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Biochemical correlates of astrocytic proliferation in the mutant Staggerer mouse
Brain Research, 128 (1977) 389-392
389
© Elsevier/North-Holland Biomedical Press
Biochemical correlates of astrocytic proliferation in the mutant Staggerer mouse
VIRGINIA LEE, S.-H. YEN and MICHAEL L. SHELANSKI Department of Neuropathology, Harvard Medical School, and Department of Neurosciences, Mental Retardation Research Center, Children's Hospital Medical Center, Boston, Mass. 02115 (U.S.A.)
(Accepted February 23rd, 1977)
The cerebellum of the mouse is an attractive system for the study of neural development due to the monotony of its architecture 8, the synchrony of its postnatal development 1° and the existence of mutations causing specific deficits in this developmentlL These features allow us to attempt to dissect the biochemical events in development by a comparison of normal and mutant animals. In the study described herein we have compared the electrophoretic patterns of the proteins in the particulate fraction of homogenates of cerebella of mutant Staggerer mice (sg/sg) 12 and their normal ( ÷ / 6 - ) l ittermates. The Staggerer mutation was placed along with marker genes (dilute and short ear) onto the C57BL/6J background. Homozygous sg/sg aninmls were obtained by intercrossing the heterozygous sg/6- mice and distinguished from heterozygous littermates on the basis of a syndrome featuring hypotaxia and ataxia and a small cerebellum deficient in granule cell neuronslL The 6-/6- control littermates can be easily distinguished at postnatal day 4 (P4) by the markers dilute and short ear. Except for the developmental study, animals were sacrificed at P21. Subcellular fractionation was carried out as described by Mallet et al. 6 with minor modifications. The cerebella were homogenized by hand in a teflon-glass potter homogenizer with 20 up and down strokes. After centrifugation in a Beckman 152 microfuge for 10 min (approx. 10,000 × g) the supernatant was removed, the pellet washed once in the homogenization buffer, solubilized in 10 % sodium lauryl sulfate containing 1% fl-mercaptoethanol and boiled for 3 min. The samples, which contained 120 #g of total protein were run on the polyacrylamide gel electrophoresis system of Studied 4, as modified by Ames ~ and Vaughan and FliesterlL The 9 % separation gels were pre-run for 16 h at 40 V prior to pouring the 5 % stacking gel. Comparison of the protein electrophoretic patterns of cerebella of P21 6-/6- (n) and sg/sg (sg) littermates (Fig. I, extreme left) shows the presence of a band with an apparent molecular weight of 51,000 daltons in the Staggerer which is faint in the normal. When this band is followed as a function of development from P4 to P21, it is found to be present, though faint, in both normal and Staggerer animals from P4 through P10. At P12-P13 one begins to see an accentuation of this band in the Staggerer (Fig. 1) which increases until P21, after which its level appears unchanged.
390
]Q
........
17
21. . . .
Fig. 1. Comparative electrophoretic protein patterns of the SDS-solubilized pellets of normal (n) and Staggerer (sg) cerebella on 9 ~ NaDodSO4 polyacrylamide gels as a function of development. SgA signifies the 51,000 dalton component in question.
The possibility that this difference is simply the accentuation of a normal constituent caused by the loss of the proteins normally contributed by the granule cells is not likely since similar experiments on the Weaver mutant which also lacks granule cells at P21 do not show this change. It is also unlikely to be a result of the dendritic abnormalities 13 in Staggerer since the Purkinje cell degeneration mutant (pcd) 7 treated in the same way at PI5, P21 and P31 does not show this difference. Examination of the supernatants showed the presence of a faint band with the same molecular weight in both normal and Staggerer which does not vary significantly as a function of age. The molecular weight of this protein suggested to us that it might be related to the brain intermediate filament2,9,16, which is the presumptive subunit of the 9 nm filaments seen in neurons and glia 11. Molecular weight comparisons of brain filament protein (BF) and the anomalous band from the Staggerer mutant (sgA) in a number of gel systems showed that the sgA band always migrated with or just fractionally ahead
391
Fig. 2. Ouchterlony plate showing cross-reaction between antiserum against brain filament (3), brain filament protein (1) and sgA protein (2). Coomassie blue stain.
of the BF. Moreover, double immunodiffusion studies using bovine anti-BF showed immunological lines of identity between BF 19 and the sgA band from P12 on (Fig. 2). Radioimmunoassays indicate that the failure to see a precipitin line prior to P12 in the Staggerer and at any age in the normal is due to low protein concentrations, since BFlike activity can be detected both in extracts and in the eluted bands at all ages in both q-/q- and sg/sg, but the marked increase in seen only in Staggerer. The bands of identical molecular weight in the soluble fraction also react against anti-BF in the radioimmunoassay. These data strongly suggest an increased accumulation of a brain-filament-like protein in the Staggerer mutant. However, a preliminary electron microscopic study of Staggerer cerebella at P4, 5, 15 and 16 failed to show any increase in the number of neurofilaments in axons or neuronal cell bodies. There is, as had previously been reported 3,4, a massive overgrowth of fibrous astroglia whose processes are filled with 9 nm filaments. The glia response in Staggerer has not been recorded in detail during the developmental period when the sgA band increases, but the electron microscopic study by Landis and Sidman 4 contains relevant data. They found that parallel fiber degeneration had begun by P7 and that by P14 granule cell degeneration was sufficiently advanced that a reduction of thickness of molecular and granule cell layers was recognizable. The maximum rate of degeneration occurred at postnatal days 16-21. In the molecular layer, the rapid loss of parallel fibers during this period was accompanied by an abrupt increase in the number of filament-packed astrocytic processes. The failure of a previous study 5 to detect this change is most likely due to a greater resolution of our electrophoretic system in the 50,000 dalton region. These data strongly suggest that the increase in the sgA protein is due to the increase of glial cell intermediate filaments and that it is therefore a reactive change and not a direct result of the genetic mutation in this animal. This work was supported by USPHS Grant NS-11,504 and Grant 1-478 from the National Foundation, March of Dimes.
392 1 Ames, G. F. L., Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs, J. biol. Chem., 249 (1974) 634-644. 2 Davidson, P. F. and Winslow, B., The protein subunit of calf brain neurofilament, J. NeurobioL, 5 (1974) 119-133. 3 Hirano, A. and Dembitzer, H. M., The fine structure ofastrocytes in the adult Staggerer, J. Neuropath. exp. Neurol., 35 (1976) 63-74. 4 Landis, D. M. D. and Sidman, R. L., Electron microscope analyses of postnatal histogenesis in the cerebellar cortex of Staggerer mutant mice, In preparation. 5 Mallet, J., Huchet, M., Pougeois, R. and Changeux, J. P., Anatomical, physiological and biochemical studies on the cerebellum from mutant mice. Ili. Protein differences associated with the Weaver, Staggerer and nervous mutations, Brain Research, 103 (1976) 291-312. 6 Mallet, J., Huchet, M., Shelanski, M. L. and Changeux, J. P., Protein differences associated with the absence of granule cells in the cerebella from the mutant Weaver mouse and from X-irradiated rat, FEBS Lett., 46 (1974) 243-246. 7 Mullen, R. J., Eicker, E. M. and Sidman, R. L., Purkinje cell degeneration, a new neurological mutation in the mouse, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 529-533. 8 Palay, S. L. and Chan-Palay, V., Cerebellar Cortex Cytology and Organization, 1974, pp. 5-10. 9 Shelanski, M. L., Albert, S., De Vries, G. H. and Norton, W. T., Isolation of filaments from brain, Science, 174 (1971) 1241. 10 Sidman, R. L., Cell-cell recognition in the developing central nervous system. In F. O. Schmitt and F. G. Worden (Eds.), The Neuroscience Third Study Program, MIT Press, Cambridge, Mass., 1974, pp. 743-758. 11 Sidman, R. L., Greene, M. C. and Appel, S. H., Catalog of the Neurological Mutants of the Mouse, Harvard University Press, Cambridge, Mass., 1965. 12 Sidman, R. L., Lane, P. and Dickie, M., Staggerer, a new mutation in the mouse affecting the cerebellum, Science, 137 (1962) 610-612. 13 Sotelo, C., Dendritic abnormalities of Purkinje cells in the cerebellum of neurological mutant mice (Weaver and Staggerer). In Proceedings of the Symposium on the Physiology and Pathology of Dendrites, Raven Press, New York, 1975. 14 Studier, F. W., Analysis of bacteriophage T7 early RNA's and proteins on slab gels, J. molec. BioL, 79 (1973) 237-248. 15 Vaughan, W. J. and Fliester, S. J., Electrophoretic resolution and comparison of brain proteins, J. Neurochem., 23 (1974) 279-281. 16 Yen, S. H., Dahl, D., Schachner, M. and Shelanski, M. L., Biochemistry of the filaments of brain, Proc. nat. Acad. ScL(Wash.), 73 (1976) 529-533.
389
© Elsevier/North-Holland Biomedical Press
Biochemical correlates of astrocytic proliferation in the mutant Staggerer mouse
VIRGINIA LEE, S.-H. YEN and MICHAEL L. SHELANSKI Department of Neuropathology, Harvard Medical School, and Department of Neurosciences, Mental Retardation Research Center, Children's Hospital Medical Center, Boston, Mass. 02115 (U.S.A.)
(Accepted February 23rd, 1977)
The cerebellum of the mouse is an attractive system for the study of neural development due to the monotony of its architecture 8, the synchrony of its postnatal development 1° and the existence of mutations causing specific deficits in this developmentlL These features allow us to attempt to dissect the biochemical events in development by a comparison of normal and mutant animals. In the study described herein we have compared the electrophoretic patterns of the proteins in the particulate fraction of homogenates of cerebella of mutant Staggerer mice (sg/sg) 12 and their normal ( ÷ / 6 - ) l ittermates. The Staggerer mutation was placed along with marker genes (dilute and short ear) onto the C57BL/6J background. Homozygous sg/sg aninmls were obtained by intercrossing the heterozygous sg/6- mice and distinguished from heterozygous littermates on the basis of a syndrome featuring hypotaxia and ataxia and a small cerebellum deficient in granule cell neuronslL The 6-/6- control littermates can be easily distinguished at postnatal day 4 (P4) by the markers dilute and short ear. Except for the developmental study, animals were sacrificed at P21. Subcellular fractionation was carried out as described by Mallet et al. 6 with minor modifications. The cerebella were homogenized by hand in a teflon-glass potter homogenizer with 20 up and down strokes. After centrifugation in a Beckman 152 microfuge for 10 min (approx. 10,000 × g) the supernatant was removed, the pellet washed once in the homogenization buffer, solubilized in 10 % sodium lauryl sulfate containing 1% fl-mercaptoethanol and boiled for 3 min. The samples, which contained 120 #g of total protein were run on the polyacrylamide gel electrophoresis system of Studied 4, as modified by Ames ~ and Vaughan and FliesterlL The 9 % separation gels were pre-run for 16 h at 40 V prior to pouring the 5 % stacking gel. Comparison of the protein electrophoretic patterns of cerebella of P21 6-/6- (n) and sg/sg (sg) littermates (Fig. I, extreme left) shows the presence of a band with an apparent molecular weight of 51,000 daltons in the Staggerer which is faint in the normal. When this band is followed as a function of development from P4 to P21, it is found to be present, though faint, in both normal and Staggerer animals from P4 through P10. At P12-P13 one begins to see an accentuation of this band in the Staggerer (Fig. 1) which increases until P21, after which its level appears unchanged.
390
]Q
........
17
21. . . .
Fig. 1. Comparative electrophoretic protein patterns of the SDS-solubilized pellets of normal (n) and Staggerer (sg) cerebella on 9 ~ NaDodSO4 polyacrylamide gels as a function of development. SgA signifies the 51,000 dalton component in question.
The possibility that this difference is simply the accentuation of a normal constituent caused by the loss of the proteins normally contributed by the granule cells is not likely since similar experiments on the Weaver mutant which also lacks granule cells at P21 do not show this change. It is also unlikely to be a result of the dendritic abnormalities 13 in Staggerer since the Purkinje cell degeneration mutant (pcd) 7 treated in the same way at PI5, P21 and P31 does not show this difference. Examination of the supernatants showed the presence of a faint band with the same molecular weight in both normal and Staggerer which does not vary significantly as a function of age. The molecular weight of this protein suggested to us that it might be related to the brain intermediate filament2,9,16, which is the presumptive subunit of the 9 nm filaments seen in neurons and glia 11. Molecular weight comparisons of brain filament protein (BF) and the anomalous band from the Staggerer mutant (sgA) in a number of gel systems showed that the sgA band always migrated with or just fractionally ahead
391
Fig. 2. Ouchterlony plate showing cross-reaction between antiserum against brain filament (3), brain filament protein (1) and sgA protein (2). Coomassie blue stain.
of the BF. Moreover, double immunodiffusion studies using bovine anti-BF showed immunological lines of identity between BF 19 and the sgA band from P12 on (Fig. 2). Radioimmunoassays indicate that the failure to see a precipitin line prior to P12 in the Staggerer and at any age in the normal is due to low protein concentrations, since BFlike activity can be detected both in extracts and in the eluted bands at all ages in both q-/q- and sg/sg, but the marked increase in seen only in Staggerer. The bands of identical molecular weight in the soluble fraction also react against anti-BF in the radioimmunoassay. These data strongly suggest an increased accumulation of a brain-filament-like protein in the Staggerer mutant. However, a preliminary electron microscopic study of Staggerer cerebella at P4, 5, 15 and 16 failed to show any increase in the number of neurofilaments in axons or neuronal cell bodies. There is, as had previously been reported 3,4, a massive overgrowth of fibrous astroglia whose processes are filled with 9 nm filaments. The glia response in Staggerer has not been recorded in detail during the developmental period when the sgA band increases, but the electron microscopic study by Landis and Sidman 4 contains relevant data. They found that parallel fiber degeneration had begun by P7 and that by P14 granule cell degeneration was sufficiently advanced that a reduction of thickness of molecular and granule cell layers was recognizable. The maximum rate of degeneration occurred at postnatal days 16-21. In the molecular layer, the rapid loss of parallel fibers during this period was accompanied by an abrupt increase in the number of filament-packed astrocytic processes. The failure of a previous study 5 to detect this change is most likely due to a greater resolution of our electrophoretic system in the 50,000 dalton region. These data strongly suggest that the increase in the sgA protein is due to the increase of glial cell intermediate filaments and that it is therefore a reactive change and not a direct result of the genetic mutation in this animal. This work was supported by USPHS Grant NS-11,504 and Grant 1-478 from the National Foundation, March of Dimes.
392 1 Ames, G. F. L., Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs, J. biol. Chem., 249 (1974) 634-644. 2 Davidson, P. F. and Winslow, B., The protein subunit of calf brain neurofilament, J. NeurobioL, 5 (1974) 119-133. 3 Hirano, A. and Dembitzer, H. M., The fine structure ofastrocytes in the adult Staggerer, J. Neuropath. exp. Neurol., 35 (1976) 63-74. 4 Landis, D. M. D. and Sidman, R. L., Electron microscope analyses of postnatal histogenesis in the cerebellar cortex of Staggerer mutant mice, In preparation. 5 Mallet, J., Huchet, M., Pougeois, R. and Changeux, J. P., Anatomical, physiological and biochemical studies on the cerebellum from mutant mice. Ili. Protein differences associated with the Weaver, Staggerer and nervous mutations, Brain Research, 103 (1976) 291-312. 6 Mallet, J., Huchet, M., Shelanski, M. L. and Changeux, J. P., Protein differences associated with the absence of granule cells in the cerebella from the mutant Weaver mouse and from X-irradiated rat, FEBS Lett., 46 (1974) 243-246. 7 Mullen, R. J., Eicker, E. M. and Sidman, R. L., Purkinje cell degeneration, a new neurological mutation in the mouse, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 529-533. 8 Palay, S. L. and Chan-Palay, V., Cerebellar Cortex Cytology and Organization, 1974, pp. 5-10. 9 Shelanski, M. L., Albert, S., De Vries, G. H. and Norton, W. T., Isolation of filaments from brain, Science, 174 (1971) 1241. 10 Sidman, R. L., Cell-cell recognition in the developing central nervous system. In F. O. Schmitt and F. G. Worden (Eds.), The Neuroscience Third Study Program, MIT Press, Cambridge, Mass., 1974, pp. 743-758. 11 Sidman, R. L., Greene, M. C. and Appel, S. H., Catalog of the Neurological Mutants of the Mouse, Harvard University Press, Cambridge, Mass., 1965. 12 Sidman, R. L., Lane, P. and Dickie, M., Staggerer, a new mutation in the mouse affecting the cerebellum, Science, 137 (1962) 610-612. 13 Sotelo, C., Dendritic abnormalities of Purkinje cells in the cerebellum of neurological mutant mice (Weaver and Staggerer). In Proceedings of the Symposium on the Physiology and Pathology of Dendrites, Raven Press, New York, 1975. 14 Studier, F. W., Analysis of bacteriophage T7 early RNA's and proteins on slab gels, J. molec. BioL, 79 (1973) 237-248. 15 Vaughan, W. J. and Fliester, S. J., Electrophoretic resolution and comparison of brain proteins, J. Neurochem., 23 (1974) 279-281. 16 Yen, S. H., Dahl, D., Schachner, M. and Shelanski, M. L., Biochemistry of the filaments of brain, Proc. nat. Acad. ScL(Wash.), 73 (1976) 529-533.