Dev. Ncurosci. 2: 254 275 (1979)
Biochemical and Immunological Studies on the P400 Protein, a Protein Characteristic of the Purkinje Cell from Mouse and Rat Cerebellum Kaisuhiko Mikoshiba, Monique Ihu hei ami Jean-Pierre Changea* Neurobiologie moléculaire et Laboratoire associé Centre national tic la Recherche sciemiiiquc. Interactions moléculaires et cellulaires. Institut Pasteur, Paris
Key Words. P lon protein • Immunology • Neuronal-specific proteins • Cerebellum • Purkinje cell
Introduction Among the complex sequence of events which take place during the formation of a synaptic contact one may distinguish at least two major processes [7, 8): a ‘recognition'
between cell surfaces and the subsequent ‘selective stabilization' of the labile contacts thus formed. This raises two questions: (1)1 low specific is the early recognition step? Should it be viewed more as a compatibility and/or an adhesion [43. 44, 53) between cell
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:47:42 AM
Abstract. The P lmi protein is a glycoprotein of high apparent molecular weight which is abundant in isolated cerebellar Purkinje cells. On SOS polyacrylamide gels, the P p r o t e i n reacts with 14il plant lectins such a s J- '1 Con A. This reaction is used to increase the level of detec tion of the protein. The P,„„ protein is purified by successive extraction of synaptosomal and microsomal membranes with 2”., Triton X-100 and 25"., Na-cholate and preparative gel electro phoresis in SOS. The specific content of P4„„ protein decreases in the cerebella from homozyg ous nervous and Purkinje eell degeneration mutant mice, where the total number of Purkinje cells is markedly reduced, and increases in those of the reeler and weaver mice where a deficit of the granule cells exists. In the cerebellum from the homozygous staggerer mouse, a small amount of P,,m protein persists. During postnatal development the specific content of P,„„ protein per net weight does not change up to the 12th day after birth then increases up to the 25th day when it reaches the adult level. Antisera have been raised against the purified P„m protein. They give precipitation lines by the immunodiffusion reaction of Ouchtcrlonv against a preparation of P„mprotein submitted to mild proteolytic attack. Indirect immunofluorescence performed on slices of rat cerebellum with purified anti-P,,,,, immunoglobulin G. absorbed or not on rat cerebrum membranes, reveals that both the soma and the dendritic arborization of the Purkinje cells are labelled. The neurons from the deep cerebellar nuclei are not stained.
P,„„ Protein Characteristic of the Purkinje Cells from the Cerebellum
missing [33. 34]. The data reported in this paper confirm that the P p r o t e i n is indeed characteristic of the Purkinje cell. Moreover the P,„n protein i. localized in situ on both the soma and the dendrites of the Purkinje cell as revealed by using antibodies raised against the purified protein by an indirect immunofluorescence technique.
Materials and Methods Animals Mouse Neuropalhological Mutants Staggerer t sg) Mutant Manse [3.4. 10.11. 18, IV. 25.27.28. 51.54.02.04.65.741. A stock of mice hetero zygous for the staggerer (sg) mutation C57BL6 ( sg dse-l established by the Jackson Laboratory (Bar Harbor. Me.) was raised at the Pasteur Institute. Homozygous sg sg were obtained by intercrossing the heterozygous mice. The C57BI.O (dse rise I animals were used as controls. Servons i hi : Mutant Manse [1A 26.33. 34.51.60]. C3HcB .1 mice originating from the Jackson Labora tory and either heterozygous or homozygous for the nervous (nr) mutation were intercrossed to produce homo/; gous nr nraninials. Animals from the C3I IcB J strain were used as controls. H eater in ; Matant Manse |5 .4. 12. 27. 32, 46 4lJ, 51. 63 651. B6CBA Rl (wv i mice originating from the Jackson Laboratory and heterozygous for the weaver (wv) mutation were intercrossed to produce homozy gous w\ wv mice. The wild t> pe strain B6CBA RI ( ) was obtained by intercrossing animals which no longer gave the weaver phenotype in their progeny after several back crosses: with wv animals. Keeler rl) Mutant Manse 117. 35. 45. 5S|. The reeler mutation was maintained on inbred strains of C57BL6 mice from the Jackson Laboratory . Only the homozygous rl rl mice have been studied. Litters containing rl. rl individuals were obtained by inter crossing heterozygous (rl ) fertile animals. Purkinje Cell Degeneration />." Mann) and in the absence (indicated as Con A) of methyl a-£>-mannoside. PK = Purkinje cells; Gr = granule cells. Note that many bands arc not common between the two classes of cells - both after Coomassie bril liant blue staining and l21l-Con A binding.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:47:42 AM
Absorbance»
■Protein
result from the survival of a significant frac tion (about 15-20",,) of the Purkinje cells in the cerebellum from this mutant. To test this point gels were run with membrane prepara tions of cerebella from the Purkinje cell de generation (ped) mutant mouse in which only 5-IO"„ of the Purkinje cells persist in theadult. Figure 7 shows that the P40l) protein markedly decreases in the high and low speed membrane pellets from ped/ped cerebellum. In addition, only small changes are observed in wet weight and DNA-RNA content of the cerebellum as a consequence of the ped mutation (table II). The P.,00 protein is therefore characteristic of the Purkinje cells. In figure 8 a gel where crude membrane preparations from cerebella of various mouse mutants are compared is presented. The de tection of eventual differences between the protein patterns has been amplified by the 125I Con A autoradiographic method. In ad dition, the same absolute quantity of mem branes, in terms of total protein content, was applied to the gel to render a quantitative comparison possible. In agreement with pre vious results [34, 35], the relative content of the P.,un protein per mass of protein is signi-
264
Mikoshiba/Huchct/Changcux
6a Con A i-------------1
ficantly enhanced in the homozygous reeler and weaver cerebella where a marked deficit of the granule cell exists. The relative increases in P4no estimated by integration of the densitoinelric scans of the gels were, respectively, 22 and 42 of the control per mass of protein, whereas total P.,(l0 contents were 30 and 49% of the control per cerebellum. On the other hand, the sensitivity of the method becomes sufficient to demonstrate the presence of a significant amount of P4#0 pro tein in the cerebellum from the homozygous staggerer mouse where the most evident defect is a marked decrease in the number of parallel fiber-Purkinje cell synapses [58, 62, 67] ac companied. also, by a decrease of the number of Purkinje cells. The content in P400 protein is only 62"„ control on a protein basis but only 12% con trol per cerebellum if one takes into account the marked decrease in wet weight of the sg/sg cerebellum. Another interesting feature of the sg/sg gels, which becomes particularly evident on the densitometric scans, is that while the P40(, band decreases other bands significantly increase. These bands are de tected almost exclusively in the 1251 Con A treated gels and have an apparent molecular weight higher than that of the P4no protein. Their Rf ranges from 2/ 3 to 1/ 3 that of the P4U0
Fig. 6. Protein pattern of separated molecular (ML) and granular layers (GL) from bovine cere bellum. The separated molecular and granular layers were applied to the 5-15% linear polyacrylamide-gel electrophoresis in SDS. After electrophoresis, parallel gels were used for incubation with ,isI-Con A, while others were fixed and stained by Coomassie brilliant blue (protein).
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:47:42 AM
A b so rb an c e
6b
Protein i------------1
265
Table II. DNA and RNA content of the cerebel lum from Purkinje cell degeneration homozygous mutant mice and wild type mice Days
mg wet weight
ped/ 35 ped 35
33.25 34.25
+/+ 35 49.30 35 50.40
Mg DNA / mg wet weight 7.47 7.90
Mg DNA/ Cerebellum 248.4 270.4
Mg RNA 1 mg wet weight 4.19 4.23
Mg RNA / Cerebellum 139.3 144.9
5.64 6.13
278.1 309.0
3.99 4.51
196.7 227.3
The data for DNA and RNA represent the value for I animal from an average of 2-3 determinations.
Absorbance
7b
Fig. 7. Protein patterns of mem brane fractions from cerebella of homozygous Purkinje cell degenera tion (ped) and wild type (+/+) mice. LSP = low-speed pellet; MSP = high-speed pellet; P = Purkinje cell degeneration (ped); C = control wild type (
Biochemical and Immunological Studies on the P400 Protein, a Protein Characteristic of the Purkinje Cell from Mouse and Rat Cerebellum Kaisuhiko Mikoshiba, Monique Ihu hei ami Jean-Pierre Changea* Neurobiologie moléculaire et Laboratoire associé Centre national tic la Recherche sciemiiiquc. Interactions moléculaires et cellulaires. Institut Pasteur, Paris
Key Words. P lon protein • Immunology • Neuronal-specific proteins • Cerebellum • Purkinje cell
Introduction Among the complex sequence of events which take place during the formation of a synaptic contact one may distinguish at least two major processes [7, 8): a ‘recognition'
between cell surfaces and the subsequent ‘selective stabilization' of the labile contacts thus formed. This raises two questions: (1)1 low specific is the early recognition step? Should it be viewed more as a compatibility and/or an adhesion [43. 44, 53) between cell
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:47:42 AM
Abstract. The P lmi protein is a glycoprotein of high apparent molecular weight which is abundant in isolated cerebellar Purkinje cells. On SOS polyacrylamide gels, the P p r o t e i n reacts with 14il plant lectins such a s J- '1 Con A. This reaction is used to increase the level of detec tion of the protein. The P,„„ protein is purified by successive extraction of synaptosomal and microsomal membranes with 2”., Triton X-100 and 25"., Na-cholate and preparative gel electro phoresis in SOS. The specific content of P4„„ protein decreases in the cerebella from homozyg ous nervous and Purkinje eell degeneration mutant mice, where the total number of Purkinje cells is markedly reduced, and increases in those of the reeler and weaver mice where a deficit of the granule cells exists. In the cerebellum from the homozygous staggerer mouse, a small amount of P,,m protein persists. During postnatal development the specific content of P,„„ protein per net weight does not change up to the 12th day after birth then increases up to the 25th day when it reaches the adult level. Antisera have been raised against the purified P„m protein. They give precipitation lines by the immunodiffusion reaction of Ouchtcrlonv against a preparation of P„mprotein submitted to mild proteolytic attack. Indirect immunofluorescence performed on slices of rat cerebellum with purified anti-P,,,,, immunoglobulin G. absorbed or not on rat cerebrum membranes, reveals that both the soma and the dendritic arborization of the Purkinje cells are labelled. The neurons from the deep cerebellar nuclei are not stained.
P,„„ Protein Characteristic of the Purkinje Cells from the Cerebellum
missing [33. 34]. The data reported in this paper confirm that the P p r o t e i n is indeed characteristic of the Purkinje cell. Moreover the P,„n protein i. localized in situ on both the soma and the dendrites of the Purkinje cell as revealed by using antibodies raised against the purified protein by an indirect immunofluorescence technique.
Materials and Methods Animals Mouse Neuropalhological Mutants Staggerer t sg) Mutant Manse [3.4. 10.11. 18, IV. 25.27.28. 51.54.02.04.65.741. A stock of mice hetero zygous for the staggerer (sg) mutation C57BL6 ( sg dse-l established by the Jackson Laboratory (Bar Harbor. Me.) was raised at the Pasteur Institute. Homozygous sg sg were obtained by intercrossing the heterozygous mice. The C57BI.O (dse rise I animals were used as controls. Servons i hi : Mutant Manse [1A 26.33. 34.51.60]. C3HcB .1 mice originating from the Jackson Labora tory and either heterozygous or homozygous for the nervous (nr) mutation were intercrossed to produce homo/; gous nr nraninials. Animals from the C3I IcB J strain were used as controls. H eater in ; Matant Manse |5 .4. 12. 27. 32, 46 4lJ, 51. 63 651. B6CBA Rl (wv i mice originating from the Jackson Laboratory and heterozygous for the weaver (wv) mutation were intercrossed to produce homozy gous w\ wv mice. The wild t> pe strain B6CBA RI ( ) was obtained by intercrossing animals which no longer gave the weaver phenotype in their progeny after several back crosses: with wv animals. Keeler rl) Mutant Manse 117. 35. 45. 5S|. The reeler mutation was maintained on inbred strains of C57BL6 mice from the Jackson Laboratory . Only the homozygous rl rl mice have been studied. Litters containing rl. rl individuals were obtained by inter crossing heterozygous (rl ) fertile animals. Purkinje Cell Degeneration />." Mann) and in the absence (indicated as Con A) of methyl a-£>-mannoside. PK = Purkinje cells; Gr = granule cells. Note that many bands arc not common between the two classes of cells - both after Coomassie bril liant blue staining and l21l-Con A binding.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:47:42 AM
Absorbance»
■Protein
result from the survival of a significant frac tion (about 15-20",,) of the Purkinje cells in the cerebellum from this mutant. To test this point gels were run with membrane prepara tions of cerebella from the Purkinje cell de generation (ped) mutant mouse in which only 5-IO"„ of the Purkinje cells persist in theadult. Figure 7 shows that the P40l) protein markedly decreases in the high and low speed membrane pellets from ped/ped cerebellum. In addition, only small changes are observed in wet weight and DNA-RNA content of the cerebellum as a consequence of the ped mutation (table II). The P.,00 protein is therefore characteristic of the Purkinje cells. In figure 8 a gel where crude membrane preparations from cerebella of various mouse mutants are compared is presented. The de tection of eventual differences between the protein patterns has been amplified by the 125I Con A autoradiographic method. In ad dition, the same absolute quantity of mem branes, in terms of total protein content, was applied to the gel to render a quantitative comparison possible. In agreement with pre vious results [34, 35], the relative content of the P.,un protein per mass of protein is signi-
264
Mikoshiba/Huchct/Changcux
6a Con A i-------------1
ficantly enhanced in the homozygous reeler and weaver cerebella where a marked deficit of the granule cell exists. The relative increases in P4no estimated by integration of the densitoinelric scans of the gels were, respectively, 22 and 42 of the control per mass of protein, whereas total P.,(l0 contents were 30 and 49% of the control per cerebellum. On the other hand, the sensitivity of the method becomes sufficient to demonstrate the presence of a significant amount of P4#0 pro tein in the cerebellum from the homozygous staggerer mouse where the most evident defect is a marked decrease in the number of parallel fiber-Purkinje cell synapses [58, 62, 67] ac companied. also, by a decrease of the number of Purkinje cells. The content in P400 protein is only 62"„ control on a protein basis but only 12% con trol per cerebellum if one takes into account the marked decrease in wet weight of the sg/sg cerebellum. Another interesting feature of the sg/sg gels, which becomes particularly evident on the densitometric scans, is that while the P40(, band decreases other bands significantly increase. These bands are de tected almost exclusively in the 1251 Con A treated gels and have an apparent molecular weight higher than that of the P4no protein. Their Rf ranges from 2/ 3 to 1/ 3 that of the P4U0
Fig. 6. Protein pattern of separated molecular (ML) and granular layers (GL) from bovine cere bellum. The separated molecular and granular layers were applied to the 5-15% linear polyacrylamide-gel electrophoresis in SDS. After electrophoresis, parallel gels were used for incubation with ,isI-Con A, while others were fixed and stained by Coomassie brilliant blue (protein).
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:47:42 AM
A b so rb an c e
6b
Protein i------------1
265
Table II. DNA and RNA content of the cerebel lum from Purkinje cell degeneration homozygous mutant mice and wild type mice Days
mg wet weight
ped/ 35 ped 35
33.25 34.25
+/+ 35 49.30 35 50.40
Mg DNA / mg wet weight 7.47 7.90
Mg DNA/ Cerebellum 248.4 270.4
Mg RNA 1 mg wet weight 4.19 4.23
Mg RNA / Cerebellum 139.3 144.9
5.64 6.13
278.1 309.0
3.99 4.51
196.7 227.3
The data for DNA and RNA represent the value for I animal from an average of 2-3 determinations.
Absorbance
7b
Fig. 7. Protein patterns of mem brane fractions from cerebella of homozygous Purkinje cell degenera tion (ped) and wild type (+/+) mice. LSP = low-speed pellet; MSP = high-speed pellet; P = Purkinje cell degeneration (ped); C = control wild type (
