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Encephalitogenic properties of myelin from the quaking mutant
Brain Research, 107 (1976) 633-637
633
© Elsevier ScientificPublishingCompany,Amsterdam- Printed m The Netherlands
Encephalitogenic properties of myelin from the quaking mutant
S. W. BROSTOFF, J. M. POWERS, H. M. WISNIEWSKI* AND E. L. HOGAN Departments of Neurology, Biochemistry and Pathology, The Medical University of South Carolina and the Veterans Administration Hospital, Charleston, S.C. 29401, and the Department of Pathology ( Neuropathology), Albert Einstein College of Medicine, Bronx, N. Y. 10461 (U.S.A.)
(Accepted February4th, 1976)
The quaking mutation in mice is an autosomal recessive trait associated with disorder in myelin formation. It manifests itself clinically 11-12 days after birth with the appearance of axial tremors. The affected animals will usually live a normal life span, but with tonic extensor seizures throughout. Morphologic studies of the CNS reveal a marked reduction in the number of lamellae for a given axon diameter with a lack of compaction of the myelin and formation of cytoplasmic pockets3,n, 12. The similarity of the morphologic appearance of mutant myelin to that observed in young normal mice supports the idea that the adult quaking mutant survives in an interrupted state of myelin development, an immature state resembling the early phase of myelination. Biochemical studies in which the composition of both the lipids2,s and proteins 7 of mutant myelin have been compared to normal myelin have generally supported this thesis. We have now extended this comparison to the immunological properties of myelin responsible for producing experimental allergic encephalomyelitis (EAE). This communication details the encephalitogenic properties of myelin isolated from normal, normal immature, and quaking mutant mice. All myelin was prepared by subcellular fraction of whole mouse brain homogenates on discontinuous sucrose gradients 10. Each preparation was processed through the gradient ultracentrifugation at least twice. From 6 to 18 mouse brains were used for each preparation. Adult myelin was prepared from animals at least three months of age. The assay for encephalitogenic activity of each myelin sample was carried out using randomly bred guinea pigs weighing 400-500 g. Each animal received an intradermal injection into each hind footpad of 0.05 ml of the test emulsion for a total of 0.1 ml per animal. The test emulsion was prepared by suspending the myelin sample in normal saline and mixing with an equal volume of adjuvant containing either 0.5 mg/ml Mycobacterium butyricum (complete Freunds adjuvant, Difco) or 1.0 mg/ml Mycobacterium tuberculosis (Difco, H 37 ra). The animals were observed * Director, MRC, DemyelinatingDiseases Unit, Newcastle General Hospital, Westgate Road, Newcastle-upon-TyneNE4 6BE, Great Britain.
634 TABLE I ENCEPHALITOGENIC .
.
.
.
.
.
.
.
.
A C T I V I T Y IN G U I N E A PIGS OF M Y E L I N F R O M Q U A K I N G A N D N O R M A L MICE .
.
.
.
.
.
.
Test Antigen* animal
.
.
.
.
.
.
.
.
Symptoms
.
.
.
.
.
Day of saertfice
Amount of per~Nature of vascular cuflfin~g infiltrates (cellpopulation) * *
1 2
Quaking myehn Quaking myelin
Asymptomatic 30 Asymptomatlc 30
-t /--
3 4 5 6
Quaking myehn Quaking myelin Quaking myehn Quaking myehn
Asymptomatic 30 Aysmptomatic 30 Asymptomatic 30 Asymptomatic 30
w ~ -t t I /--
7
Quaking myehn
Asymptomatlc
30
: /--
8
Quaking myehn
AsymptomatJc
30
-'.- t-
Mononuclear (pial region) Mononuclear Mononuclear Mononuclear Mononuclear (pial region) Mononuclear (pial region) Mononuclear
9
Normal adult myelin
30
~ ~
Mononuclear
10 11 12 13 14 15 16 17
Normal adult myelin Normal adult myelin Normal adult myelin Normaladultmyelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin
Asymptomatic (wt. loss) Asymptomatlc Asymptomatic Asymptomatlc Asymptomatic Asymptomatic Mild, day 22-26 Asymptomatic Asymptomatic
30 30 30 30 30 30 30 30
~ + ~-t / - ~~ T ~ ÷ ? t /--
Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear (pial region)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
* The dose was 2 mg of myelin in Dlfco complete Freunds adjuvant, containing 0.5 mg/ml of Mycobactertum butyricum.
** The system used to grade the histology is similar to that used by Atvord and Kies 1. The signs used are: --, no abnormality; --/--, a few small foci of perivenular leukocytes within neuraxls; + , many small foci of perivenular leukocytes within neuraxis; + ÷ , large and numerous foci of perivenular leukocytes within neuraxls.
daily for clinical s y m p t o m s o f disease such as incontinence, ataxia, paresis o r p a r a l ysis. T h e y were sacrificed either u p o n a p p e a r a n c e o f severe clinical s y m p t o m s or after a p e r i o d o f at least 27 days following sensitization. Tissue fixation was initiated by m t r a c a r d i a c perfusion o f neutral buffered f o r m a l i n . A f t e r several days o f fixation in f o r m a l i n the neuraxis f r o m each a n i m a l was dissected free o f its osseous a n d m u s c u l a r coverings a n d sectioned b o t h transversely and longitudinally. F o l l o w i n g d e h y d r a t i o n a n d paraffin e m b e d d i n g , sections were stained with h e m a t o x y l i n a n d eosin to delineate the n a t u r e and e x t e n t o f perivasc u l a r i n f l a m m a t o r y infiltration. The g r a d i n g o f the lesions basically c o n f o r m e d to the system p r o p o s e d by A l v o r d and Kies 1. In the imtial experiment, a n i m a l s were injected with 2 nag o f myelin f r o m either n o r m a l a d u l t s o r q u a k i n g m u t a n t adults. T h e results using c o m p l e t e F r e u n d s a d j u v a n t (Difco) are shown in T a b l e I. N o n e o f the animals in either group, with the exception
635 TABLE II ENCEPHALITOGENIC ACTIVITY IN GUINEA PIGS OF MYELIN FROM QUAKING AND NORMAL MICE
Test Antigen* animal
Symptoms
1 2 3 4 5 6 7 8 9 10 I1
Asymptomatic 27 Asymptomatic 27 Asymp~omatic 27 Asymptomatic 27 Asymptomatic 27 Symptomatic 12 Symptomatic 12 Symptomatic 12 Symptomatic 11 Symptomatic 21 Asymptomatic 27
Quaking myelin Quaking myehn Quaking myelin Quaking myelin Quaking myelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin
Day of sacrifice
Amount of perivascularcuffing (cellpopulation)
-+ +/-+/--++ ++ ++ ++ + +/--
Nature of infiltrates
Mononuclear Mononuclear Mononuclear Mixed Mixed Mixed Mixed Mononuclear Mononuclear (plal region)
* The dose was 2 mg of myelin in Difco H 37 ra adjuvant containing 1 mg/ml of Mycobacterium tuberculosis.
** The system used to grade the histology is similar to that used by Alvord and KiesI. For signs, see Table I.
of one animal receiving normal myelin, exhibited any symptoms of allergic encephalomyelitis. The symptomatic animal exhibited mild weakness between day 22 and 26 postsensitization. Another animal exhibited weight loss but no other symptoms. There was histological evidence, however, of CNS disease in all but one animal. The histological lesions were more severe in those guinea pigs receiving myelin isolated from normal mice. Severe lesions were found in the symptomatic animal and in the one with weight loss. The only animal without detectable lesions had received myelin prepared from the quaking mutant. The experiment was repeated using a more potent adjuvant, Difco H 37 ra (Table II). Under these conditions all but one of the guinea pigs receiving normal mouse myelin developed clinical symptoms of disease while none of those receiving myelin isolated from quaking mice became symptomatic. The histology also revealed the greater severity of the disease in animals receiving normal myelin compared to those receiving myelin from quaking mutants. Analysis of myelin protein content has shown that the large basic protein is present in relatively greater amount in normal mouse myelin (12 ~ of total protein) 9 than in myelin from quaking mice (10 ~)7. To insure that the effect of comparable quantities of antigen were studied, 5 additional guinea pigs were injected with larger doses (4 mg) of quaking myelin. Even at this larger dose (Table III) and with the more potent adjuvant (Difco H 37 ra), only one animal receiving mutant myelin exhibited symptoms or severe histological lesions. Thus the difference in severity of the disease caused by myelin isolated from normal and quaking mice cannot be explained solely by differences in content of basic protein.
636 TABLE ill ENCEPHALITOGENIC ACTIVITY IN GUINEA PIGS OF MYELIN FROM QUAKING AND YOUNG NORMAL ( 18 DAYS OLD) MICE
Test Antigen* animal
Dose Symptoms (rag)
Day oJ sacrifice
Amount oJ periNature oj vascular cuffing infiltrates ( cell population) * *
1 2 3 4 5 6 7 8 9 10 1!
4 4 4 4 4 2 2 2 2 4 4
30 30 30 13 30 12 12 13 30 13 13
-t ./-- to ~ ~ ! 4 -+÷ ~ -~+÷ ~ ~ . -+ + ÷
Quaking myelin Quaking myelin Quaking myelin Quaking myelin Quaking myelin Normal young myelin Normal young myelin Normal young myelin Normal young myelin Normal young myelin Normal young myelin
Asymptomatic Asymptomatic Asymptomatlc Paresis Asymptomatic Paralysis Paresis Dead Mild Paresis Paresis Paresis
Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mixed Mononuctear Mononuclear Mononuclear
* The adjuvant used was DlfCO H 37 ra which contains 1.0 mg/ml of Mycobacterium tuberculosis. ** The system used to grade the histology is similar to that used by Alvord and Kies1. For signs, see Table I.
The proposed similarity between myelin from quaking mice and normal young mice was investigated by testing the behavior of these membranes in the EAE assay. Guinea pigs were injected with either 2 mg or 4 mg of normal myelin from young (18-day-old) mice (Table III). All of the guinea pigs receiving myelin prepared from normal young mice exhibited disease, and histological evaluation revealed that all these ammals developed severe lesions. It should be noted that myelin from an 18-day-old mouse contains the same amount of the larger or encephalitogenic basic protein a as does myehn from an adult quaking mutant 7. Yet, despite the similar content and distribution of basic proteins in myelin from quaking mice and normal young mice, there is a significant difference in the encephalitogenic potential of the two membranes. One approach to studying a membrane product such as myelin from a mutant strain is to compare its antigenic properties to those of the normal myelin membrane. A difference m such properties would reflect a difference in the structure of the membrane. In this study we have compared the antigenic determinant of myelin responsible for producing EAE in guinea pigs in order to probe structural differences between normal myelin and myelin from the quaking mutant. The encephalitogenic determinant of myelin active in the guinea pig resides in the region (residues 113-121) of the large myelin basic protein which contains the only tryptophan residue 8. Based upon the sequence of the small rat basic protein 5, it is likely that this tryptophan region is incomplete in the small mouse basic protein and as a result the small protein would not be encephatitogenic. No other myelin component and no other region of the basic protein is active in this immunologic assay 6.
637 Our experiments indicate that the lessened encephalitogenicity of the myelin isolated from quaking mutants is not due to a reduced quantity of basic protein in the sample. The large or encephalitogenic (for the guinea pig) basic protein amounts to 10 % of myelin protein in the quaking mutant 7 and in the young (18 days) mouse 9 and 12 % in adult mice 9. Thus, the higher dose (4 mg) of myelin isolated from quaking mice contains almost double the amount of large basic protein as does 2 mg of normal myelin and, yet, is only mildly encephalitogenic. This suggests either that a genetic alteration has occurred in the tryptophan region of the mutant's basic protein or that the altered composition of the myelin membrane in the mutant has affected the antigenicity of the basic protein by altering its conformation or lessening its availability. Recent work in our laboratory 4 bears on the latter possibility. We have observed that the lipid environment in which the basic protein exists can alter the encephalitogenie nature of the basic protein. This study employed artificial membranes containing cerebroside and basic protein; the addition of the cerebroside altered the encephalitogenic response to the basic protein. We thank Mrs. Katherine Cowart, Melissa Crabtree and Beverly Robinson for their capable technical assistance and Mrs. Lynda Hills for typing the manuscript. This work was supported in part by G r a n t NS 12044 from the U S P H S and G r a n t 692 from the National Multiple Sclerosis Society. 1 ALVORD,E. C., ANDIOes, M. W., Clinic.o-pathologiccorrelation in experimental allergic encephalomyelitis. V. Development of an index for quantitative assay of encephalitogenic activity of antigens, J. Neuropath. exp. Neurol., 18 (1959) 447-457. 2 BAUMANN,N., JACQUE,C. M., POLLET,S. A., ANDHARPIN,M. L., Fatty acid and lipid composition of the brain of a myelin deficient mutant, the (quaking) mouse, Europ. J. Biochem., 4 (1968) 340-344. 3 BERGER,B., Quelques aspects ultrastructuraux de la substance blanche chez la souris Quaking, Brain Research, 25 (1971) 35-53. 4 BROSTOFF,S. W., AND POWERS,J. M., Allergic encephalomyelitis: modification of the response by synthetic membrane structures containing bovine myelin basic protein and cerebroside, Brain Research, 93 (1975) 175-181. 5 DUNKL~Y,P. R., AND CARNEGIE,P. R., Amino acid sequence of the smaller basic protein from rat brain myelin, Biochem J., 141 (1974) 243-255. 6 EYLAR,E. H., CACCAM,J., JACKSON,J. J., WESTALL,F. C., AND ROBINSON,A. B., Experimental allergic encephalomyelitis: synthesis of disease inducing sight of the basic protein, Science, 168 (1970) 1220-1223. 7 GREENFIELD,S., NORTON,W. T., ANDMORELL,P., Quaking mouse: isolation and characterization of myehn proteins, J. Neurochem., 18 (1971) 2119-2128. 8 HOGAN, E. L., AND JOSEPH, K.C., Composition of cerebral lipids in murine leucodystrophy: the quaking mutant, J. Neurochem., 17 (1970) 1209-1214. 9 MORELL,P., GREENFIELD,S., CONSTANTINO=CECCARINI,E., ANDWISNIEWSKI,H., Changes in the protein composition of mouse brain myelin during development, J. Neurochem., 19 (1972) 2545-2554. 10 NORTON, W. T., AND PODUSLO, S. E., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-757. I 1 SAMORAJSKI,T., FREIDE, R. L., AND REIMER,P. R., Hypomyelination in the quaking mouse. A model for the analysis of disturbed myelin formation, J. Neuropath. exp. Neurol., 29 (1970) 507. 12 WIS~aEWSKI,H., ANDMORELL,P., Quaking mouse: ultrastructural evidence for arrest of myelinogenesis, Brain Research, 29 (1971) 63-73.
633
© Elsevier ScientificPublishingCompany,Amsterdam- Printed m The Netherlands
Encephalitogenic properties of myelin from the quaking mutant
S. W. BROSTOFF, J. M. POWERS, H. M. WISNIEWSKI* AND E. L. HOGAN Departments of Neurology, Biochemistry and Pathology, The Medical University of South Carolina and the Veterans Administration Hospital, Charleston, S.C. 29401, and the Department of Pathology ( Neuropathology), Albert Einstein College of Medicine, Bronx, N. Y. 10461 (U.S.A.)
(Accepted February4th, 1976)
The quaking mutation in mice is an autosomal recessive trait associated with disorder in myelin formation. It manifests itself clinically 11-12 days after birth with the appearance of axial tremors. The affected animals will usually live a normal life span, but with tonic extensor seizures throughout. Morphologic studies of the CNS reveal a marked reduction in the number of lamellae for a given axon diameter with a lack of compaction of the myelin and formation of cytoplasmic pockets3,n, 12. The similarity of the morphologic appearance of mutant myelin to that observed in young normal mice supports the idea that the adult quaking mutant survives in an interrupted state of myelin development, an immature state resembling the early phase of myelination. Biochemical studies in which the composition of both the lipids2,s and proteins 7 of mutant myelin have been compared to normal myelin have generally supported this thesis. We have now extended this comparison to the immunological properties of myelin responsible for producing experimental allergic encephalomyelitis (EAE). This communication details the encephalitogenic properties of myelin isolated from normal, normal immature, and quaking mutant mice. All myelin was prepared by subcellular fraction of whole mouse brain homogenates on discontinuous sucrose gradients 10. Each preparation was processed through the gradient ultracentrifugation at least twice. From 6 to 18 mouse brains were used for each preparation. Adult myelin was prepared from animals at least three months of age. The assay for encephalitogenic activity of each myelin sample was carried out using randomly bred guinea pigs weighing 400-500 g. Each animal received an intradermal injection into each hind footpad of 0.05 ml of the test emulsion for a total of 0.1 ml per animal. The test emulsion was prepared by suspending the myelin sample in normal saline and mixing with an equal volume of adjuvant containing either 0.5 mg/ml Mycobacterium butyricum (complete Freunds adjuvant, Difco) or 1.0 mg/ml Mycobacterium tuberculosis (Difco, H 37 ra). The animals were observed * Director, MRC, DemyelinatingDiseases Unit, Newcastle General Hospital, Westgate Road, Newcastle-upon-TyneNE4 6BE, Great Britain.
634 TABLE I ENCEPHALITOGENIC .
.
.
.
.
.
.
.
.
A C T I V I T Y IN G U I N E A PIGS OF M Y E L I N F R O M Q U A K I N G A N D N O R M A L MICE .
.
.
.
.
.
.
Test Antigen* animal
.
.
.
.
.
.
.
.
Symptoms
.
.
.
.
.
Day of saertfice
Amount of per~Nature of vascular cuflfin~g infiltrates (cellpopulation) * *
1 2
Quaking myehn Quaking myelin
Asymptomatic 30 Asymptomatlc 30
-t /--
3 4 5 6
Quaking myehn Quaking myelin Quaking myehn Quaking myehn
Asymptomatic 30 Aysmptomatic 30 Asymptomatic 30 Asymptomatic 30
w ~ -t t I /--
7
Quaking myehn
Asymptomatlc
30
: /--
8
Quaking myehn
AsymptomatJc
30
-'.- t-
Mononuclear (pial region) Mononuclear Mononuclear Mononuclear Mononuclear (pial region) Mononuclear (pial region) Mononuclear
9
Normal adult myelin
30
~ ~
Mononuclear
10 11 12 13 14 15 16 17
Normal adult myelin Normal adult myelin Normal adult myelin Normaladultmyelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin
Asymptomatic (wt. loss) Asymptomatlc Asymptomatic Asymptomatlc Asymptomatic Asymptomatic Mild, day 22-26 Asymptomatic Asymptomatic
30 30 30 30 30 30 30 30
~ + ~-t / - ~~ T ~ ÷ ? t /--
Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear (pial region)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
* The dose was 2 mg of myelin in Dlfco complete Freunds adjuvant, containing 0.5 mg/ml of Mycobactertum butyricum.
** The system used to grade the histology is similar to that used by Atvord and Kies 1. The signs used are: --, no abnormality; --/--, a few small foci of perivenular leukocytes within neuraxls; + , many small foci of perivenular leukocytes within neuraxis; + ÷ , large and numerous foci of perivenular leukocytes within neuraxls.
daily for clinical s y m p t o m s o f disease such as incontinence, ataxia, paresis o r p a r a l ysis. T h e y were sacrificed either u p o n a p p e a r a n c e o f severe clinical s y m p t o m s or after a p e r i o d o f at least 27 days following sensitization. Tissue fixation was initiated by m t r a c a r d i a c perfusion o f neutral buffered f o r m a l i n . A f t e r several days o f fixation in f o r m a l i n the neuraxis f r o m each a n i m a l was dissected free o f its osseous a n d m u s c u l a r coverings a n d sectioned b o t h transversely and longitudinally. F o l l o w i n g d e h y d r a t i o n a n d paraffin e m b e d d i n g , sections were stained with h e m a t o x y l i n a n d eosin to delineate the n a t u r e and e x t e n t o f perivasc u l a r i n f l a m m a t o r y infiltration. The g r a d i n g o f the lesions basically c o n f o r m e d to the system p r o p o s e d by A l v o r d and Kies 1. In the imtial experiment, a n i m a l s were injected with 2 nag o f myelin f r o m either n o r m a l a d u l t s o r q u a k i n g m u t a n t adults. T h e results using c o m p l e t e F r e u n d s a d j u v a n t (Difco) are shown in T a b l e I. N o n e o f the animals in either group, with the exception
635 TABLE II ENCEPHALITOGENIC ACTIVITY IN GUINEA PIGS OF MYELIN FROM QUAKING AND NORMAL MICE
Test Antigen* animal
Symptoms
1 2 3 4 5 6 7 8 9 10 I1
Asymptomatic 27 Asymptomatic 27 Asymp~omatic 27 Asymptomatic 27 Asymptomatic 27 Symptomatic 12 Symptomatic 12 Symptomatic 12 Symptomatic 11 Symptomatic 21 Asymptomatic 27
Quaking myelin Quaking myehn Quaking myelin Quaking myelin Quaking myelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin Normal adult myelin
Day of sacrifice
Amount of perivascularcuffing (cellpopulation)
-+ +/-+/--++ ++ ++ ++ + +/--
Nature of infiltrates
Mononuclear Mononuclear Mononuclear Mixed Mixed Mixed Mixed Mononuclear Mononuclear (plal region)
* The dose was 2 mg of myelin in Difco H 37 ra adjuvant containing 1 mg/ml of Mycobacterium tuberculosis.
** The system used to grade the histology is similar to that used by Alvord and KiesI. For signs, see Table I.
of one animal receiving normal myelin, exhibited any symptoms of allergic encephalomyelitis. The symptomatic animal exhibited mild weakness between day 22 and 26 postsensitization. Another animal exhibited weight loss but no other symptoms. There was histological evidence, however, of CNS disease in all but one animal. The histological lesions were more severe in those guinea pigs receiving myelin isolated from normal mice. Severe lesions were found in the symptomatic animal and in the one with weight loss. The only animal without detectable lesions had received myelin prepared from the quaking mutant. The experiment was repeated using a more potent adjuvant, Difco H 37 ra (Table II). Under these conditions all but one of the guinea pigs receiving normal mouse myelin developed clinical symptoms of disease while none of those receiving myelin isolated from quaking mice became symptomatic. The histology also revealed the greater severity of the disease in animals receiving normal myelin compared to those receiving myelin from quaking mutants. Analysis of myelin protein content has shown that the large basic protein is present in relatively greater amount in normal mouse myelin (12 ~ of total protein) 9 than in myelin from quaking mice (10 ~)7. To insure that the effect of comparable quantities of antigen were studied, 5 additional guinea pigs were injected with larger doses (4 mg) of quaking myelin. Even at this larger dose (Table III) and with the more potent adjuvant (Difco H 37 ra), only one animal receiving mutant myelin exhibited symptoms or severe histological lesions. Thus the difference in severity of the disease caused by myelin isolated from normal and quaking mice cannot be explained solely by differences in content of basic protein.
636 TABLE ill ENCEPHALITOGENIC ACTIVITY IN GUINEA PIGS OF MYELIN FROM QUAKING AND YOUNG NORMAL ( 18 DAYS OLD) MICE
Test Antigen* animal
Dose Symptoms (rag)
Day oJ sacrifice
Amount oJ periNature oj vascular cuffing infiltrates ( cell population) * *
1 2 3 4 5 6 7 8 9 10 1!
4 4 4 4 4 2 2 2 2 4 4
30 30 30 13 30 12 12 13 30 13 13
-t ./-- to ~ ~ ! 4 -+÷ ~ -~+÷ ~ ~ . -+ + ÷
Quaking myelin Quaking myelin Quaking myelin Quaking myelin Quaking myelin Normal young myelin Normal young myelin Normal young myelin Normal young myelin Normal young myelin Normal young myelin
Asymptomatic Asymptomatic Asymptomatlc Paresis Asymptomatic Paralysis Paresis Dead Mild Paresis Paresis Paresis
Mononuclear Mononuclear Mononuclear Mononuclear Mononuclear Mixed Mononuctear Mononuclear Mononuclear
* The adjuvant used was DlfCO H 37 ra which contains 1.0 mg/ml of Mycobacterium tuberculosis. ** The system used to grade the histology is similar to that used by Alvord and Kies1. For signs, see Table I.
The proposed similarity between myelin from quaking mice and normal young mice was investigated by testing the behavior of these membranes in the EAE assay. Guinea pigs were injected with either 2 mg or 4 mg of normal myelin from young (18-day-old) mice (Table III). All of the guinea pigs receiving myelin prepared from normal young mice exhibited disease, and histological evaluation revealed that all these ammals developed severe lesions. It should be noted that myelin from an 18-day-old mouse contains the same amount of the larger or encephalitogenic basic protein a as does myehn from an adult quaking mutant 7. Yet, despite the similar content and distribution of basic proteins in myelin from quaking mice and normal young mice, there is a significant difference in the encephalitogenic potential of the two membranes. One approach to studying a membrane product such as myelin from a mutant strain is to compare its antigenic properties to those of the normal myelin membrane. A difference m such properties would reflect a difference in the structure of the membrane. In this study we have compared the antigenic determinant of myelin responsible for producing EAE in guinea pigs in order to probe structural differences between normal myelin and myelin from the quaking mutant. The encephalitogenic determinant of myelin active in the guinea pig resides in the region (residues 113-121) of the large myelin basic protein which contains the only tryptophan residue 8. Based upon the sequence of the small rat basic protein 5, it is likely that this tryptophan region is incomplete in the small mouse basic protein and as a result the small protein would not be encephatitogenic. No other myelin component and no other region of the basic protein is active in this immunologic assay 6.
637 Our experiments indicate that the lessened encephalitogenicity of the myelin isolated from quaking mutants is not due to a reduced quantity of basic protein in the sample. The large or encephalitogenic (for the guinea pig) basic protein amounts to 10 % of myelin protein in the quaking mutant 7 and in the young (18 days) mouse 9 and 12 % in adult mice 9. Thus, the higher dose (4 mg) of myelin isolated from quaking mice contains almost double the amount of large basic protein as does 2 mg of normal myelin and, yet, is only mildly encephalitogenic. This suggests either that a genetic alteration has occurred in the tryptophan region of the mutant's basic protein or that the altered composition of the myelin membrane in the mutant has affected the antigenicity of the basic protein by altering its conformation or lessening its availability. Recent work in our laboratory 4 bears on the latter possibility. We have observed that the lipid environment in which the basic protein exists can alter the encephalitogenie nature of the basic protein. This study employed artificial membranes containing cerebroside and basic protein; the addition of the cerebroside altered the encephalitogenic response to the basic protein. We thank Mrs. Katherine Cowart, Melissa Crabtree and Beverly Robinson for their capable technical assistance and Mrs. Lynda Hills for typing the manuscript. This work was supported in part by G r a n t NS 12044 from the U S P H S and G r a n t 692 from the National Multiple Sclerosis Society. 1 ALVORD,E. C., ANDIOes, M. W., Clinic.o-pathologiccorrelation in experimental allergic encephalomyelitis. V. Development of an index for quantitative assay of encephalitogenic activity of antigens, J. Neuropath. exp. Neurol., 18 (1959) 447-457. 2 BAUMANN,N., JACQUE,C. M., POLLET,S. A., ANDHARPIN,M. L., Fatty acid and lipid composition of the brain of a myelin deficient mutant, the (quaking) mouse, Europ. J. Biochem., 4 (1968) 340-344. 3 BERGER,B., Quelques aspects ultrastructuraux de la substance blanche chez la souris Quaking, Brain Research, 25 (1971) 35-53. 4 BROSTOFF,S. W., AND POWERS,J. M., Allergic encephalomyelitis: modification of the response by synthetic membrane structures containing bovine myelin basic protein and cerebroside, Brain Research, 93 (1975) 175-181. 5 DUNKL~Y,P. R., AND CARNEGIE,P. R., Amino acid sequence of the smaller basic protein from rat brain myelin, Biochem J., 141 (1974) 243-255. 6 EYLAR,E. H., CACCAM,J., JACKSON,J. J., WESTALL,F. C., AND ROBINSON,A. B., Experimental allergic encephalomyelitis: synthesis of disease inducing sight of the basic protein, Science, 168 (1970) 1220-1223. 7 GREENFIELD,S., NORTON,W. T., ANDMORELL,P., Quaking mouse: isolation and characterization of myehn proteins, J. Neurochem., 18 (1971) 2119-2128. 8 HOGAN, E. L., AND JOSEPH, K.C., Composition of cerebral lipids in murine leucodystrophy: the quaking mutant, J. Neurochem., 17 (1970) 1209-1214. 9 MORELL,P., GREENFIELD,S., CONSTANTINO=CECCARINI,E., ANDWISNIEWSKI,H., Changes in the protein composition of mouse brain myelin during development, J. Neurochem., 19 (1972) 2545-2554. 10 NORTON, W. T., AND PODUSLO, S. E., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-757. I 1 SAMORAJSKI,T., FREIDE, R. L., AND REIMER,P. R., Hypomyelination in the quaking mouse. A model for the analysis of disturbed myelin formation, J. Neuropath. exp. Neurol., 29 (1970) 507. 12 WIS~aEWSKI,H., ANDMORELL,P., Quaking mouse: ultrastructural evidence for arrest of myelinogenesis, Brain Research, 29 (1971) 63-73.