SOLUBLE PROTEINS IN NORMAL and DISEASED HUMAN BRAIN

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 age- dependant. 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

IN A PREVIOUS study of experimental neurological disorders in rodents (BOWEN et d., 1974), changes in lysosomal activity appeared to 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 (BOWEN et al., 1973). In the present study of a large number of diseased and control human brains we de- scribe 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 con- dition of the patient and post-mortem storage effects.

MATERIALS AND METHODS

Specimens. After death, corpses were maintained at 4°C until autopsy. The time between death and whole body refrigeration was routinely within the range 0.75-2.5 h. The brain was excised within 24 h of death and halved sagit- tally; one half was preserved for histopathology (Dr.

Present address: Laboratory of Biochemical Genetics, National Institutes of Health, Bethesda, MD 20014, U.S.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 extrac- tion. 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 hyd- rate. 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 sub- divided on the basis of morphological criteria (e.g. inci- dence 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 evidence of a lingering mode of death, coma or ‘cerebral hypoxia’. For the classification of specimens from patients with neurological diseases morphological criteria were decisive. The traditional classification (CORSELLIS, 1962) of mental illnesses into ‘organic’ and ‘functional’ has been retained. Accordingly, the brains were assigned to an appropriate group (Table 2; most of the brains in Table 1 could be placed within these groups: exceptions are in the text).

1521

1522 CAROLYN B . SMITH and D. M. BOWEN

TABLE 1. CONCENTRATION OF TOTAL SOLUBLE. AND TWO SOLUBLE ACIDIC PROTEINS IN PRE-FRONTAL CORTEX

Solublc protcins

Period Acidic before

Diagnostic No .Age storage Total Neuronin S - h Neuronin I and 2 group* Disease casest ly1 (h) (", total protein) I",, sol. protein1 (". sol. protein)

~-

I Normal adult X 60 2 6 19 f 10 28.8 f 3.3 (41 3.16 + 1.31 1.30 0.38 Alzheimer's disease 4 6 7 k 3 X + I 1 19.2 * 4.9 0.33 k 0.654 2.76 k 0.17 Significance (PI NS NS < 0.02 < 0.01 < 0.001

-:

2 Normal elderl) 5 7 9 + 5 1 8 * X 24.8 2 0.9 (41 2.63 f 0.62 1.59 It 0.93 14 Semle dementia x 7 x i 7 9 + 7 19.3 k 44 ( 5 ) 0.76 k l .4l$ 2.05 k 1.03

Significance (PI NS N S < 0.05 < 0.02 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 used to analyse the protein composition of buffered-hypotonic extracts (BOWEN er a/.. 1973) from the 6 brain regions examined. In order to increase the resolution. porosity- gradient gels were routinely used (GROSSFELD & SHOOTER. 1971). This technique revealed a number of well-separated protein bands (Fig. 1, gel on the left) including 6 bands (neuronins 1 4 ) that migrated rapidly to the anode ahead of albumin. We have proposed (BOWFN rr ul.. in press)

the generic name neirroniii foi- this group of soluble acidic proteins. for the prealbumin portion of gels of extraneural tissues do not contain such conspicuous amounts of these proteins. The results obtained with the porosity-gradient gels are compared in Fig. 1 with those obtained with 7.5",, (wjv) acrylamide gels.

The neuronin-type proteins were quantified on the gels by direct densitometry. The protein stain, Procion Brilliant Blue R.S.. was chosen as the dye because of its reproducibi-

TABLE 2. F'RE-ASSAY HISTORY OF BRAIN SPECIMENS ANALYZED FOR PROTEIN CONTENT IN SIX BRAIN REGIONS

Period between

death Cause Diagnostic No. Age & storage Sex of

group Disease cdses' I! I or assay IM : F) deatht Source: Brain morphology Comments

I

3 4 5 6

7

8 9

10 11 12 13

14 15

Non-neurological

Normal adult Normal rldrrl) Functional disabilit! P l a q u e Plaques & depression Neuronal loss

GllOSlS

Cerebral atroph! Schizophrenia Lingering death Coma 'Ccrebral hypoxia' CO poisoning

Neurological Senile dementia Mixed senile and

vascular dementia

h

9 1

hi I ' x3 2 10 70. 85

i s * 3 77 >> ..

X ?

72 60

4x.m 76 67 ?J

X6 5 h 7x 5 x

20 + x 23 i 2

I ? . 20 23 * 5

7 24

5

I S 13

20. 9 24

7 2 1

21 + 7 1 6 2 3

7 . 1 ? : 5 2 .O I : ? 0 : I 0 - 1

I :0

0 : l 0 . 1 O : ? 0 : I I :o I ,0

3 : 6 0 : 3

6012 0210

710 430 002 300 001 010

010

010 001 020 100 100 100

009 003

normal normal normal

plaques appreciable neuronal

loss scvere and bizarre ghosts

deep sulci. thin cortex severe brain surgery normal normal markcd oedemas severe anoxic changes

plaques

age range' 50-69 y age range: over 70 y had depressive illness no functional illness short depressive illness (ECT) alcoholic (liver flap)

atypical myeloproliferative

no terminal ileum hospitalized 37 y cancer (breast. colon)

lung surgery!, suicide case

disorder

~

Diagnostic division 0.2 (CORSFLLIS, 1962) Diagnostic division 0.3 (Ci)nSFLLiS, 1962)

* Different cases from 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

/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).

state.

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 1523

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 inte- grator. 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, BOWEN et al., in press). The other protein (neuronin S-6) migrates just ahead of human serum albumin. but has not as yet been identified (BOWEN et 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, BOWEN et al., in press) and two unidentified bands neuronin S-3 and 4.

TABLE 3. EFFECT OF TIME BETWEEN DEATH AND FREEZING ON CONCENTRATION OF NEURONIN S-5 AND S-6 IN PRE-

FRONTAL CORTEX

Time death to - 2 o c

( h ) Diagnosis

Control (30)’ 0.25 Control (601.

Control (62)’ 7.5 Control (68)

Senile dementia (79) 15.0 Control (67)

Senile dementia (80) 18.5 Control 176)



Senile dementia (88)

Neuronin S-6 (ME)

215 310 I82 245

0 155 45

230 0

307 50

110 0

Neuronin S-5 lPgig)

45 140

15 I20 70

105 54 89 67

190 65

175

89

~~

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 in- tervals 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 post- operative 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, BOWEN et 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 post- mortem 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 de- crease 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

I524 CAROLYN B. SMITH and D. M. BOWEN

TABLE 4. 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

Neuronin S-6 Neuronin S - 5 I,'p f uct a t 1 ills f net -11

Senile Ssnile Region dementia Control dementia Control

Pre-frontal CortCX

Caudate nuclew

Putamen

Globus pallidus

Substantia nigra

Thalamus

79 * 42 151

YJ r 44

79 +_ 27 171

19 f 15 I51

13 c 16 171

81 t 1 O 161

171

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). Prelimi- nary 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 objec- tive comparison made from observations by the same pathologist assessing all the cases showed the pre-

TABLE 5. NEURONIN S-5 AND S-6 I N CASES OF SENILE DEMEN- TIA AND 'C.OSTRO1.S' DYING OF BRONCHOPNEUMONIA

Ncurontn S-6 Neuronin S-5 l,iE g \\el W l i ( p p i g wet wtl

Senile Senile Regions derneiitie 'Conlrol' dementia 'Control'

Pre-frontal cnrtc\ 29 ? 31- 163 T 6Yt 114 f 60 83 f 56 171 (6) 171 (6)

Caudate nucleui 5 i 14 60 i 97: I28 ?c 34 79 i 54 171 171 (7) (71

~

The cases of senile dementia and 'control' specimens (2 cases of motor neurone disease, 1 case each of Hunting- ton'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

t P < 0.07. $ P < 0.01

(died of heart disease).

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


140

I20

2.

0 - L

n w 100 - 0

E b 80

.\"

C

a- 60 I v)

C C .- $ 40

2

20

0

Senile dementia,mean: ------,range 0 Normal elderly ,mean'-

3 13

3

6

1. 3

Ecortex

5

5

I5

Puturnen G.pallldus

I

5

3 I

5

4[

15'

6-13 S. nigra 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. Abso- lute 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.

mean concentration group 2 x loo.

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 Be, BOWEN et d., in preparation). These findings sug- 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.

Functional psjchiatric disorders.

Samples of prefrontal cortex were analysed from 10 elderly inmates of psychiatric hospitals with clinical 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 endo- carditis of the mitral valves). The concentrations in

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.

Organic dementia

Since the effect of the terminal illness may be con- founded 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 ad- ditional 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

1526 CAROLYN B. SMITH and D. M. BOWEN

330

312

240

%

Q

- L

; 200

E

s

- 0

160 r

Q 120 v) c c ._ e 2 80 z

40

C

6 Senile dementia.mean: -----,range 13

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 age- matched 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 (GROSSFELD & SHOOTER, 1971). Application of this technique to human brain demonstrates (Fig. 1) that grey matter contains conspicuous amounts of well- separated 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


should give useful data. However, since biopsy specimens from demented patients were not available, we have not been able to rule out the possibility that post-mortem changes are exacerbated in the diseased brains.

In order to establish the normal ranges in concentration of the neuronin-type proteins hypotonic extracts of prefrontal cortex, substantia nigra, thalamus and 3 parts of the basal ganglia have been routinely analysed. The cases that were investigated included 38 potential control patients with a clinical diagnosis that was other than a neurological or functional psychiatric illness; the ages of the patients, who had died either at home or in general hospitals, were between 48 and 100 yrs. The specimens were further evaluated in order to allow for factors that might affect brain proteins (i.e. terminal state and abnormal brain morphology). Six brains were eliminated as normal controls for they exhibited abnormal morphology (a relatiwly high senile plaque count, cerebral atrophy, focal neuronal loss or an unusual type of gliosis see 4 and 6 8 , Table 2 ) ; 4 specimens were excluded for there was evidence of either coma, ‘cerebral hypoxia’ or a lingering mode of death (10-12, Table 2). In every region examined 26 of the remaining 28 specimens contained all of the neuronin-type proteins (Fig. 1). Apart from the possible exception of neuronin S-1 and 2 in frontal cortex (which tends to increase in concentration with advancing age, BOWEN et al.. 1973) other findings (SMITH, 1976) indicate that in adulthood the pattern of the neuronin-type proteins is not age-dependent in any of the regions examined. The levels of the neuronin-type proteins were within the normal control range in the majority of the brains from patients in hospital with functional psychiatric disorders. The atypical specimens were depleted in neuronin S-6 and exhibited evidence of either morphological abnormalities, including cerebral vascular disease, or coma. Thus after elimination of these atypical patients normal adult grey matter obtained at post-mortem appears to have a consistent pattern of 6 major acidic protein bands (Fig. 1). These findings concur with those of CAIN et al. (1974) for they demonstrated that histologically normal cortical biopsies give a similar pattern. The results of CAPLAN et crl. (1974) indicate that the extracts from the post- mortem samples that they investigated did not contain neuronin s-6 (which has a similar mobility to band No. 6 of CAIN et al. 1974). It is difficult to interpret this anomaly for no description was given of the clinical, histological or terminal state of the specimens examined. Furthermore, the conditions that were used to prepare the protein extract were not optimal for neuronin S-6 (SMITH, 1976).

The concentration of neuronin S-1 and 2 (identical to glial S-100 protein, BOWEN et a/., in press) in prefrontal cortex was usually elevated in the cases of dementia. This is probably due to the gliosis which was often quite marked in the specimens examined. Neur- onin S-3 and 4 has not yet been characterized, for

in neurological diseases the two protein bands rarely appear to change in concentration. The apparent differences in the concentration of neuronin S-5 (Table 4; Fig. 3) are difficult to interpret because the protein appears to be particularly sensitive to variation in the interval between death and refrigeration of the corpse. For example, none of the normal elderly patients died in the psychiatric hospitals from which the cases of dementia were obtained. This suggests that the differences in levels of neuronin S-5 (Table 4) may be related to differences in post-mortem hand- ling for the precise mean interval between death and refrigeration of the corpse may have been greater in the normal elderly group ( i t . since some of these patients died at home). In contrast to neuronin S-5 the concentration of neuronin S-6 appears to be remarkably stable post-mortem. However, as judged by the results obtained with the non-demented patients that either died of bronchopneumonia, had evidence of coma, ‘cerebral hypoxia’ or had lingering modes of death the concentration of neuronin S-6 is affected by the agonal state. This and other evidence (BOWEN et nl., 1976a) suggests that neuronin S-6 is a relatively sensitive index of at least terminal ‘cerebral hypoxia’. We were unable to establish with any precision whether or not there were differences in the degree of terminal coma (MCGEER & MCGEER, 1976) between groups of patients. However, since patients in terminal coma probably have ‘cerebral hypoxia’ (i.e. secondary to reduced cerebral blood flow, INGVAR, 1976) our findings and those of MCGEER & MCGEER (1976) demonstrate the importance of controlling for the terminal state.

We have previously reported (BOWEN et al., 1973) that neuronin S-6 is depleted in the cortical grey matter from a small group of patients with organic dementias. In the current study we have analysed cortex from 30 cases of dementia (aged 60-97 yrs). The concentration of neuronin S-6 was markedly depleted (usually undetectable) in 26 cases. The atypical specimens (i.e. those with normal control levels of neuronin S-6) were 1 case each of Alzheimer’s disease (6 cases examined), and mixed senile and vascular dementia (7 cases examined), and 2 cases of senile dementia (17 cases examined). Three of the 4 atypical cases died of either renal or ventricular failure while almost all of the cases depleted in neuronin S-6 died of bronchopneumonia. Despite this apparent correla- tion the level of neuronin S-6 was lower in cases of senile dementia dying of bronchopneumonia than in ‘controls’ dying of the same cause (Table 5 ) . One explanation for this is that the effects of bronchopneumonia are accentuated by reduced cerebral blood flow (INGVAR & GUSTAFSON, 1970) and a defec- tive respiratory reflex (ALLISON, 1962) in the terminal stages of the disease. The concentration of neuronin S-6 was also markedly reduced in the thalamus, putamen, caudate nucleus, globus pallidus and substantia nigra which are regions that usually exhibit little evidence of light microscopic changes in this dis-

1528 CAROLYN B. SMITH and D. M. &WEN

ease (CORSELLIS J. A. N., personal communication). Despite this, there is biochemical and clinical evidence that neurotransmitter metabolism may be dis- turbed in basal ganglia in senile dementia. and Alz- heimer's disease (GOTTFRIES er d., 1969. 1974; PEARCE, 1974; DRACHMAN & STAHL. 1975).

Acknowledgements-We wish to express our gratitude to Professor A. N. DAVISON for his encouragment and advice throughout this investigation. Dr. J. A. N. CORSELLIS gener- ously provided most of the pathological specimens and assessed almost all of the brains histologically. We also thank the numerous pathologists who supplied the control material. The work was suppported by the MRC. the Mir- iam Marks Charitable Trust and the Wellcome Trust.

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SOLUBLE PROTEINS IN NORMAL and DISEASED HUMAN BRAIN