0022-3042/79/030l-0817502W/O
Journal of NeurochrniislrJ. Vui. 32 pp. 817 to 821. Pergamon Press Ltd 1979. Printed in Great Britain. 8 International Society for Ncurochemislry Ltd
THE DISTRIBUTION OF MYELIN BASIC PROTEIN IN SUBCELLULAR FRACTIONS OF DEVELOPING JIMPY MOUSE BRAIN' THOMAS R. ZIMMERMAN, JR. and STEVENR. COHEN' Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U S A . (Received 7 August 1978. Accepted 23 October 1978)
Abstract-The amount of myelin basic protein in jimpy mutants and unaffected littermates was measured by radioimmunoassay during the period of most active myelination (11-21 days). This protein was examined in whole brain homogenates and in four subcellular fractions (nuclear, 900g pellet; heavy mernbranc, 11,500 y pellet; microsomal, 100,000 y pellet; and cytosol, 100,000y supernatant solution). At all ages examined, the mutants, which have very little myelin in the CNS, had only about 2% the amount of basic protein found in controls. As expected, the amount of myelin basic protein increased 4-fold in the control animals during the developmental period studied. This was not the case in the jimpy mutants, where little increase in the whole brain basic protein was observed. In the jimpy mutants, all of the fractions had significantly less basic protein than control fractions, except the cytosol. where the amounts of basic protein were similar in controls and mutants. These results are discussed with respcct to possible mechanisms of myelination and the site of the genetic lesion.
THESYNTHESIS of the myelin sheath occurs during a precise time in the development of the mammalian nervous system. In the mouse and rat most of this sheath is laid down between 10 and 25 days after birth. It has been calculated that during this period of rapid myelination, each oligodendrocyte synthesizes more than three times its weight in myelin per 1973). Since the myelin day (NORTON& PODUSLO, membrane is composed of a variety of lipids and proteins, the mechanism of assembly must be under precise control. The site and mechanism of this synthesis, however, remain unknown. Myelin basic protein is unique to the myelin membrane and represents about 30% of the myelin protein (MOSCARELLO, 1976). Its accumulation in the brain parallels the morphological appearance of myelin as well as the accumulation of cerebrosides and sulfatides (TENNEKOON et al., 1977). Basic protein is, therefore, a specific marker for myelin. W e have developed a radioimmunoassay for myelin basic protein (COHEN et al., 1975) which permits measurement of the small quantities of this protein found both in early development and in subcellular fractions. The jimpy mouse is a mutant in which myelinogenesis is arrested early in development (SIDMANet al., 1964). This animal has been the subject of investigation for two main reasons. First, the precise result
A preliminary account was presented at the Ninth Meeting of the American Society for Neurochemistry at Washington, DC. March 1978. *Send all correspondence to S . R. Cohen at above address.
of the genetic defect is most likely to be a n alteration in the primary structure of a protein or in a genetic control mechanism. Identification of this defect may shed light o n a key step in myelin biosynthesis. Second, in these animals, myelinogenesis is perturbed without axonal, inflammatory, gliotic or necrotic changes (HOGAN,1977). Thus, a comparison of the mutant with its unaffected littermate can be used t o probe morphological and biochemical aspects of the assembly and maintenance of myelin. To facilitate these studies, the myelin basic protein was used as a specific marker for myelin. The present paper describes studies on changes in the amount of basic protein in whole brain and subcellular fractions of jimpy and littermate control mice during development. MATERIALS AND METHODS Animals. Mice of the jimpy strain were brcd in our laboratories from a stock obtained from Jackson laboratories. Bar Harbor, ME. All animals were kept with the mother until used. The mothers wcrc fed a commercial pellet diet and water ad lib. Mutants were identified by the observation of an axial body tremor beginning 1&11 days after birth. A t specified ages from 11 to 20 days. mutants and littermate controls were decapitated, the brains were removed, and each brain was homogenized by hand in 20 vol of 0.32 M-sucrose with a Dounce homogenizer. The wet weight of the brains averaged 350 mg, with no observable difference between mutants and controls. A total of 68 mice were examined with at least three mutants and three controls from each age. Fracfionafion and assay. The flow chart in Fig. I describes the fractionation procedure. For a typical fractionation, a volume of about 7ml of homogcnate was used,
817
THOMAS R. ZIMMERMAN JR. and STEVEN R. COHEN
818
900 xg; 10 min
Supernatant
w Pellet rehomogenized in 2
m l 0.32 M sucrose
\1
900 xg; 10 min
!I
I
11,500 xg; 20 min Pellet-
d HEAVY MEMBRANE
NUCLEI
Supernatant
100,000 xg; 60 min
and four fractions were obtained. The first was a nuclear fraction consisting of cell nuclei, cell debris, and unbroken cells. The second, the heavy membrane fraction, consisted of mitochondria and myelin. Since myelin in the jimpy mice may have a density and composition different from normal myelin (MATTHIEU et al., 1974), this fraction was not further purified. The last centrifugation yielded a microsomal pellet, and a final supernatant solution, which was designated the cytosol fraction. The amount of total protein in each fraction was measured by the method of LOWRYet al. (1951). Requisite dilutions were made in water. As previously demonstrated, it was necessary to boil the homogenates before assaying (COHENet al.. 1975). The radioimmunoassay is sensitive to basic protein in a range of 2-20ng. For all of the fractions from control mice, except the cytosol. samples were diluted 1s or 100-fold. In mutants, it was only necessary to dilute the heavy membrane fraction. A 2 0 4 sample of the diluted (in 0.2 M-Trisacetate buffer, pH 7.4-7.5) or undiluted specimen was added to 0.5 ml of 0.2 M-Tris-acetate buffer at pH 7.4-7.5 and incubated with an appropriately diluted antiserum to basic protein at 37°C for 1 h, after which lop1 of '251-Iabeled basic protein solution (1&20 pCi/pg) were added. The samples were then left overnight at room temperature. Next, 0.5ml of cold ethanol was added to each sample tube to precipitate the basic protein antibody complex, and the tubes were maintained for 30 min at 4°C. The samples were centrifuged for 40min at 1660y. The pellet and supernatant solution were counted separately in a Searle 1197 gamma counter, and the per cent bound 1251-labeledbasic protein was calculated. This was compared with a standard curve obtained with purified basic protein. RESULTS
We first compared the quantitative changes in distribution of total protein a n d basic protein in whole brain homogenates of control and mutant mice
between the ages of 11 and 20 days (Fig. 2). Within experimental error, there was no observable difference in the amount of total protein between the controls a n d mutants a t all ages examined. However, the amount of basic protein in the mutants was only 2% of the amount found in controls. Consequently, in this and subsequent graphs these values are expressed on two different scales on the ordinates, one for the control group [left, closed circles) and one for the mutant group (right, open circles). During the period from 11 t o 20 days, the amount of basic protein in the whole brain of control animals increased 4-fold [from 3 t o 12 pg/mg). The mutants, however, were already deficient in basic protein a t
T Control 0
Jimpy
T
P '
II
12
1
13
I
I
14
15
--F 1
16 Age (Days)
I
17
I
18
4
19
I
20
FIG.2. Basic protein in whole brain of mutant and control mice during development. Each point represents duplicate determinations of at least three control and three jimpy mice at each age. The bar is the S.D. Note that the values are presented on two different scales, closed circles (left ordinate) for controls, and open circles (right ordinate) for mutants.
Myelin basic protein in jimpy mouse brain
TABLE1. DISTRIBUTION OF BASIC
PROTEIN IN SUBCELLULAR FRACTIONS
Ratio of basic protein Control Mutant
Total basic protein in fraction (pg) Control Mutant
Fraction Nuclear Heavy membrane Microsomes Cytosol
1.29
0.20 0.077 j, 0.02
21.89 & 5.38
1.53
17 14
0.28
0.47 & 0.08 1.01 f 0.11
4.95 0.34 1.67 f 0.43
11 1.4
Each value represents the average ( ~ s . E .of) determinations from 15 animals between 15 and 17 days old. Fractions were prepared and assayed as described in the text.
11 days, having only 0.05pg/mg. This amount increased with age, reaching a maximum at approx 16 days and then declining to about 0.1 pg/ml at 20 days. A comparison of the distribution of basic protein among the subcellular fractions of the control animals and mutants is shown in Table 1 for mice 15-17 days old. The heavy membrane fraction which contains myelin had the greatest amount of basic protein in both groups of animals. This fraction represented
819
nearly 75% of the total basic protein in the controls and 50% in the mutants. The nuclear fraction accounted for similar proportions of the total basic protein in normal and jimpy brain, about 3%. Likewise, the microsomal fraction in both normal and mutant brain had 17% of the total basic protein. In sharp contrast, the cytosol from normal brain accounted for only 5% of the basic protein, whereas the amount from the jimpy mice cytosol was 33% of the total brain basic protein. Figure 3 describes the changes in basic protein that took place during development for each of the four fractions. The amount of basic protein in the nuclear fractions in control animals was only about 0.8 pg/mg at 11 days, which increased 1.5-fold to about 1.2pg/mg in 20-day animals. The mutants, which at 11 days contained only O.OSpg/mg of basic protein in this fraction, showed an increase in basic protein to a maximal value of 0.075 pg/mg at 16 days, which then declined to a value of O.OSpg/mg at 20 days. The heavy membrane fraction is the fraction in which myelin or myelin-like membranes sediment. The control animals exhibited a 4-fold increase in basic protein from 0.6 pg/mg at 11 days to 2.8 pg/mg
Nucleor Fraction
.I
Heavy Membrane Fraction
t 2.0 2.4
E"
2a
t
1.6-
&
0
$
1.2-
"7
m" 0.8-
,0.2
i
I
1
I
I
I
II
12
13
14
I
,
I
15
16
17
,
18
,
19
, ) 20
II
12
13
14
Age (Days)
15
16
17
18
19
Age (Doys)
Micrasomol Froction Cytosol Fraction
t
g 2.4a
Control
0.3 - o Jirnpy
2.0-
E" G
1.6-
0
;1.2-
czp 0.80.4 -
I # I1
I
I
12
13
I
8
I
I
I
15
16
17
I8
I9
I
20
Age (Days)
FIG.3. Basic protein in subcellular fractions of jimpy and control mice during development. Subcellular fractions were prepared and assayed for myelin basic protein as described. Each point represents duplicate determinations of at least three control or jimpy mice at each age. The bar is the standard deviation. For the nuclear, heavy membrane and rnicrosomal fractions data from jimpy and control mice are presented on different scales on the ordinate. For the cytosol fraction, data from mutant and control mice are presented on the same scale.
20
820
THOMAS R . ZIMMERMAN J R . and STEVEN R . COHEN
at 20 days. The amount of basic protein in the mutant at I 1 days (0.05pg/mg) increased only 2-fold and remained relatively constant at about 0.1 pg/mg. In the microsomal fraction the amount of basic protein in the mutant was also reduced at 11 days; mutants had only 0.1 pg/mg compared to l.Opg/mg in the controls. This is the only fraction, however, in which the amount of basic protein in the mutants increased steadily for the entire developmental period. Moreover, for both mutants and controls, the amount of basic protein in this fraction at 20 days was twice the initial 11-day values. In the cytosol fraction the amounts of basic protein in both mutants and controls are presented on the same scale. This fraction represented a small proportion of the total brain basic protein (Table 1) in the controls, but 33% in the jimpy. Clearly, in the jimpy mouse the amount of basic protein in this fraction was least affected by the mutation. The developmental distribution of basic protein in this fraction was similar for both groups of animals. The control animals contained 0.15pg/mg at 16 days and then declined to approx 0.22pg/mg at 20 days. This is the only fraction among control animals in which there was a decline in basic protein during the developmental period. The mutants exhibited a similar 2-fold increase in basic protein from 11 to 16 days. At 20 days, however, the value for basic protein fell below the initial 11-day value of 0.1 pg/mg.
support the view that biosynthesis of myelin-associated components is coordinately controlled (MORELL & COSTATINO-CECCARINI, 1972). The amount of basic protein in the cytosol relative to other fractions is very small. It is possible that this basic protein is the result of contamination from other fractions during the fractionation procedure; however, because of the significant change with development, this is unlikely. In addition, the amount of basic protein in the cytosol of the mutants is nearly normal; this may constitute a small but important ‘pool’ which is the source of basic protein for the initial formation of myelin. In this context, STERNBERGER et al. (1978) have recently shown by immunocytochemical techniques that the cytoplasm of oligodendrocytes stains with antibody to myelin basic protein. Furthermore, this staining intensity increases during the period of early myelination, and then decreases. That both controls and mutants exhibit the same 2-fold accumulation of basic protein in this fraction during the initial period from 11 to 16 days is an indication that the mechanism of basic protein synthesis and movement into this fraction is nearly normal and is not directly affected by the genetic lesion. The decline in the amount of basic protein in the cytosol which begins at 16 days may be related to the continued increase in basic protein in the microsomes of the mutants and controls. Since the concentration of total protein is the same for both controls and mutants, this decrease of basic protein in the cytosol is not attributable merely to a dilution DISCUSSION of basic protcin by an increase in the amount of total In control mice the amount of myelin basic protein protein. The perturbation of myelin biosynthesis, therefore, in the whole brain increases between 10 and 20 days. As expected, most of this basic protein is present in may exist at the point in the pathway at which basic the heavy membrane fraction, which contains myelin. protein combines with myelin lipids and other proIn the mutants, however, basic protein increases only teins from the lighter membranes of the microsomes from 10 to 16 days and then decreases. As in the to form the heavier myelin membranes. A comparison controls, the greatest amount of basic protein is in of the developmental changes of the microsomes with those in the heavy membrane fraction tends to supthe heavy membrane fraction. Other investigators have reported that myelin-asso- port this view. During the period from 16 to 20 days ciated lipids such as sulfatides and cerebrosides are the amount of basic protein increases in both the severely reduced in the jimpy mutant, whereas other microsomes and the heavy membrane fraction of the lipids, not specific to myelin, are only slightly affected controls. Thc mutants, however, d o not show the (HOGAN, 1977). Similarly, we find that while basic same pattern: although the basic protein in the microprotein is reduced in the whole brains of mutants, somes increases, the amount of basic protein in the there is no observable difference in the amount of heavy membrane remains relatively constant. Hence, total protein between mutants and controls. Hogan there appears to be little movement of basic protein also found that the amounts of cerebroside in whole from the microsomes to the heavy membrane. Failure of basic protein to enter the heavier membrain increased in the jimpy mutant between 10 and 15 days, followed by a decrease. We find a similar brane might result in an accumulation of basic propattern for the changes in amounts of basic protein tein in the microsomes. This accumulation might then in whole brain of jimpy mutants. Furthermore, in a result in feedback inhibition on earlier processes of study of sphingolipid biosynthesis, microsomal prep- myelin assembly, a consequence which is reflected by arations from jimpy mutants had only 1&20% the the reduced amount of basic protein in the microamount of galactosylceramide present in the micro- somes. We are currently investigating these possibilities. somes of controls (MORELL& COSTATINO-CECCARINI, 1972). The amount of basic protein in the microsomes of mutants is only 15% the amount in controls. There- Acknowledgements-The authors are grateful to Dr. GUY fore. our data parallel those for lipids and tend to MCKHANNfor helpful discussions and to Dr. PAMELA
Myelin basic protein in jimpy mouse brain
821
E. C. & BRODYR. 0. (1974) Characterization of the fraction obtained from the CNS of jimpy mice by a procedure for myelin isolation. J . Neurochem. 23, 517-523. E. (1972) Jimpy MORELLP. & COSTATINO-CECCARINI mouse: in vitro studies of brain sphingolipid biosynthesis. Lipids 7, 266-268. MOSCARELLO M. A. (1976) Chemical and physical properties of myelin proteins, in Current Topics in Membranes REFERENCES and Transport, Vol. 8, pp. 1-28, Academic Press, New M. (1975) COHENS. R., MCKHANNG. M. & GUARNIERI York. A radioimmunoassay for myelin basic protein and its NORTONW. T. & PODUSLO S. E. (1973) Myelination in use for quantitative measurements. J . Neurocheni. 25, rat brain: changes in composition during brain matur371-376. ation. J . Neurochem. 21, 759-773. DERORERTtS E., PELLICRINO DE IRALDI A., RODRIGUEZ DE M. M. & APPELS. H. (1964) Mutant SIDMANR. L., DICKIE LORESARNAIZG. & SALGANICOFF L. (1962) Cholinergic mice (quaking and jimpy) with deficient myelination in and non-cholinergic nerve endings in rat brain I. J . the central nervous system. Science 144, 309-311. Neurocheni. 9, 23-25. Y.,KIESM. W. & WEBSTER STERNRERGER N. H., ITOYAMA HOGANE. L. (1977) Animal models of genetic disorders H. DEF. (1978) Myelin basic protein demonstrated imof myelin, in Myelin .(MORELLP., ed), pp. 489-520. munocytochemically in oligodendroglia prior to myelin Plenum Press, New York. sheath formation. Proc. natn. Acad. Sci., U . S . A . 75, LOWRY0. H., ROSENBROUGH N. J., FARR A. L. & RANDALL 2521-2527. R. J. (1951) Protein measurement with the Fohn phenol TENNEKOON G . I., COHEN S. R., PRICED. L. & MCKHANN reagent. J . biol. Chem. 193, 165-175. G . M. (1977) Myelinogenesis in optic nerve. J . Cell B i d . J. M.. QUARLES R. H., WEBSTER H. DEF.,HOGAN MATTHIEL 72, 604616. TALALAY for assistance in preparing the manuscript. Supported in part by a fellowship (S.R.C. 1975-1977) and grants from the National Multiple Sclerosis Society (1052-B-3) and the United States Public Health Service (grant No. NS14167 and Research Career Development Award to Dr. COHEN,NSOO315).
Journal of NeurochrniislrJ. Vui. 32 pp. 817 to 821. Pergamon Press Ltd 1979. Printed in Great Britain. 8 International Society for Ncurochemislry Ltd
THE DISTRIBUTION OF MYELIN BASIC PROTEIN IN SUBCELLULAR FRACTIONS OF DEVELOPING JIMPY MOUSE BRAIN' THOMAS R. ZIMMERMAN, JR. and STEVENR. COHEN' Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U S A . (Received 7 August 1978. Accepted 23 October 1978)
Abstract-The amount of myelin basic protein in jimpy mutants and unaffected littermates was measured by radioimmunoassay during the period of most active myelination (11-21 days). This protein was examined in whole brain homogenates and in four subcellular fractions (nuclear, 900g pellet; heavy mernbranc, 11,500 y pellet; microsomal, 100,000 y pellet; and cytosol, 100,000y supernatant solution). At all ages examined, the mutants, which have very little myelin in the CNS, had only about 2% the amount of basic protein found in controls. As expected, the amount of myelin basic protein increased 4-fold in the control animals during the developmental period studied. This was not the case in the jimpy mutants, where little increase in the whole brain basic protein was observed. In the jimpy mutants, all of the fractions had significantly less basic protein than control fractions, except the cytosol. where the amounts of basic protein were similar in controls and mutants. These results are discussed with respcct to possible mechanisms of myelination and the site of the genetic lesion.
THESYNTHESIS of the myelin sheath occurs during a precise time in the development of the mammalian nervous system. In the mouse and rat most of this sheath is laid down between 10 and 25 days after birth. It has been calculated that during this period of rapid myelination, each oligodendrocyte synthesizes more than three times its weight in myelin per 1973). Since the myelin day (NORTON& PODUSLO, membrane is composed of a variety of lipids and proteins, the mechanism of assembly must be under precise control. The site and mechanism of this synthesis, however, remain unknown. Myelin basic protein is unique to the myelin membrane and represents about 30% of the myelin protein (MOSCARELLO, 1976). Its accumulation in the brain parallels the morphological appearance of myelin as well as the accumulation of cerebrosides and sulfatides (TENNEKOON et al., 1977). Basic protein is, therefore, a specific marker for myelin. W e have developed a radioimmunoassay for myelin basic protein (COHEN et al., 1975) which permits measurement of the small quantities of this protein found both in early development and in subcellular fractions. The jimpy mouse is a mutant in which myelinogenesis is arrested early in development (SIDMANet al., 1964). This animal has been the subject of investigation for two main reasons. First, the precise result
A preliminary account was presented at the Ninth Meeting of the American Society for Neurochemistry at Washington, DC. March 1978. *Send all correspondence to S . R. Cohen at above address.
of the genetic defect is most likely to be a n alteration in the primary structure of a protein or in a genetic control mechanism. Identification of this defect may shed light o n a key step in myelin biosynthesis. Second, in these animals, myelinogenesis is perturbed without axonal, inflammatory, gliotic or necrotic changes (HOGAN,1977). Thus, a comparison of the mutant with its unaffected littermate can be used t o probe morphological and biochemical aspects of the assembly and maintenance of myelin. To facilitate these studies, the myelin basic protein was used as a specific marker for myelin. The present paper describes studies on changes in the amount of basic protein in whole brain and subcellular fractions of jimpy and littermate control mice during development. MATERIALS AND METHODS Animals. Mice of the jimpy strain were brcd in our laboratories from a stock obtained from Jackson laboratories. Bar Harbor, ME. All animals were kept with the mother until used. The mothers wcrc fed a commercial pellet diet and water ad lib. Mutants were identified by the observation of an axial body tremor beginning 1&11 days after birth. A t specified ages from 11 to 20 days. mutants and littermate controls were decapitated, the brains were removed, and each brain was homogenized by hand in 20 vol of 0.32 M-sucrose with a Dounce homogenizer. The wet weight of the brains averaged 350 mg, with no observable difference between mutants and controls. A total of 68 mice were examined with at least three mutants and three controls from each age. Fracfionafion and assay. The flow chart in Fig. I describes the fractionation procedure. For a typical fractionation, a volume of about 7ml of homogcnate was used,
817
THOMAS R. ZIMMERMAN JR. and STEVEN R. COHEN
818
900 xg; 10 min
Supernatant
w Pellet rehomogenized in 2
m l 0.32 M sucrose
\1
900 xg; 10 min
!I
I
11,500 xg; 20 min Pellet-
d HEAVY MEMBRANE
NUCLEI
Supernatant
100,000 xg; 60 min
and four fractions were obtained. The first was a nuclear fraction consisting of cell nuclei, cell debris, and unbroken cells. The second, the heavy membrane fraction, consisted of mitochondria and myelin. Since myelin in the jimpy mice may have a density and composition different from normal myelin (MATTHIEU et al., 1974), this fraction was not further purified. The last centrifugation yielded a microsomal pellet, and a final supernatant solution, which was designated the cytosol fraction. The amount of total protein in each fraction was measured by the method of LOWRYet al. (1951). Requisite dilutions were made in water. As previously demonstrated, it was necessary to boil the homogenates before assaying (COHENet al.. 1975). The radioimmunoassay is sensitive to basic protein in a range of 2-20ng. For all of the fractions from control mice, except the cytosol. samples were diluted 1s or 100-fold. In mutants, it was only necessary to dilute the heavy membrane fraction. A 2 0 4 sample of the diluted (in 0.2 M-Trisacetate buffer, pH 7.4-7.5) or undiluted specimen was added to 0.5 ml of 0.2 M-Tris-acetate buffer at pH 7.4-7.5 and incubated with an appropriately diluted antiserum to basic protein at 37°C for 1 h, after which lop1 of '251-Iabeled basic protein solution (1&20 pCi/pg) were added. The samples were then left overnight at room temperature. Next, 0.5ml of cold ethanol was added to each sample tube to precipitate the basic protein antibody complex, and the tubes were maintained for 30 min at 4°C. The samples were centrifuged for 40min at 1660y. The pellet and supernatant solution were counted separately in a Searle 1197 gamma counter, and the per cent bound 1251-labeledbasic protein was calculated. This was compared with a standard curve obtained with purified basic protein. RESULTS
We first compared the quantitative changes in distribution of total protein a n d basic protein in whole brain homogenates of control and mutant mice
between the ages of 11 and 20 days (Fig. 2). Within experimental error, there was no observable difference in the amount of total protein between the controls a n d mutants a t all ages examined. However, the amount of basic protein in the mutants was only 2% of the amount found in controls. Consequently, in this and subsequent graphs these values are expressed on two different scales on the ordinates, one for the control group [left, closed circles) and one for the mutant group (right, open circles). During the period from 11 t o 20 days, the amount of basic protein in the whole brain of control animals increased 4-fold [from 3 t o 12 pg/mg). The mutants, however, were already deficient in basic protein a t
T Control 0
Jimpy
T
P '
II
12
1
13
I
I
14
15
--F 1
16 Age (Days)
I
17
I
18
4
19
I
20
FIG.2. Basic protein in whole brain of mutant and control mice during development. Each point represents duplicate determinations of at least three control and three jimpy mice at each age. The bar is the S.D. Note that the values are presented on two different scales, closed circles (left ordinate) for controls, and open circles (right ordinate) for mutants.
Myelin basic protein in jimpy mouse brain
TABLE1. DISTRIBUTION OF BASIC
PROTEIN IN SUBCELLULAR FRACTIONS
Ratio of basic protein Control Mutant
Total basic protein in fraction (pg) Control Mutant
Fraction Nuclear Heavy membrane Microsomes Cytosol
1.29
0.20 0.077 j, 0.02
21.89 & 5.38
1.53
17 14
0.28
0.47 & 0.08 1.01 f 0.11
4.95 0.34 1.67 f 0.43
11 1.4
Each value represents the average ( ~ s . E .of) determinations from 15 animals between 15 and 17 days old. Fractions were prepared and assayed as described in the text.
11 days, having only 0.05pg/mg. This amount increased with age, reaching a maximum at approx 16 days and then declining to about 0.1 pg/ml at 20 days. A comparison of the distribution of basic protein among the subcellular fractions of the control animals and mutants is shown in Table 1 for mice 15-17 days old. The heavy membrane fraction which contains myelin had the greatest amount of basic protein in both groups of animals. This fraction represented
819
nearly 75% of the total basic protein in the controls and 50% in the mutants. The nuclear fraction accounted for similar proportions of the total basic protein in normal and jimpy brain, about 3%. Likewise, the microsomal fraction in both normal and mutant brain had 17% of the total basic protein. In sharp contrast, the cytosol from normal brain accounted for only 5% of the basic protein, whereas the amount from the jimpy mice cytosol was 33% of the total brain basic protein. Figure 3 describes the changes in basic protein that took place during development for each of the four fractions. The amount of basic protein in the nuclear fractions in control animals was only about 0.8 pg/mg at 11 days, which increased 1.5-fold to about 1.2pg/mg in 20-day animals. The mutants, which at 11 days contained only O.OSpg/mg of basic protein in this fraction, showed an increase in basic protein to a maximal value of 0.075 pg/mg at 16 days, which then declined to a value of O.OSpg/mg at 20 days. The heavy membrane fraction is the fraction in which myelin or myelin-like membranes sediment. The control animals exhibited a 4-fold increase in basic protein from 0.6 pg/mg at 11 days to 2.8 pg/mg
Nucleor Fraction
.I
Heavy Membrane Fraction
t 2.0 2.4
E"
2a
t
1.6-
&
0
$
1.2-
"7
m" 0.8-
,0.2
i
I
1
I
I
I
II
12
13
14
I
,
I
15
16
17
,
18
,
19
, ) 20
II
12
13
14
Age (Days)
15
16
17
18
19
Age (Doys)
Micrasomol Froction Cytosol Fraction
t
g 2.4a
Control
0.3 - o Jirnpy
2.0-
E" G
1.6-
0
;1.2-
czp 0.80.4 -
I # I1
I
I
12
13
I
8
I
I
I
15
16
17
I8
I9
I
20
Age (Days)
FIG.3. Basic protein in subcellular fractions of jimpy and control mice during development. Subcellular fractions were prepared and assayed for myelin basic protein as described. Each point represents duplicate determinations of at least three control or jimpy mice at each age. The bar is the standard deviation. For the nuclear, heavy membrane and rnicrosomal fractions data from jimpy and control mice are presented on different scales on the ordinate. For the cytosol fraction, data from mutant and control mice are presented on the same scale.
20
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THOMAS R . ZIMMERMAN J R . and STEVEN R . COHEN
at 20 days. The amount of basic protein in the mutant at I 1 days (0.05pg/mg) increased only 2-fold and remained relatively constant at about 0.1 pg/mg. In the microsomal fraction the amount of basic protein in the mutant was also reduced at 11 days; mutants had only 0.1 pg/mg compared to l.Opg/mg in the controls. This is the only fraction, however, in which the amount of basic protein in the mutants increased steadily for the entire developmental period. Moreover, for both mutants and controls, the amount of basic protein in this fraction at 20 days was twice the initial 11-day values. In the cytosol fraction the amounts of basic protein in both mutants and controls are presented on the same scale. This fraction represented a small proportion of the total brain basic protein (Table 1) in the controls, but 33% in the jimpy. Clearly, in the jimpy mouse the amount of basic protein in this fraction was least affected by the mutation. The developmental distribution of basic protein in this fraction was similar for both groups of animals. The control animals contained 0.15pg/mg at 16 days and then declined to approx 0.22pg/mg at 20 days. This is the only fraction among control animals in which there was a decline in basic protein during the developmental period. The mutants exhibited a similar 2-fold increase in basic protein from 11 to 16 days. At 20 days, however, the value for basic protein fell below the initial 11-day value of 0.1 pg/mg.
support the view that biosynthesis of myelin-associated components is coordinately controlled (MORELL & COSTATINO-CECCARINI, 1972). The amount of basic protein in the cytosol relative to other fractions is very small. It is possible that this basic protein is the result of contamination from other fractions during the fractionation procedure; however, because of the significant change with development, this is unlikely. In addition, the amount of basic protein in the cytosol of the mutants is nearly normal; this may constitute a small but important ‘pool’ which is the source of basic protein for the initial formation of myelin. In this context, STERNBERGER et al. (1978) have recently shown by immunocytochemical techniques that the cytoplasm of oligodendrocytes stains with antibody to myelin basic protein. Furthermore, this staining intensity increases during the period of early myelination, and then decreases. That both controls and mutants exhibit the same 2-fold accumulation of basic protein in this fraction during the initial period from 11 to 16 days is an indication that the mechanism of basic protein synthesis and movement into this fraction is nearly normal and is not directly affected by the genetic lesion. The decline in the amount of basic protein in the cytosol which begins at 16 days may be related to the continued increase in basic protein in the microsomes of the mutants and controls. Since the concentration of total protein is the same for both controls and mutants, this decrease of basic protein in the cytosol is not attributable merely to a dilution DISCUSSION of basic protcin by an increase in the amount of total In control mice the amount of myelin basic protein protein. The perturbation of myelin biosynthesis, therefore, in the whole brain increases between 10 and 20 days. As expected, most of this basic protein is present in may exist at the point in the pathway at which basic the heavy membrane fraction, which contains myelin. protein combines with myelin lipids and other proIn the mutants, however, basic protein increases only teins from the lighter membranes of the microsomes from 10 to 16 days and then decreases. As in the to form the heavier myelin membranes. A comparison controls, the greatest amount of basic protein is in of the developmental changes of the microsomes with those in the heavy membrane fraction tends to supthe heavy membrane fraction. Other investigators have reported that myelin-asso- port this view. During the period from 16 to 20 days ciated lipids such as sulfatides and cerebrosides are the amount of basic protein increases in both the severely reduced in the jimpy mutant, whereas other microsomes and the heavy membrane fraction of the lipids, not specific to myelin, are only slightly affected controls. Thc mutants, however, d o not show the (HOGAN, 1977). Similarly, we find that while basic same pattern: although the basic protein in the microprotein is reduced in the whole brains of mutants, somes increases, the amount of basic protein in the there is no observable difference in the amount of heavy membrane remains relatively constant. Hence, total protein between mutants and controls. Hogan there appears to be little movement of basic protein also found that the amounts of cerebroside in whole from the microsomes to the heavy membrane. Failure of basic protein to enter the heavier membrain increased in the jimpy mutant between 10 and 15 days, followed by a decrease. We find a similar brane might result in an accumulation of basic propattern for the changes in amounts of basic protein tein in the microsomes. This accumulation might then in whole brain of jimpy mutants. Furthermore, in a result in feedback inhibition on earlier processes of study of sphingolipid biosynthesis, microsomal prep- myelin assembly, a consequence which is reflected by arations from jimpy mutants had only 1&20% the the reduced amount of basic protein in the microamount of galactosylceramide present in the micro- somes. We are currently investigating these possibilities. somes of controls (MORELL& COSTATINO-CECCARINI, 1972). The amount of basic protein in the microsomes of mutants is only 15% the amount in controls. There- Acknowledgements-The authors are grateful to Dr. GUY fore. our data parallel those for lipids and tend to MCKHANNfor helpful discussions and to Dr. PAMELA
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E. C. & BRODYR. 0. (1974) Characterization of the fraction obtained from the CNS of jimpy mice by a procedure for myelin isolation. J . Neurochem. 23, 517-523. E. (1972) Jimpy MORELLP. & COSTATINO-CECCARINI mouse: in vitro studies of brain sphingolipid biosynthesis. Lipids 7, 266-268. MOSCARELLO M. A. (1976) Chemical and physical properties of myelin proteins, in Current Topics in Membranes REFERENCES and Transport, Vol. 8, pp. 1-28, Academic Press, New M. (1975) COHENS. R., MCKHANNG. M. & GUARNIERI York. A radioimmunoassay for myelin basic protein and its NORTONW. T. & PODUSLO S. E. (1973) Myelination in use for quantitative measurements. J . Neurocheni. 25, rat brain: changes in composition during brain matur371-376. ation. J . Neurochem. 21, 759-773. DERORERTtS E., PELLICRINO DE IRALDI A., RODRIGUEZ DE M. M. & APPELS. H. (1964) Mutant SIDMANR. L., DICKIE LORESARNAIZG. & SALGANICOFF L. (1962) Cholinergic mice (quaking and jimpy) with deficient myelination in and non-cholinergic nerve endings in rat brain I. J . the central nervous system. Science 144, 309-311. Neurocheni. 9, 23-25. Y.,KIESM. W. & WEBSTER STERNRERGER N. H., ITOYAMA HOGANE. L. (1977) Animal models of genetic disorders H. DEF. (1978) Myelin basic protein demonstrated imof myelin, in Myelin .(MORELLP., ed), pp. 489-520. munocytochemically in oligodendroglia prior to myelin Plenum Press, New York. sheath formation. Proc. natn. Acad. Sci., U . S . A . 75, LOWRY0. H., ROSENBROUGH N. J., FARR A. L. & RANDALL 2521-2527. R. J. (1951) Protein measurement with the Fohn phenol TENNEKOON G . I., COHEN S. R., PRICED. L. & MCKHANN reagent. J . biol. Chem. 193, 165-175. G . M. (1977) Myelinogenesis in optic nerve. J . Cell B i d . J. M.. QUARLES R. H., WEBSTER H. DEF.,HOGAN MATTHIEL 72, 604616. TALALAY for assistance in preparing the manuscript. Supported in part by a fellowship (S.R.C. 1975-1977) and grants from the National Multiple Sclerosis Society (1052-B-3) and the United States Public Health Service (grant No. NS14167 and Research Career Development Award to Dr. COHEN,NSOO315).
