Download BIOCHEMICAL STUDIES ON MYEL1N ISOLATED

Brain (1975) 98, 213-218
BIOCHEMICAL STUDIES ON MYEL1N ISOLATED FROM THE
BRAINS OF PATIENTS WITH DOWN'S SYNDROME
BY
THE regional development of the nervous system proceeds through various stages
at different times (Davison and Dobbing, 1968). In man, prenatal establishment of
neuronal population is followed by later multiplication of glia, while arborization
of dendrites and probably formation of synapses continues over the first two years of
the human growth spurt (Dobbing and Smart, 1974). Myelination is largely a
postnatal event extending for a much more prolonged period after birth. Conditions
which interfere with postnatal brain development and growth may not affect nerve
or glial cell formation although myelination is a much more vulnerable process.
Amyelination has been observed in cases of malnutrition and in several inborn errors
of amino-acid metabolism associated with mental retardation (Davison, 1974).
Reduced myelination is also seen in the brain of hypothyroid animals (Balazs et al,
1969) and in certain neurological mutants (Sidman et al, 1964; Nussbaum et al,
1969; Meier and MacPike, 1970; Schneck et al., 1971). In Down's syndrome, brain
weights are less than normal although none show the extreme microencephaly found
in other low-grade defectives (Crome and Stem, 1972). The density of synapses and
neurons from the cerebral cortex of mongols appears to be similar to that in controls
(Cragg, 1975) but there is preliminary evidence to suggest abnormal myelination.
In addition a number of morphological abnormalities have been observed in the brains
of mongols; for example, gliosis of the white matter without demyelination has been
reported (Meyer and Jones, 1939) as well as accumulation of undifferentiated foetal
and subependymal cells (Norman, 1966).
Plaques and neurofibrillary tangles have also been recently found in the brains of
mongols (Ohara, 1972) similar to those previously described in Alzheimer's disease
(Terry, 1963; Kidd, 1964; Ellis et al, 1974). In contrast to the studies on aminoacidurias little work has been reported on the biochemistry of the affected mongol
brain (Stephens and Menkes, 1969). Palo and Savolainen (1973) suggested that there
is a deficiency of the specific myelin basic protein in Down's syndrome. This present
paper describes a collaborative study of a number of Finnish and British cases, in
which separation and analysis of myelin have been independently investigated.
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N. L. BANIK, A. N. DAVISON, J. PALO AND H. SAVOLAINEN
(From Miriam Marks Department of Neurochemistry, Institute of Neurology, The National Hospital,
Queen Square, London WC1N 3BC, England, and Laboratory of Neurochemistry, Department of
Neurology, University of Helsinki, Haartmaninkatu 4, SF-00290 Helsinki 29, Finland)
214
N. L. BANK, A. N. DAVISON, J. PALO AND H. SAVOLATNHN
MATERIALS AND METHODS
Details of the patients used in this study are given in Table I. The tissues were
obtained twenty-four to forty-eight hours after death and kept at —25°C before
TABLE I.—DETAILS OF THE PATIENTS WITH DOWN'S SYNDROME USED IN THIS STUDY
Sex Age (yrs.)
Female 22
Female 50
Male
14
Female
18
Female 37
Male
18
Female 40
Male
60+
Karyotypes
21-trisomy
21-trisomy
Not determined
21-trisomy
Not determined
21-trisomy
21-trisomy
Cause of death
Bronchiectasis with diabetes mellitus
Bronchopneumonia
Not known
Thrombosis, cystitis
Infarctus cerebri
Volvulus intestini
Pneumonia
Heart failure
analysis. Myelin was isolated from the cortical white matter of British patients (1-3)
according to the method of Norton (1971) and from the Finnish patients (4-7) with
the " A " version of the method of Rumsby et al. (1970), 0-8 M sucrose solution being
used in place of the 0-65 M sucrose. All chemicals used were Analar grade and
were obtained from British Drug House Chemicals Ltd. (Poole, Dorset, England).
Determination of Protein and Enzyme Activity
Protein was determined according to the method of Lowry et al. (1951) with bovine
serum albumin as standard. Adenosine 2', 3'-cyclic nucleotide 3'-phosphohydrolase
has been assayed according to Banik and Davison (1969).
Gel Electrophoresis
Electrophoresis of myelin proteins in a sodium dodecylsulphate (SDS) system
and the scanning of gels have been carried out as described by Banik et al. (1974).
Purified human myelin basic protein (Banik and Davison, 1973) was used as standard.
Gel electrophoresis of myelin from the Finnish cases was carried out according to the
method of Mehl (1968) and cytochrome C was used as standard.
Analysis ofUpids
The lipids were separated by TLC and analysed by the method described by
Ramsey et al. (1974).
RESULTS
As judged by protein estimations, recovery of myelin from white matter homogenates prepared from brains of patients of different ages was reduced in all cases,
particularly samples 1 and 3 (Table II). Measurement of activity of the myelin
marker enzyme 2', 3'-cyclic nucleotide 3'-phosphohydrolase also indicates a significant
reduction in the myelin content of the mongol brain.
Similarly, the yield of myelin (based on myelin protein dry weights) from Finnish
patients 6 and 7 was about half that of controls, but recovery of myelin in the other
two brains, 4 and 5, was normal. Myelin protein was separated by gel electrophoresis
in a SDS system (fig. 1). No change in the proportion of myelin basic protein to total
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Case
1
2
3
4
5
6
7
Normal
BIOCHEMISTRY OF MYELIN IN MONGOLS
215
protein was observed (Table II). The Finnish samples of myelin protein were
separated by the method of Mehl (1968) in a phenol-formic acid system. In most
samples of the Finnish series, no change in the proportion of basic protein was
observed, but where there appeared to be a loss (Case 6) fast-moving protein bands
were also present in the gels, suggesting post-mortem degradation of the basic protein.
PLP
BP
Fio. 1.—Scan of polyacrylamide gel electrophorcsis pattern of myelin protein. A, control myelin;
B, (bars) myelin from Down's syndrome; WP, Wolfgram and high molecular weight protein;
PLP, proteolipid protein; BP, basic protein.
The lipid content of myelin in 5 cases together with a typical control is presented
in Table III. No cholesterol ester could be detected in any sample. In agreement
with data on the myelin protein content, the amount of phospholipid, cholesterol and
cerebroside present in the myelin prepared from the white matter of patients was
found to be lowered in comparison with that of controls. When the results are
expressed as molar ratios of phospholipid:cholesterol:cerebroside we found little
difference in 3 cases (1-3). However, in the two samples (4, 5) of myehn from Finnish
patients, abnormal proportions of lipid were detected.
In preliminary experiments, synaptosomal fractions were isolated from whole
homogenate (occipital lobe). The protein content showed a reduced amount of
synaptosomes present in brains of patients with Down's syndrome (Cases 2 and 3;
11-61 and 9-83 mg protein/g wet wt respectively) compared to control (18-0 mg/g).
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WP
w_
216
N. L. B A N K , A. N . DAVISON, J. PALO AND H. SAVOLAINEN
TABLE n.—PROTEIN AND 2', 3'-CYCUC NUCLEOTIDE 3'-PHOSPHOHYDROLASE A C U V H Y IN
DOWN'S SYNDROME MYEUN ISOLATED FROM WHITE MATTER
¥, 3'-cyclic nucleotide
Basic protein]
White matter Total protein Basic protein
3'-phospho. act.
studied
\unolesjglh sp.act.\mg protein Total protein
mgjg wet wt.
0-22
Normal (3)
38,000 ±2,885
970
39-35 ±3-46
8-63
0-20
Casel
12,300
637
19-35
3-80
0-25
Case2
21,450
792
27-13
6-89
0-21
Case3
14,520
686
21-20
4-56
Methods of assaying enzyme activity and gel electrophoresis have been described in the text.
Results are expressed as mg or jamoles/g±standard deviation where applicable.
Tissues
Normal
Casel
Case2
Case3
Case4
Case5
Phospholipid
62-2
34-8
37-5
28-6
62-6
55-5
mglg wet wt.
Molar ratio
Cholesterol Cerebroside Phospholipid Cholesterol
1
4000
23-70
1-29
1
16-90
12-90
0-98
1
20-90
10-90
1-10
14-20
10-90
1
100
13-40
14-80
1
0-43
1
13-90
31-60
0-50
Cerebroside
0-35
0-33
0-26
0-34
0-21
0-51
Methods of separation and determination of lipids have been described in the text
DISCUSSION
It appears that amyelination is frequently found in certain inherited disorders of
amino-acid metabolism and other conditions associated with mental retardation
(Diezel and Martin, 1964; Prensky et ah, 1968; Menkes and Solcher, 1967; Shah
et ah, 1972; Davison, 1974). The lower yield of myelin now recovered from white
matter homogenates of most of our cases of Down's syndrome suggests that the
reduced deposition of myelin may also be a common feature of the disorder. No
evidence of demyelination was found in our study, for cholesterol ester could not be
detected in the brain correlating with histological studies indicating absence of
progressive and generalized demyelination in Mongol brain (Crome and Stern, 1972).
Since it was previously reported that the amount of the basic encephalitogenic
protein was reduced in the CNS myelin of patients with Down's syndrome (Palo
and Savolainen, 1973) myelin was separated by two other techniques and specimens
from Great Britain compared with some from Finland.
Despite the low yield of myelin obtained from British patients (1-3) the proportion
of basic protein to total myelin protein remained unaltered when compared to that
found in control samples.
In 3 out of 4 Finnish cases, the proportion of the basic encephalitogenic protein
also remained the same as controls, but a marked reduction of this protein was
found in one patient (Case 6) as previously reported (Palo and Savolainen, 1973).
It is possible that the reduced concentration of basic protein could well be due to
post-mortem autolysis in this case, for there was evidence on the gel of breakdown
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TABLE in.—Ln>m COMPOSITION OF MYELIN IN DOWN'S SYNDROME
BIOCHEMISTRY OF MYELIN IN MONGOLS
217
SUMMARY
Amyelination has been deduced from the data on chemical studies of myelin
isolated from the brains of Down's syndrome. The lack of cholesterol and much
reduced phosphohydrolase activity in mongol myelin possibly suggest a fault in the
structure of myelin.
ACKNOWLEDGMENTS
The authors wish to thank the Multiple Sclerosis Society of Great Britain and Northern Ireland,
the National Research Council for Medical Sciences of Finland and Kehitysvammaliitto r.y. for
financial support, and Mr. Kishor Gohil and Miss Kaarina Iindberg for excellent technical
assistance. The authors also thank Mr. H. Goodwin for lipid analysis, Mrs. I. Linnoila for her
assistance in isolating myelin and Dr. P. E. Sylvester and Dr. L. Crome for supplying specimens
and details of the patients.
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