoxidase method. A and B: phase contrast photomicrograph of neurons (arrows with darkly stained reaction product in the cytoplasm and processes). Other unlabeled neurons (N) are present in the same brain stem area. x 250. C: phase contrast photomicrograph in which control, blocked antiserum, replaced the NSP-R antiserum. Unstained neuron (N). x 250.
186 rabbit antisera to NSP-R, goat antirabbit IgG (Miels Laboratories) and PAP. Each step was followed by two (8 min) washes in Tris-saline. The bound peroxidase was reacted for l0 rain with 0.05 ~ 3,3'-diaminobenzidine (Sigma Chemical Company) and 0.01 ~ hydrogen peroxide in Tris buffer (pH 7.6) to form a characteristic brown reaction product 12,13. After a 15 min wash in distilled water, the sections were dehydrated and mounted in Permount. As controls for non-specific staining, adjacent tissue sections were stained by the procedures described except for the substitution of antibody-free serum for the NSP-R specific antiserum. The antibody-free serum was either: (i) pre-immune serum, i.e., serum collected from the rabbits before immunization with NSP-R, or (ii) blocked antisera, i.e., antisera in which the specific antibodies to NSP-R were removed by precipitation with amounts of purified protein sufficient to remove reactivity in immunoelectrophoresis and double diffusion experiments. Examination of sections of rat brain stem (pons and medulla) demonstrated the presence of brown reaction product within the cytoplasm of numerous neurons and their processes (Fig. 1). Glia were never stained. The perikarya of the labeled neurons were of various sizes and were not contained in any particular brain stem area. Their distribution did not correspond to that of the monoamine-containing nerve cells3. Not all neurons were stained and often stained cells were situated adjacent to unlabeled ones (Fig. 1A). The peroxidase labeled material extended from the perikarya into the axons and dendrites of labeled neurons. Numerous stained processes were seen through the neuropil (Fig. 1A and B). In sections incubated with control serum no specific staining was observed (Fig. IC). Thus, the present study demonstrates unequivocally that the nervous system specific protein of rat, NSP-R, is exclusively neuronal. The cytoplasmic localization of NSP-R in the perikarya and processes of selective neurons is consistent with the view that this protein, like other macromolecules, is synthesized in the perikarya and transported into axons and dendrites. The axoplasmic transport of NSP-R is supported by transport studies in the optic nerve 8. The immunocytochemical localization of NSP-R within processes of specific neurons and the transport from perikarya into axons are commonly observed for neuronal specific proteins used in the synthesis of neurotransmitters, as for example, the monoamine synthesizing enzymes tyrosine hydroxylase, dopamine-fl-hydroxylase, DOPA decarboxylase and tryptophan hydroxylase4,11,14. Preliminary studies utilizing neurotropic agents (in preparation) suggest a role for NSP-R in neurotransmitter function. However, there is no evidence, at the present time, for the participation of NSP-R in the synthesis of any neurotransmitter. The definitive localization of the NSP-R protein in the perikarya and processes of specific brain stem neurons should aid in the interpretation of various biochemical, neurophysiological and pharmacological studies being carried out at the present time to further elucidate the function of NSP-R. We thank Ms. Nancy Shieh for excellent technical assistance. This research was supported by NIH Grants MH 24285 and NS 06911.
187 1 BENNETT,G. S., Immunologic and electrophoretic identity between nervous system-specific proteins antigen alpha and 14-3-2, Brain Research, 68 (1974) 365-369. 2 BENNETT, G. S., AND EDELMAN, G. M., Isolation of an acidic protein from rat brain, J. biol. Chem., 243 (1968) 6234-6241. 3 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 64, Suppl. 247 (1964) 37. 4 GOLDSTEIN,i . s FUXE, K., AND HOKFELT,T., Characterization and tissue localization of catecholamine synthesizing enzymes, Pharmacol. Rev., 24 (1972) 293-310. 5 JOH, T. H., SHIKIMI,T., PICKEL,V. M., AND REIS, n . J., Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain, Proe. nat. Acad. Sci. (Wash.), in press. 6 MARANGOS,P. J., ZOMZELY-NEORATH,C., LUK, D. C. i . , AND YORK, C., Isolation and characterization of the nervous system specific protein 14-3-2 from rat brain. Purification, subunit composition and comparison to the beef brain protein, J. biol. Chem., 250 (1975) 1884-1891. 7 MARANGOS, P. J., ZOMZELY-NEURATH,C., AND YORK, C., Immunological studies of a nerve specific protein, Arch. Biochem., 170 (1975) 289-293. 8 MARANGOS,P. J., ZOMZELY-NEURATH,C., YORK, C., AND BONDY, S. C., Axoplasmic transport of a brain specific soluble protein, Biochim. Biophys. Acta (Amst.), 392 (1975) 75-81. 9 MOORE, B. W., Chemistry and biology of two proteins, S-100 and 14-3-2, specific to the nervous system, Int. Rev. Neurobiol., 15 (1972) 215-225. 10 MOORE, B. W., Brain specific proteins. In D. J. SCHNEIDER(Ed.), Raven Press, New York, 1973, pp. 1-12. II PICKEL,V. M., JOH, T. H., FIELD, P. M., BECKER, C. G., AND REIS, n . J., Cellular localization of tyrosine hydroxylase by immunohistochemistry, J. Histochem. Cytochem., 23 (1975) 1-12. 12 STERNBERGER,L. A., In M. A. HAYATT(Ed.), Enzyme Immunocytochemistry, Electron Microscopy of Enzymes: Principles and Methods, Vol. I, Van Nostrand and Reinholt, New York, 1973, pp. 150-191. 13 STERNBERGER,L. A., HARDY, P. H., CUCULIS,J. J., AND MYER, H. G., The unlabeled antibody enzyme method by immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidtse-antip~roxidase) and its use in identification of spirochetes, J. Histoche*n. Cytschern., 18 (1970) 315-333. 14 THOENEN,H., OTTEN, U., AND OE'~CFI,F., Axoplasmic transport of enzymes involved in the synthesis of noradrenalia: relationship b,~tween the rat~ of transport and subcellular distribution, Brain Research, 62 (1973) 471475.