Evidence on hypomyelination of central nervous system in murine muscular dystrophy

Journal of the Neurological Sciences, 1985, 68:175-184

175

Elsevier JNS 2484

Evidence on Hypomyelination of Central Nervous System in Murine Muscular Dystrophy Shigekatsu Tsuji and Hiroshi Matsushita Department of Physiology, Wakayama Medical College, 9-bancho, Wakayarna 640 (Japan) (Received 27 February, 1984) (Revised, received 21 December, 1984) (Accepted 21 December, 1984)

SUMMARY

To elucidate the disturbances of myelin metabolism in the nerve tissue of murine muscular dystrophy, the lipid composition of and the developmental changes in 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) and cholesterol ester hydrolase (CEHase) activities in the purified CNS myelin of dystrophic mice were determined. Several kinds of lipids, total galactolipid and cerebroside sulfatide levels were si~ificantly reduced as compared with controls. Total cholesterol levels in the spinal cord of dystrophic mice were moderately higher. CEHase and, to a lesser degree, CNPase activities were reduced in the purified myelin of the CNS of the dystrophic mice. The reduced myelin CEHase activity in dystrophic mice suggests that impairment of hydrolysis of steryl esters may be important in the development of hypomyelination of the CNS.

Key words: Central nervous system - Cerebroside sulphatide - Cholesterol ester hydrolase - 2' ,3'-Cyclic nucleotide 3'-phosphohydroluse - Galactolipid- Hypomyelination - Murine muscular d y s t r o p h y - Muscular d y s t r o p h y - Myelin - Myelination

INTRODUCTION

Murine hereditary muscular dystrophy was originally thought to be a primary disorder of skeletal muscle involving progressive necrotising myopathy, and hence to

This work was supported by research grants for intractable diseases and for muscular dystrophy from the Ministry of Health and Welfare of Japan. 0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

176 be a good model for certain forms of human muscular dystrophy (Michelson et al. 1955). Despite the voluminous data on the pathology of murine muscular dystrophy, it is still uncertain whether it is caused by a primary myopathic lesion or a neurotrophic abnormality. Large numbers of muscle fibers in dystrophic tibialis anterior and gastrocnemius muscle are functionally denervated (McComas and Mossawy 1965; McComas and Mrozek 1967). Morphological studies have also shown reduced numbers of peripheral nerve fibers (Harris et al. 1972; Montgomery and Swenarchuk 1978), structural alterations in motor nerve terminals (Gilbert et al. 1973), and deficiencies of the myelin sheath in the spinal and cranial roots (Biscoe et al. 1975; Bradley and Jenkison 1975). The experiments of Peterson (1974), in which chimeric offspring were produced from normal and dystrophic pre-implantation embryos, and where the muscles in the offspring were normal despite over 90~o of their nuclei being genetically dystrophic, suggest that the nervous system of dystrophic mice is abnormal in its ability to support muscle growth and to maintain functional contacts with the muscle fibers. Biochemical studies were done to examine the CNS of dystrophic mice for evidence of defects in the membraneous structure of cells and to clarify the relationship between neural abnormalities and the development of muscular dystrophy. MATERIALS AND METHODS

Animals C57BL/6J-dy mice carrying the dystrophia muscularis gene (dy), obtained from the Jackson Laboratory (Bar Harbor, ME, U.S.A.), have been maintained in our animal facility since 1970. Dystrophic mice (dy/dy) were identified by their periodic dragging of their hind legs beginning 12-14 days after birth. Dystrophic mice and their normal littermates were killed at various ages by cervical dislocation, and the whole brain and the spinal cord were excised immediately after. The tissues were weighed and used in lipid analyses and enzyme assays.

Lipid analysis Extraction of the lipid components of the brain and spinal cord was done using the Folch method (1957). After washing with 0.37~ KCI solution and with the upper phase of the pure solvent, the Folch extract was used in the lipid analysis. Total phospholipids were determined by measuring the inorganic phosphorus released by digestion (Fiske and Subbarow 1925). Total cholesterol was analysed by the method of Z ak (1957). Protein in the proteolipids was determined by the Lowry method (1951) using a lipid extract after hydrolysis by NaOH. Total galactolipids were measured by measuring hexose using a micromethod variation of the orcinol reaction (Sorenson and Haugaard 1933). Among the galactolipids, cerebroside sulfatide was determined separately by the Azure A colorimetric assay method (Kean 1968). Sphingosine levels were estimated by the procedure of Lauter and Trams (1962).

177

Enzyme assays The brains and spinal cords were homogenized in 10 volumes of ice cold 0.32 M sucrose in a Teflon homogenizer. The purified myelin fraction was isolated using a conventional subcellular fractionating procedure, basically that of Norton and Poduslo (1973). For the determination of CNPase activity in the purified myelin fraction treated with 0.3 mg/ml of Triton X-100 solution, the methods of Olafson et al. (1969) and of Sogin (1976) were used. CEHase activity in the purified myelin fraction was measured by colorimetry (Tsuji 1982). Electron microscopy For electron microscopic examination, the purified myelin fraction was directly fixed in the osmium tetroxide solution described here. The fraction was first suspended in 0.33 M sucrose and centrifuged at 100000 x g for 60 min. The pellet was placed in 2~o osmium tetroxide in 0.32 M sucrose and cut into small cubes. After 2 h of fLxation the cubes were dehydrated with graded ethanol. They were then embedded in epoxy resin and ultra-thin sections were made using a LKB ultramicrotome. Electron micrographs showed that the purified myelin fraction consisted mainly of circular myelin fragments with remnants of axonal contents (Fig. 1).

Fig. I. Electron micrograph of the purified myelin fraction, x 7700.

178 RESULTS

Lipid levels We collected data on body weight, spinal cord weight, and total lipid levels in the CNS of animals with muscular dystrophy and of their normal littermates. The mean weight of the brain and spinal cord, and the total lipid level in the spinal cord of the dystrophic mice were significantly less than those of normal littermates, but the total lipid level in the brains of dystrophic mice was not changed (Table 1). The results of the quantitative analysis of the major classes of lipids in the CNS of dystrophic mice and their normal littermates are summarized in Tables 2 and 3. Amounts of all of the lipids analyzed (except cholesterol) from the spinal cord of dystrophic mice were smaller. Total galactolipids and cerebroside sulfatide levels in the spinal cord of dystrophic mice were significantly lower; the amounts of proteolipids, phospholipids TABLE 1 C57BL/6J-dy MICE USED FOR LIPID ANALYSIS Mean + SEM. In parentheses are numbers of mice used.

Age Sex Body weight (g) Brain weight (rag) Spinal cord weight (rag) Total lipids (mg/g wet weight) Brain Spinal cord

Dystrophic

Normal

6-8 weeks Both 13.5 _+ 0.7* (10) 352.4 +_ 19.7'* (18) 51.9 + 4.7** (20)

6-8 weeks Both 20.0 + 0.8 (10) 397.1 + 18.4 (18) 65.5 + 6.0 (20)

83.1 _+ 11.2 (10) 132.1 _+ 8.3** (12)

84.0 + 13.1 (10) 146.2 _+ 8.1 (12)

* P < 0.01; ** P < 0.001.

TABLE 2 COMPARISON OF MAJOR CLASSES OF LIPIDS IN SPINAL CORDS OF DYSTROPHIC AND NORMAL MICE Based on g of wet tissue.

Cholesterol (pmol) Proteolipids (pmol) Phospholipids (pmol) Galactolipids Q~mol) Sphingosine (arbitrary units) Sulfatide Q~mol)

* P < 0.05.

Dystrophic

Normal

55.8 _+ 18.4 6.6 + 3.1 101.9 + 6.5 85.7 _+9.3 43.5 + 0.2 3.1 _+0.1

42.1 + 7.6 + 126.5 + 140.9 + 51.5 + 4.4 +

D/N x 100 3,1 3,9 8,7 38.2 16.4 0.3

132.5 86.8 80.6 60.8* 84.5 70.5*

179 TABLE 3 COMPARISON OF MAJOR CLASSES OF LIPIDS IN BRAINS OF DYSTROPHIC AND NORMAL MICE Based on g of wet tissue.

Cholesterol ~mol) Proteolipids O~mol) Phospholipids Omol) Galactolipids (~mol) Sphingosine (arbitrary units) Sulfatide ~moi)

Dystrophic

Normal

D/N x 100

57.3 + 8.2 5.3 _+1.4 76.5 + 27.4 48.9 _+ 12.8 16.1 _+6.6 3.6 + 0.7

57.7 + 12.9 5.0 + 1.9 73.5 + 28.1 48.8 + 17.8 18.6 + 7.4 4.7 + 0.5

99.3 106.0 104.1 99.6 86.6 76.6*

* P < 0.05.

and cholesterol were not significantly different from normal mice. The total amount o f cerebroside sulfatide in the brains o f dystrophic mice was significantly lower than in normal mice. Total cholesterol was higher in the spinal cords o f dystrophic mice.

Myelin CNPase and CEHase activities C N P a s e activity in the C N S o f the mouse is closely associated with the myelin membrane, and can be used as a sensitive marker for the m o u n t o f myelin sheath. M u c h C E H a s e activity is found in purified myelin, so it can be used as a myelin specific enzyme marker too. Here, we investigated the changes in activity o f these two enzymes during development o f the C N S in dystrophic mice. The low activity o f total myelin C N P a s e in both brain and spinal cord of dystrophic mice at 4 - 8 weeks o f age was a highly significant difference, but the specific activity o f this enzyme based on the amount o f protein was not significantly different (Table 4). In contrast, both total and specific activities o f myelin C E H a s e in the brain TABLE 4 2',3'-CYCLIC NUCLEOTIDE 3'-PHOSPHOHYDROLASE IN THE MYELIN FRACTION OF BRAIN AND SPINAL CORD OF DYSTROPHIC AND NORMAL MICE Units: mg of NADP/mg of protein or total myelin. Mean age of mice used was 51.3 (41-60) days. Dystrophic

Normal

D/N × 100

P

Brain Total activity Specific activity

8.6 + 0.7 3.3 + 0.8

12.9 + 1.2 3.6 + 1.2

66.7 91.7

<0.01 NS

Spinal cord Total activity Specific activity

7.7 + 1.0 3.1 + 0.6

11.3 + 1.7 3.7 + 0.8

68.1 96.9

< 0.01 NS

180 TABLE 5 CHOLESTEROL ESTER HYDROLASE IN THE MYELIN FRACTION OF BRAIN AND SPINAL CORD OF DYSTROPHIC AND NORMAL MICE Units: #M Oleate/min per mg of protein or total myelin. Mean age of mice used was 57.8 (41-75) days. Dystrophic

Normal

D/N x 100

P

14.3 + 0.6 5.6 _+ 2.5

25.7 + 3.4 10.8 + 2.7

55.6 51.8

<0.01 <0.01

21.5 + 4.2 5.4 _+ 1.9

46.9 + 11.2 9.8 _+ 1.8

45.8 55.1

< 0.01 <0.01

Brain Total activity Specific activity

Spinal cord Total activity Specific activity

and spinal cord of dystrophic mice at 8 weeks of age was significantly less than those of the controls (Table 5). Changes in myelin CNPase activity during the development of the brain and spinal cord in dystrophic mice and the controls are shown in Fig. 2. During the In'st 28 postnatal days, myelin CNPase activity in the brain of both kinds of mice increased in the same way. Then, this activity in the dystrophic mice suddenly decreased, and was about two-thirds the control level at around 40 days of age. The development of this activity in the spinal cord of dystrophic mice paralleled the curve for the controls, but was always lower. At 14 days, immediately after the disease appeared, the difference was small (25~) but increased later. Myelin CEHase activity in the brain of control mice increased rapidly during the In'st 8-30 days (Fig. 3, upper half'). In dystrophic mice, the pattern was identical for the first 20 days, but then activity decreased abruptly at day 30, it was about one-half the control level. Increases in myelin CEHase activity in the developing spinal cord of dystrophic mice paralleled the curve for control animals, but the activity was always lower (Fig. 3, lower half). The myelin CEHase activity in dystrophic mice was half that of the controls in adulthood. DISCUSSION

Several histological studies of the nervous system in dystrophic mice have reported hypomyelination in the peripheral nerves and spinal and cranial roots (Bradley and Jenkison 1975; Bray and Aguayo 1975; Stirling 1975). In the study of Montgomery and Swenarchuk (1978), the numbers of myelinated axons in the nerves to the soleus and plantaris muscles were significantly smaller at all ages in dystrophic mice than those in normal controls, and there was progressive loss with increasing age. Protein synthesis in the spinal cord of dystrophic mice is increased (Kuffer et al. 1977). Phospholipid turnover is increased in the brains and spinal cords of dystrophic mice (Austin et al. 1976).

181 8ra~n J

c: c~ ~

i

Normal

2 ~

o ,a: v

i 10

iii _ 20

~ _

~--

-- --

m 30

40

50

60

~

j, 70

Dystrophic

80

Days after btrth

Spinal cord

6

GO0

0

Normal

Dystrophic

2

lo

~o

~o

4o

~o

6o

70

eo

dsys after bteth

Fig. 2. Developmentalchanges of myelin CEHase activities in brains and spinal cords of normal and dystrophic mice. We confmlaed that there is a consistent pattern of hypomyelination in the CNS of dystrophic mice. A moderate decrease in galactolipids (particularly cerebroside sulfatide) in the spinal cord of young dystrophic mice suggests that a deficiency of these lipids may be involved in inhibiting myelin formation or maturation. CNPase in the CNS is a marker for the myelin sheath and the membrane of oligodendrocytes (Kurihara et al. 1970). However, there are at least three distinct CEHases in the brain (Eto and Suzuki 1972). One, with a pH optimum of 7.2, is activated by sodium taurocholate but not by Triton X-100, and appears to exist almost exclusively in the myelin sheath and hence in the membrane of oligodendroglia (Eto and Suzuki 1973; Igarashi and Suzuki 1977). The other two enzymes are found mainly in the crude mitochondrial and microsomal fractions, respectively (Eto and Suzuki 1971).

182 20 Brain

16 Normll

12

. . . . . . . . . . . . . . .

4

Dystrophic

/; lo

2b

3b

io

so

s6

70

eb

Days after blrth

12' Spinal cord 3 o

lO,

8 Normal

6

4

2

/

/,-t'-

-- .....

"-

Dystrophic

lO Days after birth

Fig. 3. Developmentalchanges of mye~nCNPase activities in brains and spinal cords of normal and dystrophicmice. The developmental changes in CNPase and CEHase activity in the myelin fraction purified in this experiment indicate that these enzymes are localized exclusively in the myelin membrane of the mouse CNS. Not only does the sharp increase in activity coincide with the period of active myelination, but the changes also appear to reflect precisely differences in developmental characteristics of the brain and spinal cord. The reduced activity of total CNPase in the myelin fraction of the CNS of young dystrophic mice seems to be proportional to the reduced amount of total myelin rather than to any specific defect in enzyme synthesis. The extent of reduction in the myelin CEHase activity in the spinal cord of

183

dystrophic mice at 4-8 weeks of age was somewhat greater than for myelin CNPase. Since cholesterol is a major lipid component of membranous structures, including the myelin sheath in nerve tissue, the decrease in myelin CEHase activity in dystrophic mice must be important in the abnormal myelination. Rabinowitz (1960) and Kabara (1964) showed increased cholesterol synthesis and decreased levels of cholesterol esters in the brain of dystrophic mice. This, in combination with personal observation (Tsuji 1982; Tsuji and Matsushita 1983), suggests that the CNS of dystrophic mice fail to arrange the lipid constituents of myelin structures to form mature myelin membranes. There is indirect evidence pointing to the involvement of the nervous system in human muscular dystrophy (Dubowitz 1979). In Duchenne dystrophy (Marsh and Munsat 1974), some forms of congenital muscular dystrophy (Fukuyama et al. 1960), and congenital myotonic dystrophy (Harper 1975), there is well documented evidence of mental retardation and CNS involvement. Recently, Takada et al. (1983) documented that in the brains of patients with Fukuyama-type congenital muscular dystrophy, GFAP-positive glial cells were present in large numbers in the superficial layers of the cerebral cortex and in the cerebral and cerebellar white matter. Associated central and peripheral nervous system involvement in murine muscular dystrophy has important implications for the understanding of the nature and pathogenesis of the muscular dystrophies. It may help the fmding if the involvement of muscle is only one manifestation of a wider neurologic disorder, and possibly a more universal involvement of other systems and cells as well. REFERENCES Austin, L., C.T. Kwok, A.D. Kuffer and B.Y. Tang (1976) Phospholipid metabolism in murine muscular dystrophy. In: G. Porcellati, L. Amaducci and C. Galli (Eds.), Functional and Metabolism of Phospholipids in the CentralandPeripheralNervous Systems, Plenum Publishing Corporation, New York, pp. 367-372. Biscoe, T.J., K.W.T. Caddy, D.J. Pallet and U. M.M. Pehrson (1975) Investigation of cranial and other nerves in the mouse with muscular dystrophy, J. Neurol. Neurosurg. Psychiat., 38: 391-403. Bradley, W.G. and M. Jenkison (1975) Neural abnormalities in the dystrophic mouse, J. Neurol. Sci., 25: 249-255. Bray, G.M. and A.J. Aguayo (1975) Quantitative ultrastructural studies of the axon-Schwann abnormality in spinal nerve roots from dystrophic mice, J. Neuropath. Exp. Neurol,, 34: 517-530. Dubowitz, V. (1979) Involvement of the nervous system in muscular dystrophies in man. In: J.B. Harris (Ed.), Muscular Dystrophy and other Inherited Diseases of Skeletal Muscle in Animals, Ann. N. 1I. Acad. Sci., 317: 431-439. Eto, Y. and K. Suzuki (1971) Cholesterol ester metabolism in the brain - - Properties and subcellular distribution of cholesterol ¢sterifying enzymes and cholesterol ester hydrolases in adult rat brain, Biochim. Biophys. Acta, 239:293-311. Eto, Y. and K. Suzuki (1972) Cholesterol esters in developing rat brain - - Enzymes of cholesterol ester metabolism, J. Neurochem., 19:117-121. Eto, Y. and K. Suzuki (1973) Enzymes of cholesterol ester metabolism in the brains of mutant mice, quaking and jimpy, Exp. Neurol., 41: 222-226. Fiske, C.H. and R.R. Subbarow (1925)The colorimetric determination of phosphorus, J. Biol. Chem., 66: 375-400. Folch, J. M. L. and G.H. Slane-Sanley (1957) A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226: 491-509. Fukuyama, Y., H. Haruna and M. Kawazura (1960) A peculiar form of congenital progressive muscular dystrophy, Paediat. Univ. Tokyo, 4: 5-8. Gilbert, J.J., M.C. Steinberg and B.Q. Banker (1973) Ultrastructural alterations of the motor end plate in myotonic dystrophy of the mouse (dyZl/dyZl), J. Neuropath. Exp. Neurol., 32: 345-364.

184 Harper, P.S. (1975) Congenital myotonic dystrophy in Britain, Part 1 (Clinical aspect), Arch. Dis. Child., 50: 505-513. Harris, J.G., C. Wallace and J. Wing (1972) Myelinated nerve fiber counts in the nerves of normal and dystrophic mouse muscle, J. Neurol. Sci., 15: 245-249. Igarashi, M. and K. Suzuki (1977) Solubilization and characterization of the rat brain cholesterol ester hydrolase localized in the myelin sheath, d. Neurochem., 28: 729-738. Kabara, J. J. (1964) Brain cholesterol VII. The effect of hereditary dystrophia muscularis on 2-~4C mevalonic and 23H acetate incorporation, Texas Rep. Biol. Med., 22(1): 143-151. Kean, E. L. (1968) Rapid, sensitive spectrophotometric method for quantitative determination of sulfatides, J. Lipid Res., 9: 319-327. Kuffer, A. D., Y. Komiya and L. Austin (1977) Proteins of fast axoplasmic transport in the sciatic nerve of the dystrophic mouse, Exp. Neurol., 55: 74-83. Kurihara, T., J. L. Nassbaum and Mandel (1970) 2', 3'-Cyclic nucleotide 3'-phosphohydrolase in brains of mutant mice with deficient myelination, J. Neurochem., 17: 993-997. Lauter, C.J. and E.G. Trams (1962) A spectrophotometric determination of sphingosine, J. Lipid Res., 3: 136-138. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193: 265-275. McComas, A.J. and S.J. Mossawy (1965) Electrophysiologieal investigation of normal and dystrophic muscle in mice. In: 3rd Symposium on Research in Muscular Dystrophy, Pitman's Medical Publishing Co., Ltd., London, pp. 317-338. McComas, A.J. and K. Mrozek (1967) Denervated muscle fibers in hereditary mouse dystrophy, J. Neurol. Neurosurg. Psychiat., 30: 526-530. Marsh, G.G. and T.L. Munsat (1974) Evidence for early impairment of verbal intelligence in Duchenne muscular dystrophy, Arch. Dis. Child., 49: 118-122. Michelson, A.M., E.S. Russell and P.J. Harman (1955) Dystrophia muscularis - - A hereditary primary myopathy in the house mouse, Proc. Nat. Acad. Sci. (USA) 41: 1079-1084. Montgomery, A. and L. Swenarchuk (1978) Further observations on myelinated axon numbers in normal and dystrophic mice, .L Neurol. Sci., 38: 77-82. Norton, W.T. and S. E. Poduslo (1973) Myelination in rat brain - Method of myelin isolation, J. Neurochem., 21: 77-82. Olafson, R. W., G. I. Drummond and J. F, Lee (1969) Studies on 2', 3'-cyclic nucleotide 3' -phosphohydrolase from brain, Canad. J. Biochem., 47: 961-966. Peterson, A.C. (1974) Chimera mouse study shows absence of disease in genetically dystrophic muscle, Nature (Lond.) 248: 561-564. Rabinowitz, J.L, (1960) Enzymic studies on dystrophic mice and their littermates (lipogenesis and cholesterolgenesis), Biochim. Biophys. Acta, 43: 337-338. Sogin, D.C. (1976) 2', 3'-cyclic NADP as a substrate for 2', 3' -cyclic nucleotide 3' -phosphohydrolase, J. Neurochem., 27: 1333-1337. Sorenson, M. and G. Hangaard (1933) Ueber die Anwendbarkeit der Orcinreaktion zur Bestimmung der Art und Menge von Kohlenhydratgruppen in Eiweissstoffen, Biochem. Z., 260: 247-277. Stirling, C. A. (1975) Abnormalities in Sehwann cell sheaths in spinal nerve roots of dystrophic mice, J. Anat. (Lond.), 119: 169-180. Takada, K., J. Tanaka and S. Takashima (1983) An immunohistochemical study on glial proliferation in brain of Fukuyama type congenital muscular dystrophy using GFAP stain, Brain Dev., 5(2): 244. Tsuji, S. (1982) A study of myelin deficiency in central nervous system of mouse with genetic muscular dystrophy. In: Y. Tsukada (Ed.), Genetic Approaches to Developmental Neurobiology, University of Tokyo Press, Tokyo, pp. 135-148. Tsuji, S. and H. Matsushita (1983) Hypomyelination in central nervous system of murine muscular dystrophy. In: S. Ebashi and E. Ozawa (Eds.), Muscular Dystrophy - - Biomedical Aspects, Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin, pp. 161-165. Zak, B. (1957) Simple rapid microtechnique for serum total cholesterol, Amer. J. Clin. Path., 27: 583-588.