Brain Research, 535 (1990) 39-42 Elsevier
39
BRES 16099
Expression of neural cell adhesion molecule in dysmyelinating mutants Shama Bhat and Donald H. Silberberg Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, PA 19104 (U.S.A.) (Accepted 12 June 1990) Key words: Neural cell adhesion molecule; Oligodendrocyte; Neuron; Myelin; Cell-cell interaction; Jimpy; Shiverer
The possible role of neural cell adhesion molecule (NCAM) in myelination was studied in the dysmyelinating mouse mutants jimpy and shiverer, by characterizing the expression of the different molecular forms of brain NCAM as a function of age. In jimpy, the expression of NCAM-120 (120,000-Da NCAM) was low and in shiverer both NCAM-120 and NCAM-180 (180,000-Da NCAM) were reduced when compared to controls. In both jimpy and shiverer there was no significant change in the phospholipase C-sensitive NCAM-120. These data further support the possibility that NCAM may be involved in myelination. INTRODUCTION Neural cell adhesion molecule ( N C A M ) , a cell surface glycoprotein, mediates cell-cell interaction during development 7's'21. Three molecular forms of N C A M have been documented in the adult rodent brain with apparent molecular weights of 180,000, 140,000, and 120,000 D a (NCAM-180, -140, and -120, respectively) 1' 6.11,14 These molecules show regional and cell-type specificities in their expression during development 1' 5,10,16,18,20. Recent studies have indicated that N C A M 180 is expressed mainly by neurons 4'5'16 and oligodendrocytes express mainly NCAM-1201'2. We have shown that in rat and mouse brains during development, differential expression of these 3 molecular forms occurs 2' 3 The expression of NCAM-120 is closely related in timing to the development and maturation of oligodendrocytes and myelination. Based on these studies and studies of oligodendrocyte interaction with neuroblastoma cells 4 we hypothesized that during myelination and remyelination oligodendrocyte N C A M s come in contact with neuronal axons 2-4. Abnormalities in the expression of N C A M may lead to dysmyelination or prevent remyelination. A number of mouse neurological mutants have been characterized that are abnormal in the structure, composition, and metabolism of myelin (see ref. 15 for a review). In order to explore the possible role of N C A M in myelination we studied the developmental expression of different molecular forms of N C A M in 3 types of dysmyelinating mutants, whose structural and myelin components have differing abnormalities. Jimpy is a
sex-linked recessive mutation with severe abnormalities in myelin and oligodendrocytes (see ref. 15). Since oligodendrocytes express only NCAM-120, we expected low levels of NCAM-120 in jimpy brains. The shiverer mutation is an autosomal recessive, with very low levels of myelin and myelin components in the central nervous system 15. Quaking, an autosomal recessive mutant, is characterized by an absence of compaction of myelin. We found previously that in quaking brain the expression of NCAM-180 was markedly reduced 3. Therefore we expected low levels of NCAM-180 in Shiverer mutants; and because of low levels of oligodendrocytes and reduced myelin in shiverer we expected low levels of NCAM-120 in this mutant. We found that in jimpy, NCAM-120 is reduced, while in shiverer both NCAM-180 and -120 are reduced. As discussed below, this supports the possibility that N C A M may be involved during myelination, and perhaps in remyelination. MATERIALS AND METHODS Mutant brains Jimpy (jp), littermate control and normal control (B6C3) mice were obtained from the Jackson Laboratory. Breeding pairs of shiverer (shi) mutants were kind gifts of Dr. Elisa Barbarese of the University of Connecticut Health Center, Farmington. Shiverer mutants were identified by their characteristic shaking behavior. In each group there were more than 3 animals. Brains at the ages indicated were removed, different regions of the brains were dissected out and homogenized in NP-40 buffer (1% Nonidet P-40, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride in phosphate-buffered solution (PBS). Total homogenate proteins were used to analyze NCAM. Quantitation of different molecular forms of NCAM SDS-polyacrylamide gel electrophoresis and Western blot analysis
Correspondence: S. Bhat, Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, PA 19104, U.S.A.
40 were performed as in a previous study 2. One hundred micrograms of total homogenate protein a7 were used for gel electrophoresis. Immunostaining utilized anti-NCAM (1:3000 dilution) and radioiodinated [125I]goat anti-rabbit-IgG (New England Nuclear, Boston). After visualizing the results by autoradiography, the corresponding band areas of the nitrocellulose paper were cut and the radioactivity was counted in a 7 spectrometer. The % activity were calculated as follows:
incubated for 2 h at 37 °C before Western blot analysis of NCAMs of both the supernatant and the pellet.
RESULTS AND DISCUSSION
N C A M expression in jimpy brains Brains
cpm of mutant cpm of control × 100
control
Phospholipase C (PLC) treatment of brain membranes Mouse brain membranes were prepared by centrifugation of the homogenate at 15,000 g for 20 min. One hundred micrograms of protein of the membranes in 100/A of PBS were incubated for 2 h at 37 °C followed by centrifugation in a microfuge for 10 min at 4 °C, to remove any soluble NCAM. The pellet was resuspended in PBS containing 2 mM ZnCI 2 and 4 units of PLC (Sigma Chemicals) and
A
I 2 C
I
3 4 5 6 7 8 9 I0 II j I j CC I j C I
I 2 j I
5 c
4567 j I c
j
8
9
I0 II
12
I
c
j
c
I
2-
and
control
3-week-old
jimpy,
(B6C3) m i c e w e r e
littermate
removed
and
12545678 cscscscs 180K
140K 120K
12 j
--180K
B
A
from
and
-
140K
-
120K
-- 1 8 0 K
I 2 34 BSCSCSC
56
78 CS
12545678 Csc $c s c s c
-- 1 4 0 K -- 1 2 0 K
Fig. 1. Western blot analysis of NCAM in jimpy, littermate control and normal brains. A: 2-weeks, 1-3, cerebral hemispheres; 4-6, cerebellum; 7.9, brainstem; 10-12, spinal cord. B: 3-weeks, 1-3, cerebral hemispheres; 4-6, cerebellum, 7-9, brainstem; 10-12, spinal cord. In all cases 100 #g of brain homogenate protein was analyzed, c, control; 1, littermate control; j, jimpy. See text for experimental details. This is an autoradiogram.
Fig. 2. Western blot analysis of NCAMs in shiverer and normal control brains. A: 2-weeks, 1, 2, spinal cord; 3, 4, brainstem; 5, 6, cerebellum; 7, 8, cerebral hemisphere. B: 3-weeks, 1, 2, cerebral hemisphere; 3, 4, spinal cord; 5, 6, cerebellum; 7, 8, brainstem. C: 4-weeks, 1, 2, brainstem; 3, 4, cerebral hemisphere; 5, 6, spinal cord; 7, 8, cerebellum. In all cases 100/~g of brain homogenate proteins were analyzed, s, shiverer, c, control. See text for experimental details. This is an autoradiogram.
41 TABLE I
to 79% in 3-week spinal cord. The a m o u n t of NCAM-180
Quantitation of different molecular forms of NCAM in jimpy brains compared to controls
was not reduced in jimpy compared to controls; in some
Jimpy and control brain homogenates were analyzed by Western blot and quantitated as described in the text. Values expressed were % of control, mean + S.E.M. For each group 3 brains were analyzed.
Region of the brain
% of Control 2 weeks
Cerebral hemispheres NCAM-180 NCAM-140 NCAM-120 Cerebellum NCAM-180 NCAM-140 NCAM-120 Brainstem NCAM-180 NCAM-140 NCAM-120 Spinal Cord NCAM-180 NCAM-140 NCAM-120
3 weeks
96 + 14 79 + 20 61 + 4**
193 + 28 150 + 32 77 + 4*
95 + 9 77 + 16 60 + 5*
134 + 27 114 + 23 50 + 5*
113 + 3 74 + 13 53 + 4**
142 + 41 81 + 13 62 + 11"
161 + 33 109 + 18 71 + 3**
145 + 7 94 + 15 79 + 7*
different parts were dissected out, homogenized and analyzed by Western blot 2. As shown in Fig. 1 and Table I, at ages 2 and 3 weeks, NCAM-120 was significantly reduced in jimpy compared to control brain. The degree of reduction ranged from 50% in 3-week-old cerebellum
TABLE II
Quantitation of different molecular forms of NCAM in shiverer brain compared to controls Shiverer and control brain homogenates were analyzed by Western blot and quantitated as described in the text. Values expressed were % of control, mean + S.E.M. For each group 3 brains were analyzed.
% of Control 2 weeks
Cerebral hemispheres NCAM-180 NCAM-140 NCAM-120 Cerebellum NCAM-180 NCAM-140 NCAM-120 Brainstem NCAM-180 NCAM-140 NCAM-120 Spinal Cord NCAM-180 NCAM-140 NCAM-120 *P < 0.05; **P < 0.01.
sufficiently to yield data which were not statistically significant. The a m o u n t of NCAM-140 ranged from 74% of controls in 2-week-old brainstem to 150% in 3week-old cerebral hemisphere. However, variation among samples also rendered this change statistically insignificant. Littermate controls showed two levels of NCAM-120, reduced or normal (Fig. 1) compared to controls. This is predictable because jimpy is a recessive
*P< 0.05; **P> 0.01.
Region of the brain
regions it was higher than in controls, ranging from 95% in 2-week-old cerebellum to 192% in cerebral hemispheres of 3-week-old jimpy. However, samples differed
3 weeks
4 weeks
71 + 6* 82 + 13 71 + 5*
41 _+6** 78 __+17 57 + 8*
55 + 13" 84 + 15 76 + 5*
69 + 10" 120 + 32 61 + 7*
35 + 3** 99 + 9 72 + 5*
72 + 8* 97 + 10 62 + 9*
72 + 5" 83 + 10 78+1"*
44 + 2** 94 + 22 71+8"
77 + 4* 89 + 22 64+1"*
49 + 10" 77 + 21 69 + 7*
-
70 + 2** 88 + 21 61 + 3**
mutation.
N C A M expression in shiverer brains As with jimpy, shiverer brains were also removed at ages 2, 3, and 4 weeks after birth and dissected, homogenized and N C A M s quantitated. As shown in Fig. 2 and Table II, NCAM-180 and -120 were decreased significantly in shiverer brains compared to controls. The decrease was observed in all parts of the brain tested, but the degree of decrease varied from region to region. For example, NCAM-180 was reduced more in 3-week-old shiverer cerebellum and least in the brainstem at 4 weeks. NCAM-120 was reduced most in the 3-week-old cerebral hemispheres and least in the brainstem at 4 weeks. Variations in the a m o u n t of NCAM-140 in shiverer m u t a n t samples were too large to allow statistically significant interpretation. Phospholipase C ( P L C ) treatment o f N C A M NCAM-120 is anchored to the m e m b r a n e via phosphatidyl inosito112'13'z2. To investigate whether there is any abnormality in the phosphatidyl inositol anchoring of NCAM-120, mouse brain m e m b r a n e s from controls and mutants were treated with PLC and analyzed by Western blot. Both jimpy and shiverer showed no significant levels of increase or decrease in the PLC-released NCAM-120 compared to controls, suggesting that the abnormality is not with the anchoring of N C A M (data not shown). Embryonic rodent brain yields heterogeneou.s molecular forms of N C A M with apparent molecular weights ranging from 140,000 to 200,000 D a 8. Unlike the staggerer mutation 9, the maturation of N C A M (i.e. conversion of embryonic form of N C A M to adult form) is not hindered in either jimpy or shiverer mutants. At ages 3 and 4 weeks, the 3 molecular forms of N C A M can be clearly seen (Fig. 1 and 2). The primary defective genes in jimpy and shiverer are proteolipid protein and myelin basic protein, respectively. Therefore, the abnormalities in the expression of
42 NCAM
m a y result f r o m changes in o t h e r constituents
w h o s e p r e s e n c e m a y be r e q u i r e d for the expression of NCAM.
N e v e r t h e l e s s , the N C A M
and s h i v e r e r ,
r e d u c t i o n s in j i m p y
t o g e t h e r with the d e v e l o p m e n t a l
data 2
Acknowledgement. We thank M. Richardson and L. Lynch for excellent technical assistance, and N. Bridge for typing the manuscript. This work was supported by grants from the National Institutes of Health (NSl1037), and National Multiple Sclerosis Society, New York.
suggest a role for N C A M in m y e l i n a t i o n .
REFERENCES 1 Bhat, S. and Silberberg, D.H., Oligodendrocyte cell adhesion molecules are related to neural cell adhesion molecule, NCAM, J. Neurosci., 6 (1986) 3348-3354. 2 Bhat, S. and Silberberg, D.H., Developmental expression of NCAMs of oligodendrocytes in vivo and in culture, J. Neurochem., 50 (1988) 1830-1838. 3 Bhat, S. and Silberberg, D.H., NCAM-180, the largest component of neural cell adhesion molecule is reduced in dysmyelihating quaking mutant mouse brain, Brain Research, 452 (1988) 373-377. 4 Bhat, S. and Silberberg, D.H., Neuroblastoma cell-oligodendrocyte interaction is mediated by neural cell adhesion molecules, Dev. Neurosci., 10 (1988) 231-235. 5 Chuong, C.M. and Edelman, G.M., Alterations in neural cell adhesion molecules during development of different regions of the nervous system, J. Neurosci., 4 (1984) 2354-2368. 6 Chuong, C.M., McClain, D.A., Streit, P. and Edelman, G.M., Neural cell adhesion molecules in rodent brains isolated by monoclonal antibodies with cross-species reactivity, Proc. Natl. Acad. Sci. U.S,A., 79 (1982) 4234-4238. 7 Cunningham, B.A., Cell adhesion molecules: a new perspective on molecular biology, Trends Biol. Sci., II (1986) 423-426. 8 Edelman, G.M., Cell adhesion molecules in the regulations of animal form and tissue pattern, Annu. Rev. Cell Biol., 2 (1986) 81-116. 9 Edelman, G.M. and Chuong, C.M., Embryonic to adult conversion of neural cell adhesion molecules in normal and staggerer mice, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 7035-7038. 10 Gennerini, G., Hirsch, M.R., He, H.T., Hirn, M., Finne, J. and Goridis, C., Differential expression of mouse neural celladhesion molecule (N-CAM) mRNA species during brain development and in neural cell lines, J. Neurosci., 6 (1986) 1983-1990. 11 Hansen, D.C., Nybroe, O. and Bock, E., Cell-free synthesis of D2-cell adhesion molecules: evidence for three primary translation products, J. Neurochem., 44 (1985) 712-717. 12 He, H.-T., Barbet, J., Chaix, J.-C. and Goridis, C., Phospha-
13
14
15
16
17
18
19
20
21 22
tidyl-inositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule, The EMBO J., 5 (1986) 2489. Hemperly, J.J., Edelman, G.M. and Cunningham, B,A., cDNA clones of the neural cell adhesion molecule (N-CAM) lacking a membrane-spanning region consistent with evidence for membrane attachment via a phosphatidylinositol intermediate, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9822-9826. Him, J., Pierres, M., Deagostini-Bazin, J., Hirsch, M. and Goridis, C., Monoclonal antibody against cell surface glycoprotein of neurons, Brain Research, 214 (1981) 433-439. Hogan, E.R. and Greenfield, S., Animal models of genetic disorders of myelin. In D. Morell (Ed.), Myelin, Plenum, New York, 1984, p. 489. Keilhauer, G., Faissner, A. and Schachner, M., Differential inhibition of neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion by L1, L2 and N-CAM antibodies, Nature, 316 (1985) 728-730. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Noble, M., Albrechtsen, M., Moiler, C., Lyles, J., Bock, E., Goridis, C., Watanabe, M. and Rutishauer, U., Glial cells express N-CAM/D2-CAM-Iike polypeptides in vitro, Nature, 316 (1985) 725-728, Pollerberg, E.G., Sadoul, R., Goridis, C. and Schachner, M., Selective expression of the 180-KD component of the neural cell adhesion molecule N-CAM during development, J. Cell Biol., 101 (1985) 1921-1929. Rougon, G., Deagostini-Bazin, H., Him, M. and Goridis, C., Tissue- and developmental stage-specific forms of a neural cell surface antigen linked to differences in glycosylation of a common polypeptide, EMBO J., 1 (1982) 1239-1244. Rutishauser, U. and Goridis, C., N-CAM: the molecule and its genetics, Trends Genet., 2 (1986) 72-76. Sadoul, K., Meyer, A., Low, M.G. and Schachner, M., Release of the 120 kDa component of the mouse neural cell adhesion molecule NCAM from cell surfaces by phosphatylinositolspecific phospholipase C., Neurosci. Lett., 72 (1986) 341-346.
39
BRES 16099
Expression of neural cell adhesion molecule in dysmyelinating mutants Shama Bhat and Donald H. Silberberg Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, PA 19104 (U.S.A.) (Accepted 12 June 1990) Key words: Neural cell adhesion molecule; Oligodendrocyte; Neuron; Myelin; Cell-cell interaction; Jimpy; Shiverer
The possible role of neural cell adhesion molecule (NCAM) in myelination was studied in the dysmyelinating mouse mutants jimpy and shiverer, by characterizing the expression of the different molecular forms of brain NCAM as a function of age. In jimpy, the expression of NCAM-120 (120,000-Da NCAM) was low and in shiverer both NCAM-120 and NCAM-180 (180,000-Da NCAM) were reduced when compared to controls. In both jimpy and shiverer there was no significant change in the phospholipase C-sensitive NCAM-120. These data further support the possibility that NCAM may be involved in myelination. INTRODUCTION Neural cell adhesion molecule ( N C A M ) , a cell surface glycoprotein, mediates cell-cell interaction during development 7's'21. Three molecular forms of N C A M have been documented in the adult rodent brain with apparent molecular weights of 180,000, 140,000, and 120,000 D a (NCAM-180, -140, and -120, respectively) 1' 6.11,14 These molecules show regional and cell-type specificities in their expression during development 1' 5,10,16,18,20. Recent studies have indicated that N C A M 180 is expressed mainly by neurons 4'5'16 and oligodendrocytes express mainly NCAM-1201'2. We have shown that in rat and mouse brains during development, differential expression of these 3 molecular forms occurs 2' 3 The expression of NCAM-120 is closely related in timing to the development and maturation of oligodendrocytes and myelination. Based on these studies and studies of oligodendrocyte interaction with neuroblastoma cells 4 we hypothesized that during myelination and remyelination oligodendrocyte N C A M s come in contact with neuronal axons 2-4. Abnormalities in the expression of N C A M may lead to dysmyelination or prevent remyelination. A number of mouse neurological mutants have been characterized that are abnormal in the structure, composition, and metabolism of myelin (see ref. 15 for a review). In order to explore the possible role of N C A M in myelination we studied the developmental expression of different molecular forms of N C A M in 3 types of dysmyelinating mutants, whose structural and myelin components have differing abnormalities. Jimpy is a
sex-linked recessive mutation with severe abnormalities in myelin and oligodendrocytes (see ref. 15). Since oligodendrocytes express only NCAM-120, we expected low levels of NCAM-120 in jimpy brains. The shiverer mutation is an autosomal recessive, with very low levels of myelin and myelin components in the central nervous system 15. Quaking, an autosomal recessive mutant, is characterized by an absence of compaction of myelin. We found previously that in quaking brain the expression of NCAM-180 was markedly reduced 3. Therefore we expected low levels of NCAM-180 in Shiverer mutants; and because of low levels of oligodendrocytes and reduced myelin in shiverer we expected low levels of NCAM-120 in this mutant. We found that in jimpy, NCAM-120 is reduced, while in shiverer both NCAM-180 and -120 are reduced. As discussed below, this supports the possibility that N C A M may be involved during myelination, and perhaps in remyelination. MATERIALS AND METHODS Mutant brains Jimpy (jp), littermate control and normal control (B6C3) mice were obtained from the Jackson Laboratory. Breeding pairs of shiverer (shi) mutants were kind gifts of Dr. Elisa Barbarese of the University of Connecticut Health Center, Farmington. Shiverer mutants were identified by their characteristic shaking behavior. In each group there were more than 3 animals. Brains at the ages indicated were removed, different regions of the brains were dissected out and homogenized in NP-40 buffer (1% Nonidet P-40, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride in phosphate-buffered solution (PBS). Total homogenate proteins were used to analyze NCAM. Quantitation of different molecular forms of NCAM SDS-polyacrylamide gel electrophoresis and Western blot analysis
Correspondence: S. Bhat, Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, PA 19104, U.S.A.
40 were performed as in a previous study 2. One hundred micrograms of total homogenate protein a7 were used for gel electrophoresis. Immunostaining utilized anti-NCAM (1:3000 dilution) and radioiodinated [125I]goat anti-rabbit-IgG (New England Nuclear, Boston). After visualizing the results by autoradiography, the corresponding band areas of the nitrocellulose paper were cut and the radioactivity was counted in a 7 spectrometer. The % activity were calculated as follows:
incubated for 2 h at 37 °C before Western blot analysis of NCAMs of both the supernatant and the pellet.
RESULTS AND DISCUSSION
N C A M expression in jimpy brains Brains
cpm of mutant cpm of control × 100
control
Phospholipase C (PLC) treatment of brain membranes Mouse brain membranes were prepared by centrifugation of the homogenate at 15,000 g for 20 min. One hundred micrograms of protein of the membranes in 100/A of PBS were incubated for 2 h at 37 °C followed by centrifugation in a microfuge for 10 min at 4 °C, to remove any soluble NCAM. The pellet was resuspended in PBS containing 2 mM ZnCI 2 and 4 units of PLC (Sigma Chemicals) and
A
I 2 C
I
3 4 5 6 7 8 9 I0 II j I j CC I j C I
I 2 j I
5 c
4567 j I c
j
8
9
I0 II
12
I
c
j
c
I
2-
and
control
3-week-old
jimpy,
(B6C3) m i c e w e r e
littermate
removed
and
12545678 cscscscs 180K
140K 120K
12 j
--180K
B
A
from
and
-
140K
-
120K
-- 1 8 0 K
I 2 34 BSCSCSC
56
78 CS
12545678 Csc $c s c s c
-- 1 4 0 K -- 1 2 0 K
Fig. 1. Western blot analysis of NCAM in jimpy, littermate control and normal brains. A: 2-weeks, 1-3, cerebral hemispheres; 4-6, cerebellum; 7.9, brainstem; 10-12, spinal cord. B: 3-weeks, 1-3, cerebral hemispheres; 4-6, cerebellum, 7-9, brainstem; 10-12, spinal cord. In all cases 100 #g of brain homogenate protein was analyzed, c, control; 1, littermate control; j, jimpy. See text for experimental details. This is an autoradiogram.
Fig. 2. Western blot analysis of NCAMs in shiverer and normal control brains. A: 2-weeks, 1, 2, spinal cord; 3, 4, brainstem; 5, 6, cerebellum; 7, 8, cerebral hemisphere. B: 3-weeks, 1, 2, cerebral hemisphere; 3, 4, spinal cord; 5, 6, cerebellum; 7, 8, brainstem. C: 4-weeks, 1, 2, brainstem; 3, 4, cerebral hemisphere; 5, 6, spinal cord; 7, 8, cerebellum. In all cases 100/~g of brain homogenate proteins were analyzed, s, shiverer, c, control. See text for experimental details. This is an autoradiogram.
41 TABLE I
to 79% in 3-week spinal cord. The a m o u n t of NCAM-180
Quantitation of different molecular forms of NCAM in jimpy brains compared to controls
was not reduced in jimpy compared to controls; in some
Jimpy and control brain homogenates were analyzed by Western blot and quantitated as described in the text. Values expressed were % of control, mean + S.E.M. For each group 3 brains were analyzed.
Region of the brain
% of Control 2 weeks
Cerebral hemispheres NCAM-180 NCAM-140 NCAM-120 Cerebellum NCAM-180 NCAM-140 NCAM-120 Brainstem NCAM-180 NCAM-140 NCAM-120 Spinal Cord NCAM-180 NCAM-140 NCAM-120
3 weeks
96 + 14 79 + 20 61 + 4**
193 + 28 150 + 32 77 + 4*
95 + 9 77 + 16 60 + 5*
134 + 27 114 + 23 50 + 5*
113 + 3 74 + 13 53 + 4**
142 + 41 81 + 13 62 + 11"
161 + 33 109 + 18 71 + 3**
145 + 7 94 + 15 79 + 7*
different parts were dissected out, homogenized and analyzed by Western blot 2. As shown in Fig. 1 and Table I, at ages 2 and 3 weeks, NCAM-120 was significantly reduced in jimpy compared to control brain. The degree of reduction ranged from 50% in 3-week-old cerebellum
TABLE II
Quantitation of different molecular forms of NCAM in shiverer brain compared to controls Shiverer and control brain homogenates were analyzed by Western blot and quantitated as described in the text. Values expressed were % of control, mean + S.E.M. For each group 3 brains were analyzed.
% of Control 2 weeks
Cerebral hemispheres NCAM-180 NCAM-140 NCAM-120 Cerebellum NCAM-180 NCAM-140 NCAM-120 Brainstem NCAM-180 NCAM-140 NCAM-120 Spinal Cord NCAM-180 NCAM-140 NCAM-120 *P < 0.05; **P < 0.01.
sufficiently to yield data which were not statistically significant. The a m o u n t of NCAM-140 ranged from 74% of controls in 2-week-old brainstem to 150% in 3week-old cerebral hemisphere. However, variation among samples also rendered this change statistically insignificant. Littermate controls showed two levels of NCAM-120, reduced or normal (Fig. 1) compared to controls. This is predictable because jimpy is a recessive
*P< 0.05; **P> 0.01.
Region of the brain
regions it was higher than in controls, ranging from 95% in 2-week-old cerebellum to 192% in cerebral hemispheres of 3-week-old jimpy. However, samples differed
3 weeks
4 weeks
71 + 6* 82 + 13 71 + 5*
41 _+6** 78 __+17 57 + 8*
55 + 13" 84 + 15 76 + 5*
69 + 10" 120 + 32 61 + 7*
35 + 3** 99 + 9 72 + 5*
72 + 8* 97 + 10 62 + 9*
72 + 5" 83 + 10 78+1"*
44 + 2** 94 + 22 71+8"
77 + 4* 89 + 22 64+1"*
49 + 10" 77 + 21 69 + 7*
-
70 + 2** 88 + 21 61 + 3**
mutation.
N C A M expression in shiverer brains As with jimpy, shiverer brains were also removed at ages 2, 3, and 4 weeks after birth and dissected, homogenized and N C A M s quantitated. As shown in Fig. 2 and Table II, NCAM-180 and -120 were decreased significantly in shiverer brains compared to controls. The decrease was observed in all parts of the brain tested, but the degree of decrease varied from region to region. For example, NCAM-180 was reduced more in 3-week-old shiverer cerebellum and least in the brainstem at 4 weeks. NCAM-120 was reduced most in the 3-week-old cerebral hemispheres and least in the brainstem at 4 weeks. Variations in the a m o u n t of NCAM-140 in shiverer m u t a n t samples were too large to allow statistically significant interpretation. Phospholipase C ( P L C ) treatment o f N C A M NCAM-120 is anchored to the m e m b r a n e via phosphatidyl inosito112'13'z2. To investigate whether there is any abnormality in the phosphatidyl inositol anchoring of NCAM-120, mouse brain m e m b r a n e s from controls and mutants were treated with PLC and analyzed by Western blot. Both jimpy and shiverer showed no significant levels of increase or decrease in the PLC-released NCAM-120 compared to controls, suggesting that the abnormality is not with the anchoring of N C A M (data not shown). Embryonic rodent brain yields heterogeneou.s molecular forms of N C A M with apparent molecular weights ranging from 140,000 to 200,000 D a 8. Unlike the staggerer mutation 9, the maturation of N C A M (i.e. conversion of embryonic form of N C A M to adult form) is not hindered in either jimpy or shiverer mutants. At ages 3 and 4 weeks, the 3 molecular forms of N C A M can be clearly seen (Fig. 1 and 2). The primary defective genes in jimpy and shiverer are proteolipid protein and myelin basic protein, respectively. Therefore, the abnormalities in the expression of
42 NCAM
m a y result f r o m changes in o t h e r constituents
w h o s e p r e s e n c e m a y be r e q u i r e d for the expression of NCAM.
N e v e r t h e l e s s , the N C A M
and s h i v e r e r ,
r e d u c t i o n s in j i m p y
t o g e t h e r with the d e v e l o p m e n t a l
data 2
Acknowledgement. We thank M. Richardson and L. Lynch for excellent technical assistance, and N. Bridge for typing the manuscript. This work was supported by grants from the National Institutes of Health (NSl1037), and National Multiple Sclerosis Society, New York.
suggest a role for N C A M in m y e l i n a t i o n .
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