SOLUBLE PROTEINS IN NORMAL AND DISEASED HUMAN BRAIN CAROLYN B. SMITH‘and D. M. BOWEN Miriam Marks Department of Neurochemistry, Institute of Neurology, The National Hospital. Queen Square, London WClN 3BG, U.K. (Receioed 1 1 June 1976. Accepted 15 June 1976) Abstract-Six brain regions (frontal cortex, parts of the basal ganglia thalamus and substantia nigra) were examined from over 80 human brains obtained at post-mortem. After elimination of patients with evidence of either ‘cerebral hypoxia’, lingering modes of death or abnormal brain morphology brain extracts were found to contain a characteristic pattern of 6 major soluble-acidic protein bands (neuronin-type proteins). As judged by studies using cortical biopsy specimens these proteins are relatively unaffected by post-mortem changes. Moreover, in adulthood the pattern is not noticeably agedependant. Two of the protein bands have been identified as s-100 (neuronin s-1 and 2) while a third (neuronin S-5)is similar in most respects to antigen ci (14-3-2). S-100 is increased in brains with evidence of marked gliosis. The other protein bands have not been identified. Two of them (neuronin S-3 and 4)are rarely depleted while the concentration of neuronin S-6 is affected particularly in extracortical regions in controls with either lingering modes of death and/or ‘cerebral hypoxia’ and in all regions in most patients with Alzheimer’s disease, senile dementia and mixed senile and vascular dementia PREVIOUS study of experimental neurological disorders in rodents (BOWEN et d.,1974), changes in lysosomal activity appeared t o be more sensitive index of acute brain damage than the electrophoretic profile of soluble brain proteins. However, in chronic non-inflammatory human brain disease alterations in total lysosomal hydrolase activity is less evident (BOWENet al., 1973). In the present study of a large number of diseased and control human brains we describe the quantitative estimation and protein composition of six brain regions. Since there is considerable current interest in pathological changes in the basal ganglia (e.g. striatum in Huntington’s chorea) and there are clinical and biochemical indications of extrapyramidal disorder in Alzheimer’s disease and senile dementia (GOTTFRIES ef al., 1969; F’EARCE,1974) we have examined samples from the caudate nucleus, putamen and globus pallidus. Various soluble proteins have been electrophoretically separated and analysed as a possible index of alteration in cellular pathology. To facilitate this comparison all brains have been examined histologically. We have also attempted to interpret our data with respect to the terminal condition of the patient and post-mortem storage effects.
IN A
J. A. N. CORSELLIS). Samples of prefrontal grey matter (from the crests of the gyri and including all cortical layers). thalamus (medial portion), putamen, globus pallidus, caudate nucleus and substantia nigra were taken for biochemical analysis. The samples were weighed and routinely stored overnight at -20°C prior to protein extraction. Details of age, sex, cause of death and source of specimens are given in Tables 1 and 2. No selection of cases by drug treatment was made. The only noticeable consistent pattern of treatment was that many of the patients (both dements and functionally ill) in the psychiatric hospitals received the same innocuous medication, chloral hydrate. as a sleeping draught. Six brain regions from the specimens described in Table 2 were analysed for protein composition while only the protein composition of frontal cortex of those in Table 1 was examined. Nosology of the specimens. For the classification of the material examined, the initial division was based on the clinical presentation. Thus the specimens from 86 patients included 52 brains from patients who had died without obvious signs of neurological disease while the other brains were from patients with various neurological diseases. The brains in the non-neurological group were further subdivided on the basis of morphological criteria (e.g. incidence of senile or neuritic plaques and neurofibrillary degeneration, focal nerve cell loss and gliosis and degree of either cerebral vascular change or gross cerebral atrophy), presence of obvious functional disability (e.g. depressive illness or schizophrenia) and whether there was eviMATERIALS AND METHODS dence of a lingering mode of death, coma or ‘cerebral Specimens. After death, corpses were maintained at 4°C hypoxia’. For the classification of specimens from patients until autopsy. The time between death and whole body with neurological diseases morphological criteria were refrigeration was routinely within the range 0.75-2.5h. The decisive. The traditional classification (CORSELLIS, 1962) of brain was excised within 24 h of death and halved sagit- mental illnesses into ‘organic’ and ‘functional’ has been tally; one half was preserved for histopathology (Dr. retained. Accordingly, the brains were assigned to an appropriate group (Table 2; most of the brains in Table Present address: Laboratory of Biochemical Genetics, 1 could be placed within these groups: exceptions are in National Institutes of Health, Bethesda, M D 20014,U.S.A. the text). 1521
CAROLYN B . SMITHand D. M. BOWEN
1522
TABLE1. CONCENTRATION OF TOTAL
SOLUBLE. AND TWO SOLUBLE ACIDIC PROTEINS IN PRE-FRONTAL CORTEX Solublc protcins
Diagnostic group*
.Age ly1
No
Disease
Period before storage (h)
casest
Acidic Total (", total protein)
Neuronin S - h I",,sol. protein1
Neuronin I and 2 (". sol. protein)
~-
I
Normal adult Alzheimer's disease Significance (PI
-: 2
5
Normal elderl) Semle dementia Significance (PI
14
&,
X 4
x
60 2 6 67k3 NS
19 f 10
79+5 7xi7 NS
18*X 9+7 NS
28.8 f 3.3 (41 19.2 4.9 < 0.02
3.16
+ 1.31
0.33
k 0.654
24.8 2 0.9 (41 19.3 k 44 ( 5 ) < 0.05
2.63 f 0.62
*
X+I1 NS
1.30 2.76
0.38
k 0.17
< 0.001
< 0.01
1.59 It 0.93
k l.4l$ < 0.02
0.76
2.05 k 1.03 NS
S.D.
Number of samples where this differs from No. cases are shown in brackets. * See Materials and Methods. t Different cases from those in Table 2. $ Clinically established and autopsy confirmed cases. Q Neuronin S-6 detected in one brain (1.32"J. Frontal cortex from 2 other cases of Alzheimer's disease was also subject to electrophoresis. the gels were not scanned but visual examination indicated that neuronin S-6 was absent from one and within the normal range in the other specimen. Protein analpis. Polyacrylamide gel electrophoresis was the generic name neirroniii foi- this group of soluble acidic proteins. for the prealbumin portion of gels of extraneural used to analyse the protein composition of buffered-hypotonic extracts (BOWENer a/.. 1973) from the 6 brain regions tissues do not contain such conspicuous amounts of these proteins. The results obtained with the porosity-gradient examined. In order to increase the resolution. porosity& SHOOTER. gels are compared in Fig. 1 with those obtained with 7.5",, gradient gels were routinely used (GROSSFELD 1971).This technique revealed a number of well-separated (wjv) acrylamide gels. protein bands (Fig. 1, gel on the left) including 6 bands The neuronin-type proteins were quantified on the gels (neuronins 1 4 ) that migrated rapidly to the anode ahead by direct densitometry. The protein stain, Procion Brilliant of albumin. We have proposed (BOWFNrr ul.. in press) Blue R.S.. was chosen as the dye because of its reproducibiTABLE2.
Diagnostic group
F'RE-ASSAY HISTORY OF BRAIN SPECIMENS ANALYZED FOR PROTEIN CONTENT IN SIX BRAIN REGIONS
Disease
Period between death Cause & storage Sex of or assay IM : F) deatht Source:
No. cdses'
Age
h
hi I x3 2 10
I! I
Brain morphology
Comments
0-1
002 300 001 010
normal normal normal plaques plaques appreciable neuronal
age range' 50-69 y age range: over 70 y had depressive illness no functional illness short depressive illness (ECT) alcoholic (liver flap)
Non-neurological
3 4 5 6
Normal adult Normal rldrrl) Functional disabilit! Plaque Plaques & depression Neuronal loss
7
GllOSlS
8
Cerebral atroph! Schizophrenia Lingering death Coma 'Ccrebral hypoxia' CO poisoning
I
'
70. 85 3 77
is
*
.. >>
+x
7.1
23 i 2 I?. 20 23 5 7 24
?:5 2 .O I :? 0:I
20
*
710 430
loss
9 10 11 12 13
14 15
5
I :0
010
scvere and bizarre ghosts
72 60 4x.m 76
IS
0:l 0.1 O:?
010
deep sulci. thin cortex severe brain surgery normal normal markcd oedemas severe anoxic changes
13 20. 9 24 7 21
67 ?J
Neurological Senile dementia Mixed senile and vascular dementia
* Different cases from
X?
9
X6 5 h
21 + 7
1
7x 5
x
1623
001 020 100 100
0:I I :o I ,0
3 :6 0:3
100
6012 0210
009 003
atypical myeloproliferative disorder n o terminal ileum hospitalized 37 y cancer (breast. colon) ~
lung surgery!, suicide case
Diagnostic division 0.2 (CORSFLLIS,1962) Diagnostic division 0.3 (Ci)nSFLLiS, 1962)
those in Table 1.
t 1st digit identifies the number of patients dying from respiratory disease (usually bronchopneumonia, but also chronic bronchitis and pulmonary thromboembolism). 2nd from coronary thrombosis, 3rd from cancer and 4th from other causes. $ 1st digit identifies the number of specimens originating from coroner's courts, 2nd from general hospitals, 3rd from psychiatric hospitals. S.D. (age and interval between death and assay for groups 2 and 14 did not differ significantly). §This is a change not infrequently seen in persons suffering from cor pulmonale who die in a severely hypoxic state. /I Lobectomy for bronchietasis. pleuro-bronchial fistula. Plaques. indicate that the relati\e number of senile plaques was at least moderate in at least one region examined (in groups 2 and 14. the scores were zero to slight and moderate to severe. respectively. Corsellis J. A. N., personal communication).
+,
FIG.I. Tris -glyciiie polyacrylamide gels of hypotonic extracts of pre-frontal cortex from normal IiLinian brain. The gels shown are from left to right: porosity gradient gel, 7.5"" acrylamide gels 15 CIII and 5 cm. respectively.
Soluble brain proteins lity, uniformity of protein binding and the stable covalent dye-protein bond (MAURER,1971). In the pilot study
(Table I ) neuronin-type proteins were quantified as a percentage of the total soluble protein that entered the 404 acrylamide gel. The gels were scanned at 595 nm using a Fisons’ vitatron Scanning Densitometer. The areas under the peaks corresponding to neuronins S-1 and 2 and S-6 were determined (by weighing the appropriate portions of chart paper) as a percentage of the total area (limit of detection, 0.6% of the total soluble protein). In subsequent experiments the absolute amounts of the proteins were determined using a standard of bovine serum albumin (Fraction V, Sigma Chemical Co., St. Louis, MO). In these experiments the gels were scanned at 602 nm on a Gilford Model 240 spectrophotometer equipped with a linearly transported glass cuvette. Absorbances were recorded on a Servoscribe 15 chart-driven recorder fitted with an integrator. On the chart recorder traces the troughs of the peaks corresponding to neuronins S-5 and sd were joined to form a baselin. The areas under the two peaks were compared with a standard curve (limit of detection, 7 pg/g wet wt). Total protein and total soluble protein (i.e. the protein content of the buffered hypotonic extract) was measured using the method of LOWRY et nl. (1951).
RESULTS
Total and total soluble protein. In general the levels of total protein were similar in all the brains that were examined. For example, no significant differences were detected in any region when the levels of total protein in senile dementia were compared with the levels in either the normal adult or elderly control groups. In contrast, the level of total protein in prefrontal cortex from the patients in groups 1&13 (Table 2) was significantly (P= < 0.02) reduced to 84:d of the concentration in the age-matched normal controls. The levels of total protein in caudate nucleus from these patients (67.2 f 16.3 mg protein/g wet wt) was also significantly reduced ( P = < 0.05) to 74”/, of the level in the control group. Similarly, the levels of total soluble protein were usually moderately reduced (to about 75% of the control value) in the cases of dementia (Table 1). Neuronin-type proteins. Polyacrylamide gel electrophoresis was used to compare the protein composition of the soluble proteins present in the frontal grey matter of control and senile brains. This technique revealed a number of relatively well-separated protein bands (Fig. 1) and facilitated the detection of differences between normal and diseased brain in the concentration of acidic proteins. Two protein bands (neuronin S-1 and 2) have an electrophoretic mobility identical to a specific glial protein (S-100 protein, BOWENet al., in press). The other protein (neuronin S-6) migrates just ahead of human serum albumin. but has not as yet been identified (BOWENet al., in press). Other acidic protein bands visualized with this technique include neuronin S-5 (identical in most respects to antigen a or 14-3-2, BOWENet al., in press) and two unidentified bands neuronin S-3 and 4.
1523
TABLE 3. EFFECTOF
TIME BETWEEN DEATH AND FREEZING ON CONCENTRATION OF NEURONIN S-5 AND S-6 IN PREFRONTAL CORTEX Time death to -2oc (h)
Neuronin S-6 Diagnosis
Control (30)’ 0.25 Control (601. Control (62)’ 7.5 Control (68) Senile dementia 15.0 Control (67) Senile dementia 18.5 Control 176) Senile dementia 24.0 Control (70) Senile dementia 31.5 Control (67) Senile dementia
(79) (80)
(ME)
Neuronin S-5 lPgig)
215 310 I82
45 140 89
245
15 I20 70 105 54
0 155 45 230
(85)
0 307
89
(84)
50
190
110
65 175
(88)
0
67
~~
Numbers in brackets denote age (y). * Post-operative specimens, removed during therapeutic or exploratory surgery.
Post-mortem stability. Experiments on the stability of neuronins S-5 and S-6 in post-mortem tissues have been carried out. The specimens studied included human cortical tissues, removed during brain tumour surgery, and frozen as rapidly as possible. In addition post-mortem samples of prefrontal cortex, caudate nucleus, putamen, thalamus, globus pallidus and substantia nigra from both control cases and patients with senile dementia were frozen at various time intervals after death (Table 3). In all regions the concentration of both of the neuronin-type proteins were essentially independent of the period between death and freezing (results for frontal cortex are given in Table 3). Furthermore, the levels of these proteins in cortex were similar in both the post-mortem and post-operative specimens (Table 3). In order to confirm and extend these findings, portions af the postoperative tissues were incubated prior to freezing. The results of this study showed that in cortex both proteins are remarkably resistant to quite drastic treatment (e.g. incubation at body temperature for 18 h, BOWENet a/.. in preparation). Similar in oitro experiments carried out on cerebella cortex (post-operative specimen) confirm that in cortical regions neuronins S-5 and S-6 are relatively unaffected by post-mortem changes; preliminary results indicate that neuronins S-1 and 2 and S-3 and 4 are also stable. In a postmortem sample of caudate nucleus, maintained at room temperature for 18 h, the level of neuronin S-5 was decreased to 50% of the control value (the latter was obtained by assaying a sample 2.5 h after death). In a series of 7 normal control specimens a precise record was obtained of the interval between death and refrigeration of the corpse. In the caudate nucleus when the corpse was maintained at room temperature the concentration of neuronin S-5 was found to decrease at the rate of 46 pg/g wet wt/h ( P = < 0.01. linear regression analysis). Other results (SMITH. 1976) show that in the putamen the concentration decreases at half this rate (21 pg/g wet wt/h, P = < 0.02); in
CAROLYN B. SMITHand D. M. BOWEN
I524
TABLE4. CONCEXTRATIO~ OF W L I R O N I N S-5
A Y D S-6 I N CASES OF SENILE DEMEYTIA COMPARFI) WITH AGED-MATCHED COSTROLS
TABLE 5. NEURONIN S-5
AND S-6 I N CASES OF SENILE DEMENTIA A N D 'C.OSTRO1.S' DYING OF BRONCHOPNEUMONIA Ncurontn S-6
Neuronin S-6 I,'p f uct a t 1 Region
Senile dementia
l,iE g \\el
Neuronin S - 5
ills f
net -11
dementia
Pre-frontal cnrtc\
* 151
79
Caudate nuclew
YJ
Putamen
79 +_ 27 171
Globus pallidus
19 f 15 I51
Substantia
13
Thalamus
'Conlrol'
Senile dementia
'Control'
29 ? 31-
163 T 6 Y t (6)
114 f 60 171
83 f 56 (6)
I28 ?c 34
79 i 54
(7)
(71
Control
Pre-frontal CortCX
nigra
Neuronin S-5 ( p p i g wet wtl
derneiitie
Senile Regions
Ssnile
Control
Wli
171
42
r 44 171
c
5 i 14
Caudate nucleui
171
60
i 97: 171
~
16
171 81 t 1 O 161
S.D. Details of specimens in Table 7 (groups 2 and 14). Numbers in brackets are number of cases: NS. not
significantly different from control. *+Significantly different from control. where P is: *. < 0.001, t. < 0.oi. $ .= 0.02, 6. < 0.05.
the extra-striatal regions examined (prefrontal cortex, thalamus, substantia nigra and globus pallidus) provided the corpse is refrigerated within 2-3 h of death the concentration of neuronin S-5 is relatively unaffected by a minor delay in refrigeration. Efect ofage. In the 15 normal control brains (Table 2, groups 1 and 2) examined in this study there were no significant correlations with age in the levels of neuronins S-5 and S-6 in any of the six brain regions (prefrontal cortex, caudate nucleus. putamen. thalamus, substantia nigra and globus pallidus). Preliminary results indicate that the level of neuronin S-3 and 4 is independent of age (5G100 y). The absolute levels of neuronin S-5 and S-6 in normal controls are given in Table 4.
Eflect of rlie ternlirltrl disease Apart from clinical evidence of neurological or functional psychiatric disease most of the patients from which abnormal brains were obtained died of respiratory disease (e.g. bronchopneumonia) or had a lingering mode of death. In contrast. the majority of the normal 'controls' had died suddenly of myocardial infarction (Table 2). Bronc.hop,ieur,ionitr. The level of neuronin S-6 in the normal elderly patients is significantly higher than in 'control' cases dying of bronchopneumonia (Table 5, footnotes). Therefore. the levels of neuronin S-6 in cases of senile dementia and controls dying of bronchopneumonia have been compared. The results (Table 5 ) show that the concentration of neuronin S-6 in frontal cortex is significantly higher in the 'controls' that died of bronchopneumonia. A more objective comparison made from observations by the same pathologist assessing all the cases showed the pre-
The cases of senile dementia and 'control' specimens (2 cases of motor neurone disease, 1 case each of Huntington's chorea and multiple sclerosis and 3 non-neurological controls) all died of bronchopneumonia. Numbers in brackets are number of cases. * Significantly different from 'control'. P < 0.001. t,: Significantly different from the controls in Table 4
(died of heart disease). t P < 0.07. $ P < 0.01
and post-mortem state of the respiratory system in 2 cases of senile dementia to be no more markedly diseased than in 2 patients with depressive illness. Despite this. neuronin S-6 was conspicuously present in the control brains but was undetected in the cases of senile dementia. 'CerehroI hj,posia'. The brains of at least two of the patients that were examined in this study exhibited signs of severe terminal 'cerebral hypoxia'. The cortical grey matter from the patient with carbon monoxide poisoning ('group 13'. Table 2 and Fig. 2) was not depleted in neuronin 5-6. In contrast, the protein was depleted in all the brain regions from the patient with evidence of respiratory disease and profound 'cerebral hypoxia' ('group' 12, Table 2 and Fig. 2). Furthermore, in this brain the level of neuronin S-5 was also markedly depleted in some regions ('group 12'. Fig. 3). In 3 patients without obvious neurological disease or histological evidence of abnormal brain morphology neuronin S-6 was strikingly depleted (groups 10 and 11, Fig. 2). These brains were almost unique for the levels of neuronin S-5 (groups 10 and 11. Fig. 3) and S-3 and 4 were often lower than in the normal controls. The clinical data indicated that these patients had died in coma or had a lingering mode of death. The severity of coma was difficult to assess due to either incomplete records or drowsiness induced by pain-killing drug therapy. Furthermore, the division between coma and 'cerebral hypoxia' was unclear. For example, in group 10 (Table 2, cancer patients) one patient appeared to have been in a terminal drowsy state for several weeks while the other patient had post-mortem evidence of 'brain anaemia' and extensive damage to the lungs. The interpretation of the results for this patient was further complicated by a 5-year history of difficulty in controlling diabetes. Of the 6 'controls' with abnormal brain morphology ('groups' 4 and &8, Table 2) one case may have had prolonged terminal coma and
Soluble brain proteins 140
Senile dementia,mean: ------,range
0
1525
3
Normal elderly ,mean'I20
-
2.
3 13
L
0
nw
-
100
0
E
I
6
C
b
80
5
.\" a- 60
1.
3
3 I
5
I
5
v)
5
.-CC
$ 2
40
I5
4[
15'
20
0
Ecortex
Puturnen
G.pallldus
S. nigra
6-13 Thalamus
FIG. 2. Summary of the concentration of neuronin S-6 in six brain regions. The levels of neuronin S-6 in the normal elderly controls (continuous line) and cases of senile dementia (dashed line, range indicated by bar) are compared to the amounts in the other specimens. (The number identify the following groups: 1. normal adults; 3, functional disability; 4. plaques: 5. plaques and depression: 6 neuronal loss: 7. gliosis; 8, cerebral atrophy; 9, schizophrenia; 10. lingering death: 11, coma; 12, 'cerebral hypoxia'; 13. CO poisoning; 15, mixed senile and vascular dementia: see Table 2. The position of the numbers indicates the amount of the protein, e.g., the concentration of the protein in frontal cortcx from the norinal adults was 65:/, of the amount in the normal elderly cases.) The results were calculated. using mean values for each group. from concentration data expressedlg wet wt tissue. Absolute concentrations ( + S . U . ) for groups 2 & 14 (senile dementia) are given in Table 4. Bars enclose groups within the range of patients with senile dementia. The range was calculated from:
mean concentration group 14 + 1 S.D. x loo. mean concentration group 2 when the S.D. was greater than the mean the lower limit of the range is represented as 0. Two of the 3 brains examined within range of group 14: h l of the 2 brains examined within range of group 14; 'I of the brains examined within range of group 14.
three patients had circumstantial evidence of terminal defects in the systems that control the blood and oxygen supply to the brain (see patients Ja, We and in preparation). These findings sugBe, BOWENet d., gest that the depletion in neuronin S-6 in these cases (Fig. 2) may be attributable to the agonal state rather than the ongoing brain pathology.
the other 6 patients were similar to the levels in the normal control patients (e.g. groups 3 and 5. Fig. 2). Similarly, the concentration of neuronin S-6 was relatively high in the other five brain regions examined (groups 3 and 5. Fig. 2). Neuronins S-1 and 2, S-3 and 4 and S-5 were detected in all the specimens from the functionally ill patients that were analysed.
Functional psjchiatric disorders.
Organic dementia
Samples of prefrontal cortex were analysed from 10 elderly inmates of psychiatric hospitals with clini-
cal diagnoses that were usually of either schizophrenia or depressive illness (details of 4 cases are given under 'groups' 3, 5 and 9). Neuronin S-6 was depleted in the 4 cases that exhibited either severe cerebral vascular changes, cerebral atrophy or surgical lesions ('group' 9). The interpretation of the result for the case with surgical lesions cgroup' 9) is complicated, for these were defects in the state of systems that control the blood and oxygen supply to the brain (e.g. bilateral bronchopneumonia and terminal endocarditis of the mitral valves). The concentrations in
Since the effect of the terminal illness may be confounded by changes due to the neurological disease (e.g. reduced cerebral blood flow) where appropriate in this section the cause of death for individual cases is given. Senile dementia. In the pilot study all 13 normal controls contained conspicuous amounts of neuronin S-6 in the frontal cortex accounting for about 3"; of the total soluble protein (Table 1). All but one of the samples of prefrontal cortex from the additional 12 normal control brains contained about 300 pg/g wet wt. (Table 4). The atypical case died of fibrosing alveolitis. The same brain region from
CAROLYN B. SMITHand D. M. BOWEN
1526
330
6
312
Senile dementia.mean: -----,range
13
240 % L
; 200 Q
0
E r
160
s Q 120 v)
._c c
e
2 z
80
40
C
F. cortex
Caudate
Putarnen
G.pallidus
Snigra
Thalrnus
FIG. 3. Summary of the concentration of neuronin S-5 in six brain regions. Details are in Fig. 2. "One brain examined, bl brain examined within the range of group 14, '2 brains. examined, d2 of the 3 brains examined within the range of group 14.
17 cases of senile dementia have been examined: the amount of neuronin S-6 was markedly reduced in 15 of the cases (Tables 1 and 4).The atypical cases were unique because instead of dying of bronchopneumonia they died of either renal or ventricular failure. In the cases of senile dementia examined the mean levels of neuronin S-6 were also significantly reduced in the extracortical regions. The prefrontal cortex, the 3 parts of the basal ganglia, substantia nigra and the thalamus from 12 to 15 normal control brains and 9 cases of senile dementia were examined for content of neuronin S-5. All the specimens contained this protein (e.g. between 50 and 150pg/g wet wt of prefrontal cortex, Table 4).The mean levels in the putamen, globus pallidus and thalamus of the age-matched controls were significantly less than in senile dementia (i.e. 166 to 1800/, of the age-matched normal control value, Table 4). Neuronin S-3 and 4 was detected visually in all the specimens of prefrontal cortex. caudate nucleus and putamen and were present in most (i.e. in 62 of 69 specimens examined) of the samples of globus pallidus. substantia nigra and thalamus from both normal control and senile dementia brains. In most instances (i.e. in 7 to 8 specimens) where neuronin S-3 and 4 was undetected neuronin S-6 was very markedly reduced. Samples of prefrontal cortex from 13 normal control brains and 8 cases of senile dementia all contained conspicuous amounts of neuronin S-1 and 2. Although the mean concentration was 30'1; greater in senile dementia, compared with the level in the elderly normal controls, this difference is not statistically significant. Alzheimer's disease. Samples of prefrontal cortex have been examined from 6 patients afflicted with
Alzheimer's disease. Neuronin S-6 was not detected in 5 of the cases: this included one case in which the respiratory system was found to be normal at post-mortem. All specimens examined contained conspicuous amounts of neuronin S-3 and 4 and s-5, while the mean level of neuronin S-1 and 2 was significantly greater in Alzheimer's disease than in agematched controls (Table 1). Mixed senile and vascular dementia. Neuronin S-6 was not detected in the prefrontal cortex of 5 of the 7 cases examined; these cases died of respiratory disease. In the other patients the protein was markedly reduced in one case (cause of death: cerebral infarction) and within the normal control range in the other case (cause of death: myocardial infarction). In two of the 3 cases examined neuronin S-6 was depleted in all of the extra-cortical regions (group 15, Fig. 2) while in the third case the level was reduced in the striatum only. All specimens contained conspicuous amounts of neuronin S-1 and 2 and S-5 (Table 1 and Fig. 3) and neuronin S-3 and 4. DISCUSSION The development of improved gel electrophoresis techniques for examining complex mixtures of proteins has led to the detection of age-related changes in the protein composition of mouse brain extracts 1971). Application of this (GROSSFELD & SHOOTER, technique to human brain demonstrates (Fig. 1) that grey matter contains conspicuous amounts of wellseparated acidic protein bands (the neuronin-type proteins). These proteins in human brain are relatively unaffected by post-mortem changes (Table 3 and text) so that measurement of the concentrations
Soluble brain proteins
1527
should give useful data. However, since biopsy speci- in neurological diseases the two protein bands rarely mens from demented patients were not available, we appear to change in concentration. The apparent difhave not been able to rule out the possibility that ferences in the concentration of neuronin S-5 (Table post-mortem changes are exacerbated in the diseased 4; Fig. 3) are difficult to interpret because the protein brains. appears to be particularly sensitive to variation in In order to establish the normal ranges in concen- the interval between death and refrigeration of the tration of the neuronin-type proteins hypotonic corpse. For example, none of the normal elderly extracts of prefrontal cortex, substantia nigra, tha- patients died in the psychiatric hospitals from which lamus and 3 parts of the basal ganglia have been the cases of dementia were obtained. This suggests routinely analysed. The cases that were investigated that the differences in levels of neuronin S-5 (Table included 38 potential control patients with a clinical 4)may be related to differences in post-mortem handdiagnosis that was other than a neurological or func- ling for the precise mean interval between death and tional psychiatric illness; the ages of the patients, who refrigeration of the corpse may have been greater in had died either at home or in general hospitals, were the normal elderly group ( i t . since some of these between 48 and 100 yrs. The specimens were further patients died at home). In contrast to neuronin S-5 evaluated in order to allow for factors that might the concentration of neuronin S-6 appears to be reaffect brain proteins (i.e. terminal state and abnormal markably stable post-mortem. However, as judged by brain morphology). Six brains were eliminated as nor- the results obtained with the non-demented patients mal controls for they exhibited abnormal morphology that either died of bronchopneumonia, had evidence (a relatiwly high senile plaque count, cerebral atro- of coma, ‘cerebral hypoxia’ or had lingering modes phy, focal neuronal loss or an unusual type of gliosis of death the concentration of neuronin S-6 is affected see 4 and 6 8 , Table 2 ) ; 4 specimens were excluded by the agonal state. This and other evidence (BOWEN for there was evidence of either coma, ‘cerebral et nl., 1976a) suggests that neuronin S-6 is a relatively hypoxia’ or a lingering mode of death (10-12, Table sensitive index of at least terminal ‘cerebral hypoxia’. 2). In every region examined 26 of the remaining 28 We were unable to establish with any precision specimens contained all of the neuronin-type proteins whether or not there were differences in the degree (Fig. 1). Apart from the possible exception of neur- of terminal coma (MCGEER& MCGEER, 1976) onin S-1 and 2 in frontal cortex (which tends to in- between groups of patients. However, since patients crease in concentration with advancing age, BOWEN in terminal coma probably have ‘cerebral hypoxia’ (i.e. secondary to reduced cerebral blood flow, et al.. 1973) other findings (SMITH,1976) indicate that in adulthood the pattern of the neuronin-type pro- INGVAR,1976) our findings and those of MCGEER& teins is not age-dependent in any of the regions exam- MCGEER(1976) demonstrate the importance of conined. The levels of the neuronin-type proteins were trolling for the terminal state. We have previously reported (BOWENet al., 1973) within the normal control range in the majority of the brains from patients in hospital with functional that neuronin S-6 is depleted in the cortical grey matpsychiatric disorders. The atypical specimens were de- ter from a small group of patients with organic pleted in neuronin S-6 and exhibited evidence of dementias. In the current study we have analysed coreither morphological abnormalities, including cere- tex from 30 cases of dementia (aged 60-97 yrs). The bral vascular disease, or coma. Thus after elimination concentration of neuronin S-6 was markedly depleted of these atypical patients normal adult grey matter (usually undetectable) in 26 cases. The atypical speciobtained at post-mortem appears to have a consistent mens (i.e. those with normal control levels of neurpattern of 6 major acidic protein bands (Fig. 1). These onin S-6) were 1 case each of Alzheimer’s disease (6 findings concur with those of CAIN et al. (1974) for cases examined), and mixed senile and vascular they demonstrated that histologically normal cortical dementia (7 cases examined), and 2 cases of senile biopsies give a similar pattern. The results of CAPLAN dementia (17 cases examined). Three of the 4 atypical et crl. (1974) indicate that the extracts from the post- cases died of either renal or ventricular failure while mortem samples that they investigated did not con- almost all of the cases depleted in neuronin S-6 died tain neuronin s-6(which has a similar mobility to band of bronchopneumonia. Despite this apparent correlaNo. 6 of CAINet al. 1974). It is difficult to interpret tion the level of neuronin S-6 was lower in cases of this anomaly for no description was given of the clini- senile dementia dying of bronchopneumonia than in cal, histological or terminal state of the specimens ‘controls’ dying of the same cause (Table 5 ) . One examined. Furthermore, the conditions that were used explanation for this is that the effects of bronchoto prepare the protein extract were not optimal for pneumonia are accentuated by reduced cerebral blood flow (INGVAR & GUSTAFSON, 1970) and a defecneuronin S-6 (SMITH,1976). The concentration of neuronin S-1 and 2 (identical tive respiratory reflex (ALLISON,1962) in the terminal to glial S-100 protein, BOWENet a/.,in press) in prefron- stages of the disease. The concentration of neuronin tal cortex was usually elevated in the cases of demen- S-6 was also markedly reduced in the thalamus, tia. This is probably due to the gliosis which was putamen, caudate nucleus, globus pallidus and suboften quite marked in the specimens examined. Neur- stantia nigra which are regions that usually exhibit onin S-3 and 4 has not yet been characterized, for little evidence of light microscopic changes in this dis-
1528
CAROLYN B. SMITHand D. M.
ease (CORSELLIS J. A. N., personal communication).
&WEN
BOWEND. M.. SMITH C. B.. WHITEP. & DAVISON A. N. Tl7r Nrurohiolo;7~. of Ayeiiiy. Raven Press, New Despite this, there is biochemical and clinical eviYork. In press. dence that neurotransmitter metabolism may be disCAIN D. F., BALLE. D. & DEKABAN A. S. (1974) J . Neuroturbed in basal ganglia in senile dementia. and Alzchenl. 23, 561-568. heimer's disease (GOTTFRIES er d., 1969. 1974; PEARCE, CAPLAN R., CHEUNG S. C.-Y & OMENN G. S. (1974) d . 1974; DRACHMAN & STAHL.1975). Neurochem. 22, 5 17-520. CORSELLIS J. A. N. (1962) Mental Illness and the Ageing Acknowledgements-We wish to express our gratitude to Braiii. Oxford Uni\ersity Press. New York. for his encouragment and advice DRACHMAS Professor A. N. DAVISON D. A. & STAHLS. (1975) Lancet (I), 809. gener- INGVAR throughout this investigation. Dr. J. A. N. CORSELLIS D. H. (1976) Brain Res. 107, 181-197. ously provided most of the pathological specimens and INGAR D. H. & GUSTAFSON L. (1970) Acta neuw/. scand. assessed almost all of the brains histologically. We also 46, Suppl. 43, 42-73. thank the numerous pathologists who supplied the control GOTTFRIES C. G.. GOTTFRIES I. & ROOS B. E. (1969) Br. material. The work was suppported by the MRC. the MirJ . Ph!.chiut. 115, 563-574. iam Marks Charitable Trust and the Wellcome Trust. GOTTFRIES C. G.. KJALLQUIST A,. PONTINU., Roos B. E. 61 SL'VDARG G. (1974) Br. .I. Psychiot. 124, 28&287. GROSSFELD R. M. & SHOOTER E. M. (1971) J. Neurochem. REFERENCES 18, 2265-2277. ALLISON R. S. (1962) The Senile Bruin. Edward Arnold, LOWRY 0. H.. ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. b i d . Chem. 193, 269-275. London. BOWEND. M., SMITH C. B. & DAVISOX A. N. (1973) Brain MAURERH. H. (1971) Disc Electrophoresis and Related 96, 849-856. Techniques of Poljacrylamitfe Gel Electrophoresis. Walter BOWEN D. M., FLACKR. H. A,. MARTINR. 0.. SMITH de Gruyter, Berlin. C. B., WHITEP. & DAVISON A. N. (1974) J . Neurochem. MCGEERP. L. & MCGEERE. G. (1976) J . Neurochem. 22, 1099-1 107. 26,65-76. BOWEN D. M.. SMITHC. B.. WHITEP. & DAVISOS A. N. PEARCE J. ( 1974) Eirr. Nrtiro/. 12. 94103. Bruin. In preparation. SMITHC. B. (1976) PI1.D. Thesis. University of London.
IN A
J. A. N. CORSELLIS). Samples of prefrontal grey matter (from the crests of the gyri and including all cortical layers). thalamus (medial portion), putamen, globus pallidus, caudate nucleus and substantia nigra were taken for biochemical analysis. The samples were weighed and routinely stored overnight at -20°C prior to protein extraction. Details of age, sex, cause of death and source of specimens are given in Tables 1 and 2. No selection of cases by drug treatment was made. The only noticeable consistent pattern of treatment was that many of the patients (both dements and functionally ill) in the psychiatric hospitals received the same innocuous medication, chloral hydrate. as a sleeping draught. Six brain regions from the specimens described in Table 2 were analysed for protein composition while only the protein composition of frontal cortex of those in Table 1 was examined. Nosology of the specimens. For the classification of the material examined, the initial division was based on the clinical presentation. Thus the specimens from 86 patients included 52 brains from patients who had died without obvious signs of neurological disease while the other brains were from patients with various neurological diseases. The brains in the non-neurological group were further subdivided on the basis of morphological criteria (e.g. incidence of senile or neuritic plaques and neurofibrillary degeneration, focal nerve cell loss and gliosis and degree of either cerebral vascular change or gross cerebral atrophy), presence of obvious functional disability (e.g. depressive illness or schizophrenia) and whether there was eviMATERIALS AND METHODS dence of a lingering mode of death, coma or ‘cerebral Specimens. After death, corpses were maintained at 4°C hypoxia’. For the classification of specimens from patients until autopsy. The time between death and whole body with neurological diseases morphological criteria were refrigeration was routinely within the range 0.75-2.5h. The decisive. The traditional classification (CORSELLIS, 1962) of brain was excised within 24 h of death and halved sagit- mental illnesses into ‘organic’ and ‘functional’ has been tally; one half was preserved for histopathology (Dr. retained. Accordingly, the brains were assigned to an appropriate group (Table 2; most of the brains in Table Present address: Laboratory of Biochemical Genetics, 1 could be placed within these groups: exceptions are in National Institutes of Health, Bethesda, M D 20014,U.S.A. the text). 1521
CAROLYN B . SMITHand D. M. BOWEN
1522
TABLE1. CONCENTRATION OF TOTAL
SOLUBLE. AND TWO SOLUBLE ACIDIC PROTEINS IN PRE-FRONTAL CORTEX Solublc protcins
Diagnostic group*
.Age ly1
No
Disease
Period before storage (h)
casest
Acidic Total (", total protein)
Neuronin S - h I",,sol. protein1
Neuronin I and 2 (". sol. protein)
~-
I
Normal adult Alzheimer's disease Significance (PI
-: 2
5
Normal elderl) Semle dementia Significance (PI
14
&,
X 4
x
60 2 6 67k3 NS
19 f 10
79+5 7xi7 NS
18*X 9+7 NS
28.8 f 3.3 (41 19.2 4.9 < 0.02
3.16
+ 1.31
0.33
k 0.654
24.8 2 0.9 (41 19.3 k 44 ( 5 ) < 0.05
2.63 f 0.62
*
X+I1 NS
1.30 2.76
0.38
k 0.17
< 0.001
< 0.01
1.59 It 0.93
k l.4l$ < 0.02
0.76
2.05 k 1.03 NS
S.D.
Number of samples where this differs from No. cases are shown in brackets. * See Materials and Methods. t Different cases from those in Table 2. $ Clinically established and autopsy confirmed cases. Q Neuronin S-6 detected in one brain (1.32"J. Frontal cortex from 2 other cases of Alzheimer's disease was also subject to electrophoresis. the gels were not scanned but visual examination indicated that neuronin S-6 was absent from one and within the normal range in the other specimen. Protein analpis. Polyacrylamide gel electrophoresis was the generic name neirroniii foi- this group of soluble acidic proteins. for the prealbumin portion of gels of extraneural used to analyse the protein composition of buffered-hypotonic extracts (BOWENer a/.. 1973) from the 6 brain regions tissues do not contain such conspicuous amounts of these proteins. The results obtained with the porosity-gradient examined. In order to increase the resolution. porosity& SHOOTER. gels are compared in Fig. 1 with those obtained with 7.5",, gradient gels were routinely used (GROSSFELD 1971).This technique revealed a number of well-separated (wjv) acrylamide gels. protein bands (Fig. 1, gel on the left) including 6 bands The neuronin-type proteins were quantified on the gels (neuronins 1 4 ) that migrated rapidly to the anode ahead by direct densitometry. The protein stain, Procion Brilliant of albumin. We have proposed (BOWFNrr ul.. in press) Blue R.S.. was chosen as the dye because of its reproducibiTABLE2.
Diagnostic group
F'RE-ASSAY HISTORY OF BRAIN SPECIMENS ANALYZED FOR PROTEIN CONTENT IN SIX BRAIN REGIONS
Disease
Period between death Cause & storage Sex of or assay IM : F) deatht Source:
No. cdses'
Age
h
hi I x3 2 10
I! I
Brain morphology
Comments
0-1
002 300 001 010
normal normal normal plaques plaques appreciable neuronal
age range' 50-69 y age range: over 70 y had depressive illness no functional illness short depressive illness (ECT) alcoholic (liver flap)
Non-neurological
3 4 5 6
Normal adult Normal rldrrl) Functional disabilit! Plaque Plaques & depression Neuronal loss
7
GllOSlS
8
Cerebral atroph! Schizophrenia Lingering death Coma 'Ccrebral hypoxia' CO poisoning
I
'
70. 85 3 77
is
*
.. >>
+x
7.1
23 i 2 I?. 20 23 5 7 24
?:5 2 .O I :? 0:I
20
*
710 430
loss
9 10 11 12 13
14 15
5
I :0
010
scvere and bizarre ghosts
72 60 4x.m 76
IS
0:l 0.1 O:?
010
deep sulci. thin cortex severe brain surgery normal normal markcd oedemas severe anoxic changes
13 20. 9 24 7 21
67 ?J
Neurological Senile dementia Mixed senile and vascular dementia
* Different cases from
X?
9
X6 5 h
21 + 7
1
7x 5
x
1623
001 020 100 100
0:I I :o I ,0
3 :6 0:3
100
6012 0210
009 003
atypical myeloproliferative disorder n o terminal ileum hospitalized 37 y cancer (breast. colon) ~
lung surgery!, suicide case
Diagnostic division 0.2 (CORSFLLIS,1962) Diagnostic division 0.3 (Ci)nSFLLiS, 1962)
those in Table 1.
t 1st digit identifies the number of patients dying from respiratory disease (usually bronchopneumonia, but also chronic bronchitis and pulmonary thromboembolism). 2nd from coronary thrombosis, 3rd from cancer and 4th from other causes. $ 1st digit identifies the number of specimens originating from coroner's courts, 2nd from general hospitals, 3rd from psychiatric hospitals. S.D. (age and interval between death and assay for groups 2 and 14 did not differ significantly). §This is a change not infrequently seen in persons suffering from cor pulmonale who die in a severely hypoxic state. /I Lobectomy for bronchietasis. pleuro-bronchial fistula. Plaques. indicate that the relati\e number of senile plaques was at least moderate in at least one region examined (in groups 2 and 14. the scores were zero to slight and moderate to severe. respectively. Corsellis J. A. N., personal communication).
+,
FIG.I. Tris -glyciiie polyacrylamide gels of hypotonic extracts of pre-frontal cortex from normal IiLinian brain. The gels shown are from left to right: porosity gradient gel, 7.5"" acrylamide gels 15 CIII and 5 cm. respectively.
Soluble brain proteins lity, uniformity of protein binding and the stable covalent dye-protein bond (MAURER,1971). In the pilot study
(Table I ) neuronin-type proteins were quantified as a percentage of the total soluble protein that entered the 404 acrylamide gel. The gels were scanned at 595 nm using a Fisons’ vitatron Scanning Densitometer. The areas under the peaks corresponding to neuronins S-1 and 2 and S-6 were determined (by weighing the appropriate portions of chart paper) as a percentage of the total area (limit of detection, 0.6% of the total soluble protein). In subsequent experiments the absolute amounts of the proteins were determined using a standard of bovine serum albumin (Fraction V, Sigma Chemical Co., St. Louis, MO). In these experiments the gels were scanned at 602 nm on a Gilford Model 240 spectrophotometer equipped with a linearly transported glass cuvette. Absorbances were recorded on a Servoscribe 15 chart-driven recorder fitted with an integrator. On the chart recorder traces the troughs of the peaks corresponding to neuronins S-5 and sd were joined to form a baselin. The areas under the two peaks were compared with a standard curve (limit of detection, 7 pg/g wet wt). Total protein and total soluble protein (i.e. the protein content of the buffered hypotonic extract) was measured using the method of LOWRY et nl. (1951).
RESULTS
Total and total soluble protein. In general the levels of total protein were similar in all the brains that were examined. For example, no significant differences were detected in any region when the levels of total protein in senile dementia were compared with the levels in either the normal adult or elderly control groups. In contrast, the level of total protein in prefrontal cortex from the patients in groups 1&13 (Table 2) was significantly (P= < 0.02) reduced to 84:d of the concentration in the age-matched normal controls. The levels of total protein in caudate nucleus from these patients (67.2 f 16.3 mg protein/g wet wt) was also significantly reduced ( P = < 0.05) to 74”/, of the level in the control group. Similarly, the levels of total soluble protein were usually moderately reduced (to about 75% of the control value) in the cases of dementia (Table 1). Neuronin-type proteins. Polyacrylamide gel electrophoresis was used to compare the protein composition of the soluble proteins present in the frontal grey matter of control and senile brains. This technique revealed a number of relatively well-separated protein bands (Fig. 1) and facilitated the detection of differences between normal and diseased brain in the concentration of acidic proteins. Two protein bands (neuronin S-1 and 2) have an electrophoretic mobility identical to a specific glial protein (S-100 protein, BOWENet al., in press). The other protein (neuronin S-6) migrates just ahead of human serum albumin. but has not as yet been identified (BOWENet al., in press). Other acidic protein bands visualized with this technique include neuronin S-5 (identical in most respects to antigen a or 14-3-2, BOWENet al., in press) and two unidentified bands neuronin S-3 and 4.
1523
TABLE 3. EFFECTOF
TIME BETWEEN DEATH AND FREEZING ON CONCENTRATION OF NEURONIN S-5 AND S-6 IN PREFRONTAL CORTEX Time death to -2oc (h)
Neuronin S-6 Diagnosis
Control (30)’ 0.25 Control (601. Control (62)’ 7.5 Control (68) Senile dementia 15.0 Control (67) Senile dementia 18.5 Control 176) Senile dementia 24.0 Control (70) Senile dementia 31.5 Control (67) Senile dementia
(79) (80)
(ME)
Neuronin S-5 lPgig)
215 310 I82
45 140 89
245
15 I20 70 105 54
0 155 45 230
(85)
0 307
89
(84)
50
190
110
65 175
(88)
0
67
~~
Numbers in brackets denote age (y). * Post-operative specimens, removed during therapeutic or exploratory surgery.
Post-mortem stability. Experiments on the stability of neuronins S-5 and S-6 in post-mortem tissues have been carried out. The specimens studied included human cortical tissues, removed during brain tumour surgery, and frozen as rapidly as possible. In addition post-mortem samples of prefrontal cortex, caudate nucleus, putamen, thalamus, globus pallidus and substantia nigra from both control cases and patients with senile dementia were frozen at various time intervals after death (Table 3). In all regions the concentration of both of the neuronin-type proteins were essentially independent of the period between death and freezing (results for frontal cortex are given in Table 3). Furthermore, the levels of these proteins in cortex were similar in both the post-mortem and post-operative specimens (Table 3). In order to confirm and extend these findings, portions af the postoperative tissues were incubated prior to freezing. The results of this study showed that in cortex both proteins are remarkably resistant to quite drastic treatment (e.g. incubation at body temperature for 18 h, BOWENet a/.. in preparation). Similar in oitro experiments carried out on cerebella cortex (post-operative specimen) confirm that in cortical regions neuronins S-5 and S-6 are relatively unaffected by post-mortem changes; preliminary results indicate that neuronins S-1 and 2 and S-3 and 4 are also stable. In a postmortem sample of caudate nucleus, maintained at room temperature for 18 h, the level of neuronin S-5 was decreased to 50% of the control value (the latter was obtained by assaying a sample 2.5 h after death). In a series of 7 normal control specimens a precise record was obtained of the interval between death and refrigeration of the corpse. In the caudate nucleus when the corpse was maintained at room temperature the concentration of neuronin S-5 was found to decrease at the rate of 46 pg/g wet wt/h ( P = < 0.01. linear regression analysis). Other results (SMITH. 1976) show that in the putamen the concentration decreases at half this rate (21 pg/g wet wt/h, P = < 0.02); in
CAROLYN B. SMITHand D. M. BOWEN
I524
TABLE4. CONCEXTRATIO~ OF W L I R O N I N S-5
A Y D S-6 I N CASES OF SENILE DEMEYTIA COMPARFI) WITH AGED-MATCHED COSTROLS
TABLE 5. NEURONIN S-5
AND S-6 I N CASES OF SENILE DEMENTIA A N D 'C.OSTRO1.S' DYING OF BRONCHOPNEUMONIA Ncurontn S-6
Neuronin S-6 I,'p f uct a t 1 Region
Senile dementia
l,iE g \\el
Neuronin S - 5
ills f
net -11
dementia
Pre-frontal cnrtc\
* 151
79
Caudate nuclew
YJ
Putamen
79 +_ 27 171
Globus pallidus
19 f 15 I51
Substantia
13
Thalamus
'Conlrol'
Senile dementia
'Control'
29 ? 31-
163 T 6 Y t (6)
114 f 60 171
83 f 56 (6)
I28 ?c 34
79 i 54
(7)
(71
Control
Pre-frontal CortCX
nigra
Neuronin S-5 ( p p i g wet wtl
derneiitie
Senile Regions
Ssnile
Control
Wli
171
42
r 44 171
c
5 i 14
Caudate nucleui
171
60
i 97: 171
~
16
171 81 t 1 O 161
S.D. Details of specimens in Table 7 (groups 2 and 14). Numbers in brackets are number of cases: NS. not
significantly different from control. *+Significantly different from control. where P is: *. < 0.001, t. < 0.oi. $ .= 0.02, 6. < 0.05.
the extra-striatal regions examined (prefrontal cortex, thalamus, substantia nigra and globus pallidus) provided the corpse is refrigerated within 2-3 h of death the concentration of neuronin S-5 is relatively unaffected by a minor delay in refrigeration. Efect ofage. In the 15 normal control brains (Table 2, groups 1 and 2) examined in this study there were no significant correlations with age in the levels of neuronins S-5 and S-6 in any of the six brain regions (prefrontal cortex, caudate nucleus. putamen. thalamus, substantia nigra and globus pallidus). Preliminary results indicate that the level of neuronin S-3 and 4 is independent of age (5G100 y). The absolute levels of neuronin S-5 and S-6 in normal controls are given in Table 4.
Eflect of rlie ternlirltrl disease Apart from clinical evidence of neurological or functional psychiatric disease most of the patients from which abnormal brains were obtained died of respiratory disease (e.g. bronchopneumonia) or had a lingering mode of death. In contrast. the majority of the normal 'controls' had died suddenly of myocardial infarction (Table 2). Bronc.hop,ieur,ionitr. The level of neuronin S-6 in the normal elderly patients is significantly higher than in 'control' cases dying of bronchopneumonia (Table 5, footnotes). Therefore. the levels of neuronin S-6 in cases of senile dementia and controls dying of bronchopneumonia have been compared. The results (Table 5 ) show that the concentration of neuronin S-6 in frontal cortex is significantly higher in the 'controls' that died of bronchopneumonia. A more objective comparison made from observations by the same pathologist assessing all the cases showed the pre-
The cases of senile dementia and 'control' specimens (2 cases of motor neurone disease, 1 case each of Huntington's chorea and multiple sclerosis and 3 non-neurological controls) all died of bronchopneumonia. Numbers in brackets are number of cases. * Significantly different from 'control'. P < 0.001. t,: Significantly different from the controls in Table 4
(died of heart disease). t P < 0.07. $ P < 0.01
and post-mortem state of the respiratory system in 2 cases of senile dementia to be no more markedly diseased than in 2 patients with depressive illness. Despite this. neuronin S-6 was conspicuously present in the control brains but was undetected in the cases of senile dementia. 'CerehroI hj,posia'. The brains of at least two of the patients that were examined in this study exhibited signs of severe terminal 'cerebral hypoxia'. The cortical grey matter from the patient with carbon monoxide poisoning ('group 13'. Table 2 and Fig. 2) was not depleted in neuronin 5-6. In contrast, the protein was depleted in all the brain regions from the patient with evidence of respiratory disease and profound 'cerebral hypoxia' ('group' 12, Table 2 and Fig. 2). Furthermore, in this brain the level of neuronin S-5 was also markedly depleted in some regions ('group 12'. Fig. 3). In 3 patients without obvious neurological disease or histological evidence of abnormal brain morphology neuronin S-6 was strikingly depleted (groups 10 and 11, Fig. 2). These brains were almost unique for the levels of neuronin S-5 (groups 10 and 11. Fig. 3) and S-3 and 4 were often lower than in the normal controls. The clinical data indicated that these patients had died in coma or had a lingering mode of death. The severity of coma was difficult to assess due to either incomplete records or drowsiness induced by pain-killing drug therapy. Furthermore, the division between coma and 'cerebral hypoxia' was unclear. For example, in group 10 (Table 2, cancer patients) one patient appeared to have been in a terminal drowsy state for several weeks while the other patient had post-mortem evidence of 'brain anaemia' and extensive damage to the lungs. The interpretation of the results for this patient was further complicated by a 5-year history of difficulty in controlling diabetes. Of the 6 'controls' with abnormal brain morphology ('groups' 4 and &8, Table 2) one case may have had prolonged terminal coma and
Soluble brain proteins 140
Senile dementia,mean: ------,range
0
1525
3
Normal elderly ,mean'I20
-
2.
3 13
L
0
nw
-
100
0
E
I
6
C
b
80
5
.\" a- 60
1.
3
3 I
5
I
5
v)
5
.-CC
$ 2
40
I5
4[
15'
20
0
Ecortex
Puturnen
G.pallldus
S. nigra
6-13 Thalamus
FIG. 2. Summary of the concentration of neuronin S-6 in six brain regions. The levels of neuronin S-6 in the normal elderly controls (continuous line) and cases of senile dementia (dashed line, range indicated by bar) are compared to the amounts in the other specimens. (The number identify the following groups: 1. normal adults; 3, functional disability; 4. plaques: 5. plaques and depression: 6 neuronal loss: 7. gliosis; 8, cerebral atrophy; 9, schizophrenia; 10. lingering death: 11, coma; 12, 'cerebral hypoxia'; 13. CO poisoning; 15, mixed senile and vascular dementia: see Table 2. The position of the numbers indicates the amount of the protein, e.g., the concentration of the protein in frontal cortcx from the norinal adults was 65:/, of the amount in the normal elderly cases.) The results were calculated. using mean values for each group. from concentration data expressedlg wet wt tissue. Absolute concentrations ( + S . U . ) for groups 2 & 14 (senile dementia) are given in Table 4. Bars enclose groups within the range of patients with senile dementia. The range was calculated from:
mean concentration group 14 + 1 S.D. x loo. mean concentration group 2 when the S.D. was greater than the mean the lower limit of the range is represented as 0. Two of the 3 brains examined within range of group 14: h l of the 2 brains examined within range of group 14; 'I of the brains examined within range of group 14.
three patients had circumstantial evidence of terminal defects in the systems that control the blood and oxygen supply to the brain (see patients Ja, We and in preparation). These findings sugBe, BOWENet d., gest that the depletion in neuronin S-6 in these cases (Fig. 2) may be attributable to the agonal state rather than the ongoing brain pathology.
the other 6 patients were similar to the levels in the normal control patients (e.g. groups 3 and 5. Fig. 2). Similarly, the concentration of neuronin S-6 was relatively high in the other five brain regions examined (groups 3 and 5. Fig. 2). Neuronins S-1 and 2, S-3 and 4 and S-5 were detected in all the specimens from the functionally ill patients that were analysed.
Functional psjchiatric disorders.
Organic dementia
Samples of prefrontal cortex were analysed from 10 elderly inmates of psychiatric hospitals with clini-
cal diagnoses that were usually of either schizophrenia or depressive illness (details of 4 cases are given under 'groups' 3, 5 and 9). Neuronin S-6 was depleted in the 4 cases that exhibited either severe cerebral vascular changes, cerebral atrophy or surgical lesions ('group' 9). The interpretation of the result for the case with surgical lesions cgroup' 9) is complicated, for these were defects in the state of systems that control the blood and oxygen supply to the brain (e.g. bilateral bronchopneumonia and terminal endocarditis of the mitral valves). The concentrations in
Since the effect of the terminal illness may be confounded by changes due to the neurological disease (e.g. reduced cerebral blood flow) where appropriate in this section the cause of death for individual cases is given. Senile dementia. In the pilot study all 13 normal controls contained conspicuous amounts of neuronin S-6 in the frontal cortex accounting for about 3"; of the total soluble protein (Table 1). All but one of the samples of prefrontal cortex from the additional 12 normal control brains contained about 300 pg/g wet wt. (Table 4). The atypical case died of fibrosing alveolitis. The same brain region from
CAROLYN B. SMITHand D. M. BOWEN
1526
330
6
312
Senile dementia.mean: -----,range
13
240 % L
; 200 Q
0
E r
160
s Q 120 v)
._c c
e
2 z
80
40
C
F. cortex
Caudate
Putarnen
G.pallidus
Snigra
Thalrnus
FIG. 3. Summary of the concentration of neuronin S-5 in six brain regions. Details are in Fig. 2. "One brain examined, bl brain examined within the range of group 14, '2 brains. examined, d2 of the 3 brains examined within the range of group 14.
17 cases of senile dementia have been examined: the amount of neuronin S-6 was markedly reduced in 15 of the cases (Tables 1 and 4).The atypical cases were unique because instead of dying of bronchopneumonia they died of either renal or ventricular failure. In the cases of senile dementia examined the mean levels of neuronin S-6 were also significantly reduced in the extracortical regions. The prefrontal cortex, the 3 parts of the basal ganglia, substantia nigra and the thalamus from 12 to 15 normal control brains and 9 cases of senile dementia were examined for content of neuronin S-5. All the specimens contained this protein (e.g. between 50 and 150pg/g wet wt of prefrontal cortex, Table 4).The mean levels in the putamen, globus pallidus and thalamus of the age-matched controls were significantly less than in senile dementia (i.e. 166 to 1800/, of the age-matched normal control value, Table 4). Neuronin S-3 and 4 was detected visually in all the specimens of prefrontal cortex. caudate nucleus and putamen and were present in most (i.e. in 62 of 69 specimens examined) of the samples of globus pallidus. substantia nigra and thalamus from both normal control and senile dementia brains. In most instances (i.e. in 7 to 8 specimens) where neuronin S-3 and 4 was undetected neuronin S-6 was very markedly reduced. Samples of prefrontal cortex from 13 normal control brains and 8 cases of senile dementia all contained conspicuous amounts of neuronin S-1 and 2. Although the mean concentration was 30'1; greater in senile dementia, compared with the level in the elderly normal controls, this difference is not statistically significant. Alzheimer's disease. Samples of prefrontal cortex have been examined from 6 patients afflicted with
Alzheimer's disease. Neuronin S-6 was not detected in 5 of the cases: this included one case in which the respiratory system was found to be normal at post-mortem. All specimens examined contained conspicuous amounts of neuronin S-3 and 4 and s-5, while the mean level of neuronin S-1 and 2 was significantly greater in Alzheimer's disease than in agematched controls (Table 1). Mixed senile and vascular dementia. Neuronin S-6 was not detected in the prefrontal cortex of 5 of the 7 cases examined; these cases died of respiratory disease. In the other patients the protein was markedly reduced in one case (cause of death: cerebral infarction) and within the normal control range in the other case (cause of death: myocardial infarction). In two of the 3 cases examined neuronin S-6 was depleted in all of the extra-cortical regions (group 15, Fig. 2) while in the third case the level was reduced in the striatum only. All specimens contained conspicuous amounts of neuronin S-1 and 2 and S-5 (Table 1 and Fig. 3) and neuronin S-3 and 4. DISCUSSION The development of improved gel electrophoresis techniques for examining complex mixtures of proteins has led to the detection of age-related changes in the protein composition of mouse brain extracts 1971). Application of this (GROSSFELD & SHOOTER, technique to human brain demonstrates (Fig. 1) that grey matter contains conspicuous amounts of wellseparated acidic protein bands (the neuronin-type proteins). These proteins in human brain are relatively unaffected by post-mortem changes (Table 3 and text) so that measurement of the concentrations
Soluble brain proteins
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should give useful data. However, since biopsy speci- in neurological diseases the two protein bands rarely mens from demented patients were not available, we appear to change in concentration. The apparent difhave not been able to rule out the possibility that ferences in the concentration of neuronin S-5 (Table post-mortem changes are exacerbated in the diseased 4; Fig. 3) are difficult to interpret because the protein brains. appears to be particularly sensitive to variation in In order to establish the normal ranges in concen- the interval between death and refrigeration of the tration of the neuronin-type proteins hypotonic corpse. For example, none of the normal elderly extracts of prefrontal cortex, substantia nigra, tha- patients died in the psychiatric hospitals from which lamus and 3 parts of the basal ganglia have been the cases of dementia were obtained. This suggests routinely analysed. The cases that were investigated that the differences in levels of neuronin S-5 (Table included 38 potential control patients with a clinical 4)may be related to differences in post-mortem handdiagnosis that was other than a neurological or func- ling for the precise mean interval between death and tional psychiatric illness; the ages of the patients, who refrigeration of the corpse may have been greater in had died either at home or in general hospitals, were the normal elderly group ( i t . since some of these between 48 and 100 yrs. The specimens were further patients died at home). In contrast to neuronin S-5 evaluated in order to allow for factors that might the concentration of neuronin S-6 appears to be reaffect brain proteins (i.e. terminal state and abnormal markably stable post-mortem. However, as judged by brain morphology). Six brains were eliminated as nor- the results obtained with the non-demented patients mal controls for they exhibited abnormal morphology that either died of bronchopneumonia, had evidence (a relatiwly high senile plaque count, cerebral atro- of coma, ‘cerebral hypoxia’ or had lingering modes phy, focal neuronal loss or an unusual type of gliosis of death the concentration of neuronin S-6 is affected see 4 and 6 8 , Table 2 ) ; 4 specimens were excluded by the agonal state. This and other evidence (BOWEN for there was evidence of either coma, ‘cerebral et nl., 1976a) suggests that neuronin S-6 is a relatively hypoxia’ or a lingering mode of death (10-12, Table sensitive index of at least terminal ‘cerebral hypoxia’. 2). In every region examined 26 of the remaining 28 We were unable to establish with any precision specimens contained all of the neuronin-type proteins whether or not there were differences in the degree (Fig. 1). Apart from the possible exception of neur- of terminal coma (MCGEER& MCGEER, 1976) onin S-1 and 2 in frontal cortex (which tends to in- between groups of patients. However, since patients crease in concentration with advancing age, BOWEN in terminal coma probably have ‘cerebral hypoxia’ (i.e. secondary to reduced cerebral blood flow, et al.. 1973) other findings (SMITH,1976) indicate that in adulthood the pattern of the neuronin-type pro- INGVAR,1976) our findings and those of MCGEER& teins is not age-dependent in any of the regions exam- MCGEER(1976) demonstrate the importance of conined. The levels of the neuronin-type proteins were trolling for the terminal state. We have previously reported (BOWENet al., 1973) within the normal control range in the majority of the brains from patients in hospital with functional that neuronin S-6 is depleted in the cortical grey matpsychiatric disorders. The atypical specimens were de- ter from a small group of patients with organic pleted in neuronin S-6 and exhibited evidence of dementias. In the current study we have analysed coreither morphological abnormalities, including cere- tex from 30 cases of dementia (aged 60-97 yrs). The bral vascular disease, or coma. Thus after elimination concentration of neuronin S-6 was markedly depleted of these atypical patients normal adult grey matter (usually undetectable) in 26 cases. The atypical speciobtained at post-mortem appears to have a consistent mens (i.e. those with normal control levels of neurpattern of 6 major acidic protein bands (Fig. 1). These onin S-6) were 1 case each of Alzheimer’s disease (6 findings concur with those of CAIN et al. (1974) for cases examined), and mixed senile and vascular they demonstrated that histologically normal cortical dementia (7 cases examined), and 2 cases of senile biopsies give a similar pattern. The results of CAPLAN dementia (17 cases examined). Three of the 4 atypical et crl. (1974) indicate that the extracts from the post- cases died of either renal or ventricular failure while mortem samples that they investigated did not con- almost all of the cases depleted in neuronin S-6 died tain neuronin s-6(which has a similar mobility to band of bronchopneumonia. Despite this apparent correlaNo. 6 of CAINet al. 1974). It is difficult to interpret tion the level of neuronin S-6 was lower in cases of this anomaly for no description was given of the clini- senile dementia dying of bronchopneumonia than in cal, histological or terminal state of the specimens ‘controls’ dying of the same cause (Table 5 ) . One examined. Furthermore, the conditions that were used explanation for this is that the effects of bronchoto prepare the protein extract were not optimal for pneumonia are accentuated by reduced cerebral blood flow (INGVAR & GUSTAFSON, 1970) and a defecneuronin S-6 (SMITH,1976). The concentration of neuronin S-1 and 2 (identical tive respiratory reflex (ALLISON,1962) in the terminal to glial S-100 protein, BOWENet a/.,in press) in prefron- stages of the disease. The concentration of neuronin tal cortex was usually elevated in the cases of demen- S-6 was also markedly reduced in the thalamus, tia. This is probably due to the gliosis which was putamen, caudate nucleus, globus pallidus and suboften quite marked in the specimens examined. Neur- stantia nigra which are regions that usually exhibit onin S-3 and 4 has not yet been characterized, for little evidence of light microscopic changes in this dis-
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CAROLYN B. SMITHand D. M.
ease (CORSELLIS J. A. N., personal communication).
&WEN
BOWEND. M.. SMITH C. B.. WHITEP. & DAVISON A. N. Tl7r Nrurohiolo;7~. of Ayeiiiy. Raven Press, New Despite this, there is biochemical and clinical eviYork. In press. dence that neurotransmitter metabolism may be disCAIN D. F., BALLE. D. & DEKABAN A. S. (1974) J . Neuroturbed in basal ganglia in senile dementia. and Alzchenl. 23, 561-568. heimer's disease (GOTTFRIES er d., 1969. 1974; PEARCE, CAPLAN R., CHEUNG S. C.-Y & OMENN G. S. (1974) d . 1974; DRACHMAN & STAHL.1975). Neurochem. 22, 5 17-520. CORSELLIS J. A. N. (1962) Mental Illness and the Ageing Acknowledgements-We wish to express our gratitude to Braiii. Oxford Uni\ersity Press. New York. for his encouragment and advice DRACHMAS Professor A. N. DAVISON D. A. & STAHLS. (1975) Lancet (I), 809. gener- INGVAR throughout this investigation. Dr. J. A. N. CORSELLIS D. H. (1976) Brain Res. 107, 181-197. ously provided most of the pathological specimens and INGAR D. H. & GUSTAFSON L. (1970) Acta neuw/. scand. assessed almost all of the brains histologically. We also 46, Suppl. 43, 42-73. thank the numerous pathologists who supplied the control GOTTFRIES C. G.. GOTTFRIES I. & ROOS B. E. (1969) Br. material. The work was suppported by the MRC. the MirJ . Ph!.chiut. 115, 563-574. iam Marks Charitable Trust and the Wellcome Trust. GOTTFRIES C. G.. KJALLQUIST A,. PONTINU., Roos B. E. 61 SL'VDARG G. (1974) Br. .I. Psychiot. 124, 28&287. GROSSFELD R. M. & SHOOTER E. M. (1971) J. Neurochem. REFERENCES 18, 2265-2277. ALLISON R. S. (1962) The Senile Bruin. Edward Arnold, LOWRY 0. H.. ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. b i d . Chem. 193, 269-275. London. BOWEND. M., SMITH C. B. & DAVISOX A. N. (1973) Brain MAURERH. H. (1971) Disc Electrophoresis and Related 96, 849-856. Techniques of Poljacrylamitfe Gel Electrophoresis. Walter BOWEN D. M., FLACKR. H. A,. MARTINR. 0.. SMITH de Gruyter, Berlin. C. B., WHITEP. & DAVISON A. N. (1974) J . Neurochem. MCGEERP. L. & MCGEERE. G. (1976) J . Neurochem. 22, 1099-1 107. 26,65-76. BOWEN D. M.. SMITHC. B.. WHITEP. & DAVISOS A. N. PEARCE J. ( 1974) Eirr. Nrtiro/. 12. 94103. Bruin. In preparation. SMITHC. B. (1976) PI1.D. Thesis. University of London.
