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Loss of synaptophysin-like immunoreactivity in the hippocampal formation is an early phenomenon in alzheimer's disease
Neuroseienee VoL 64, No. 2, pp. 375 384, 1995
~
Pergamon
0306-4522(94)00422-6
Elsevier ScienceLtd Copyright 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.1/0
LOSS OF S Y N A P T O P H Y S I N - L I K E I M M U N O R E A C T I V I T Y IN THE H I P P O C A M P A L F O R M A T I O N IS A N E A R L Y P H E N O M E N O N IN A L Z H E I M E R ' S DISEASE O. H E I N O N E N , * H. S O I N I N E N , * H. S O R V A R I , * O. KOSUNEN,~" L. PALJ,~,RVI,'~ E. K O I V I S T O * and P. J. R I E K K I N E N Sr*++§ *Departments of Neurology and tPathology and ++A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, SF-70211 Kuopio, Finland
Abstract--We
studied a synaptophysin-like immunoreactivity in the hippocampal formation of patients with definite Alzheimer's disease, multi-infarct dementia, patients with no evidence of clinical dementia with neuropathological findings fulfilling the criteria of possible Alzheimer's disease, and age-matched nondemented controls. Possible Alzheimer's disease cases were of special interest because they were considered to represent early Alzheimer's disease. We also studied the spatial relationship of synaptophysin-like immunopositivity with amyloid-/~-protein immunopositive senile plaques and anti-paired helical filament immunopositive degenerating neurons locally as well as considering the intrinsic circuits in the hippocampal formation. The synaptophysin-like immunoreactivity was decreased in the hippocampus and the entorhinal cortex in patients with definite and possible Alzheimer's disease but not in multi-infarct dementia patients compared to controls. Equal loss of synapses in possible and definite Alzheimer's disease patients supports the hypothesis that synaptic loss is an early phenomenon in Alzheimefs disease. Unchanged synaptophysin-like immunopositivity in patients with multi-infarct dementia suggests that the loss of synapses is centrally involved in the pathogenesis of Alzheimer's disease and not dementia per se. There was no spatial correlation between loss of synapses and amyloid-,g-protein positive senile plaques, Moreover, we could not find a strict spatial relationship between senile plaques and degenerating neurons. Our results do not support the amyloid cascade hypothesis of Alzheimer's disease that local accumulation of amyloid-/~-protein leads to the loss of synapses.
Neuronal death in patients with Alzheimer's disease (AD) has been suggested to be closely associated with the extensive synapse loss in the neocortex. Although two earlier studies described no change in synaptic number in A D cortex, 1°'25 more recent studies indicate that loss of synapses occur in the neocortex and the hippocampus of A D brain. 5"6"14'2°'21"2s The synapse loss in A D has been proposed to be a relatively early phenomenon, possibly preceding the loss of neurons. Thus, synapse damage would be a primary phenomenon rather than a secondary reflection of degeneration occurring in the perikaryon. :~ Moreover, the relationship of synaptic pathology to amyloid-fl-protein (3/A4) accumulation and cytoskeletal pathology in A D is of particular interest. Several studies suggest that the neocortical synapse loss, rather than the number of senile plaques or neurofibrillary tangles, correlates with the cognitive impairment in AD. This supports the view that the loss of synapses rather than ~/A4 deposition should be considered as a fundamental lesion in AD. 6'2~'~3
§To whom correspondence should be addressed. Abbret~iations: AD, Alzheimer's disease; []/A4, amyloid-/~'protein; DAB, 3,3'-diaminobenzidine; MID, multi-infarct dementia: MMS, Mini-Mental State; OD, optical density: PBS, phosphate-buffered saline. 375
Synaptophysin (p38) is a 38,000 mol. wt calcium binding glycoprotein :7 specifically located in the membrane of presynaptic vesicles in most and probably all neocortical axonal endings. ~7 Quantification of synaptophysin-like immunoreactivity is a valuable method for studying presynaptic terminals in normal and pathologic conditions. 2° Previous studies employing this method showed a significant synaptic loss in the neocortex and in the molecular layer of the hippocampus in patients with A D . 21"22'12 The decrement in synaptic connections in critical brain areas most probably cause impairment in memory, cognitive and behavioural functions. 12 We report here quantitative immunohistochemical data on synaptophysin-like immunoreactivity in the hippocampus and the entorhinal cortex of patients with definite AD, patients with multi-infarct dementia (MID), patients with no evidence of dementia but whose neuropathological findings fulfilled the criteria of possible AD, and age-matched nondemented controls. Especially, subjects without clinical evidence of dementia, but who had a sufficient amount of senile plaques for the diagnosis of possible AD, are of interest in studying the synapse pathology, since they probably represent the very early stage of AD. In addition, we studied the spatial relationship of synaptophysin-like immunoreactivity with senile
376
O. Heinonen et al. Table 1. Clinical characteristics of patients and controls Case no. 1 2 3 4 5 6 7 8 9 l0 11 12 13 14 15 16 17
Age/Sex
Duration of the disease (years)
88 M 60 F 65 M 77 F 75 F 86 M 90 F 77 F 72 M 91 F 77 F 80 F 84 F 87 F 81 M 84 F 75 F
---1 5 7 ---18 4 18 9 3 6 12 9
MMS
-12.00 6.00 6.00 ---0.00 8.00 0.00 0.00 9.00 1.00 0.00 0.00
Neuropathologic diagnosis Control Control Control MID MID MID Possible A D Possible A D Possible A D AD AD AD AD AD AD AD AD
Cause o f death Pneumonia Myocardial infarction Aortic valve stenosis Pneumonia Pneumonia Pneumonia Pulmonar embolism Myocardial infarction Myocardial infarction Pneumonia Pneumonia Pneumonia Pneumonia Chronic pyelonephritis Pneumonia Pneumonia Pneumonia
F, female; M, male; - - , not determined. p l a q u e s a n d d e g e n e r a t i n g n e u r o n s . Senile p l a q u e s were d e t e c t e d b y e m p l o y i n g /~/A4 i m m u n o h i s t o c h e m i s t r y . C y t o s k e l e t a l a l t e r a t i o n s were d e m o n s t r a t e d b y u s i n g a n t i - p a i r e d helical f i l a m e n t a n t i b o d y C D - 1 u w h i c h is a v a r i a n t o f t h e A l z - 5 0 a n t i b o d y 36 reacting against AD associated proteins including A68.
EXPERIMENTAL PROCEDURES
Patients and controls We used brain tissue from 17 subjects whose clinical characteristics are shown in Table 1. The mean post-mortem delay ( + S.D.) was 4.5 _ 1.9 h and it did not differ across the study groups. The histopathological diagnosis of A D was based on the C E R A D guidelines and criteria described by Mirra and coworkers, z3 We used modified Bielschowsky's silver impregnation staining to detect plaques and tangles. The Congo Red staining was also used for rating cerebral amyloid, particularly in the cerebral blood vessel walls. Eight patients had definite AD. Their mean age was 82.4 _ 5.2 years, duration of the disease was 9.9 _+ 5.8 years, and the mean score of Mini-Mental Status examination (MMS) 8 was 2.3 _+ 3.9. Three patients had had recurrent strokes with dementia and they fulfilled the D S M lII criteria of MID. z The patients with M I D did not show neocortical plaques in Bielschowsky's silver staining, and thus they did not fulfil the neuropathologic criteria o f AD. Their mean age was 79.3 + 5.9 years, mean duration of dementia was 4.3 + 3.1 years, and the mean M M S score was 8.0__+ 3.6. The patients with definitive A D and M I D went through lifetime examinations including neuropsychological testing, electroencephalogram, and cerebrospinal fluid analysis. The remaining six cases derived from an autopsy series of Kuopio University Hospital. None of them had clinical evidence of dementia. However, three of these cases (mean age 79.7 ___9.3 years) showed plaques and tangles by
Bielschowsky's silver staining allowing a diagnosis of possible A D according to the C E R A D criteria. The other three subjects (mean age 71.0 + 14.9 years) without clinical dementia and without plaques and tangles in Bielschowsky's staining served as controls. Immunohistochemistry We fixed samples from the hippocampal formation (including the dentate gyrus, the hippocampus proper, the subicular complex, and the entorhinal cortex) overnight in 4% buffered formalin, sectioned at 5 # m and collected onto 3-aminopropyltriethoxysilane (Sigma, St Louis, MO) pretreated slides. After the deparaffinization, we incubated sections for /~/A4 immunohistochemistry in 90% formic acid for 10min to intensify the staining. We blocked the endogenous peroxidase activity by using 5% hydrogen peroxide in distilled water for 5 min. Before the primary serum, we incubated the sections in normal horse serum (1:67, Vector, Burlingame, CA) for 1 h to avoid nonspecific staining. We stained the first set of sections using a monoclonal anti-synaptophysin antibody (SY 38, Dako, l/~g/ml), the second set of sections with a monoclonal mouse antibody to h u m a n fl-amyloid (anti-fl/A4) (6F/30, Dako, A/S Denmark, 0.13/zg/ml), and the third set of sections employing a monoclonal anti-paired helical filament (CD-I) antibody (Abbott Laboratories, IL, 1 ~g/ml), at + 4 ° C overnight. After the primary serum, we incubated the sections in biotinylated anti-mouse serum (made in horse, 1:200, Vector) for 30 min followed by the incubation in avidin-biotin-peroxidase complex (Vectastain ABC-standard kit, Vector) for 40 min. lmmunoperoxidase reaction was developed using 0.05% 3,3'-diaminobenzidine (DAB, Sigma) and 0.03% hydrogen peroxide for 5 min. Between different steps we thoroughly washed the sections in phosphate-buffered saline (PBS), p H 7.4. Finally, we counterstained the sections with anti-fl/A4 and anti-CD-I in Meyer's haematoxylin-eosin, dehydrated, mounted with DePex (BDH, Broom Rd, Poole, U.K.) and covered by coverslips. The immunolabelling protocol for antisynaptophysin was repeated twice in adjacent sections to
Fig. 1. Synaptophysin-like immunoreactivity in the entorhinal cortex of a control case. Immunopositivity is seen as a granular staining pattern in the neuropil. Neuronal cytoplasma remain unstained or show a low granular immunostaining. The grain size and the intensity of the staining of different granules vary. Layer II of the entorhinal cortex shows a denser staining compared to layer I and layer III. Asterix shows the site of magnification. I, II and III represent entorhinal layers. Scale bars = 150 # m (A) and 45 # m (B).
Fig. 2. (A, B) Synaptophysin-like immunoreactivity in the hippocampus of a control case. The outer molecular layer shows a slighly denser staining compared to the inner molecular layer. (C, D) A patient with AD shows a visible line of demarcation in the molecular layer separating a zone of light staining in the outer half from one of denser staining in the inner half. Around plaques there are large synaptophysin-like immunopositive grains representing abnormal dilated terminals (open arrows). Asterix shows the site of magnification. H, hilus; GL, granular cell layer; IML, inner molecular layer; OML, outer molecular layer. Scale bars = 150#m (A, C) and 45/~m (B, D). 378
Synapse loss a n d / L a m y l o i d o s i s in Alzheimer's disease
379
Table 2. Synaptophysin immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients and controls
Dentate gyrus Outer molecular layer Inner molecular layer Hilus CA3 CA I Subiculum Entorhinal cortex layer II Entorhinal cortex layer Ill Entorhinal cortex layer IV V
Control n=3
MID n =3
Possible A D n=3
AD n =8
Kruskall Waltis P
0.71 _+ 0.19 1.02 ± 0.25 1.07 ± 0.34 1.57 ± 0.25 1.57 £ 0.37 1.28 _+ 0•08 1.40 ± 0.17 1.40 ± 0.08 1.16 ± 0.23
1.2 ± 0.76 1.45 -+ 0.42 .19 _+ 0•40 .56 _ 0.71 .58 _+ 0.85 .28 _+ 1.02 .25 _+ 0.42 .26 _+ 0.29 0.98 _+ 0.43
0.57_+0.23 0.93 ± 0.35 0.69_+0.11 1.07 ± 0.08 1.19 ± 0.42 0.84+0.17 0.80_+0.12 0.79 ± 0.09 0.66+0.10
0.55_+ 0.17 1.03 + 0.31 0.78_+0.31 0.99 ± 0.47 0.90 _+ 0.32 0.75_+0.27 0.90_+0.31 0.81 ± 0.34 0.75-+0.31
0.4186 0•4081 0.2179 0.1904 0•2304 0.1795 0.0786 0.0330 0.2463
MannWhitney U-test
0,2 0,3:0,2 0,3;0,2 0,3;0,2 0,2
Data are background corrected absolute O D s of the neuropil. Results are expressed as mean + S.D. The differences across the study groups were tested using Kruskall-Wallis one-way analysis of variance• The numbers in the last column indicate which two groups differ significantly in the M a n n Whitney U-test. 0, control; 1, multi-infarct dementia: 2. possible Alzheimer's disease; 3, Alzheimer's disease. assure reproducibility of results. For control staining of each case we omitted the primary serum, otherwise the procedure was similar.
Quant(fication o[' optical density We measured optical densities (OD) of synaptophysinlike immunoreactivity by using a Quantimet 570 Image Analysis System (Leica Cambridge Ltd, Cambridge, England). The image was acquired using a Sony 3CCD video camera DXC-750P. The camera was mounted in an Olympus Vanox-T Research Microscope with a x 2 0 SPlan objective, x'2.5 intermediate lens, and x I0 oculars (Olympus Optical Co., Ltd, Tokyo, Japan)• We calibrated the system for O D measurements as follows: the section was aligned in the field of view and a level of light intensity for video camera performance was adjusted under computer control; an electronic shading correction was applied to the image to compensate for possible unevenness present in the illumination; a calibration curve was established with three filters with known ODs (Kodak Wratten Gelatin Filter, 75 m m x 75 mm, Eastman Kodak Company, Rochester, N.Y.). The O D was obtained by manually positioning the area of interest of the immunostained section in the camera field of view. The neuronal perikarya, blood vessels, and senile plaques were excluded• Ten fields of each area of interest were measured and the O D measurements were averaged. The O D of the white matter on the same section served as the value of the background. The absolute OD values were calculated according to the calibration curve. The background corrected absolute OD of the neuropil was obtained
Table 3. Synaptophysin-like immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients compared to controls. The results are presented as percentage of control values
Dentate gyrus Outer molecular layer Inner molecular layer Hilus CA3 CAI Subiculum Entorhinal cortex Layer II Layer III Layer IV V
MID n =3
Possible A D n =3
AD n =8
169% 142% 111% 99% 100% 100%
80% 91% 64% 68% 76% 66%
77% 100% 73% 63% 57% 59%
89% 90% 84%
57% 56% 57%
64% 58% 65%
by subtracting the OD of the background from the OD of the neuropil.
Quantification of" amyloM-~-protein positit,e plaques and CD-I positive neurons The density of diffuse and neuritic senile plaques were counted separately per square millimeter (mm 2) in the hilus, in the CA3 and the CA1 areas, in the presubiculum and in layers I, II, Ill, IV V, and VI of the entorhinal cortex. The number of CD-I positive neurons per m m 2 were counted (including both tangle bearing and granularly stained neurons) in the same areas. The total number of neurons per m m 2 in the CA3 and the CAI areas were measured and the density of plaques and CD-1 positive neurons in the outer and the inner molecular layers of the dentate gyrus were described using the terms "~none", "moderate" and " m a n y " . The measurements were executed using Nikon Optiphot-2 microscope, x 10 or x 20 Plan objectives, x 10 oculars, and an ocular craticule (Nikon Corporation, Tokyo, Japan).
Statistical analysis Data were analysed using SPSS/PC V.3.2 software. Kruskal Wallis one-way analysis of variance was used, followed by M a n n Whitney U-test to detect differences between the study groups. Correlations were calculated by Pearson's correlation test. The level of significance was P < 0.05. RESULTS
Synaptophysin-like immunoreactivity S y n a p t o p h y s i n - l i k e i m m u n o r e a c t i v i t y w a s seen as g r a n u l a r s t a i n i n g p a t t e r n in t h e n e u r o p i l (Fig. IA, B). T h e g r a i n size a n d t h e i n t e n s i t y o f t h e s t a i n i n g o f different g r a n u l e s varied. A r o u n d senile p l a q u e s t h e r e were l a r g e s y n a p t o p h y s i n - l i k e i m m u n o p o s i t i v e g r a i n s r e p r e s e n t i n g a b n o r m a l dilated t e r m i n a l s (Fig. 2C, D). N e u r o n a l c y t o p l a s m a were n o t s t a i n e d or s h o w e d a low g r a n u l a r i m m u n o s t a i n i n g p r o b a b l y r e p r e s e n t i n g s y n a p t o p h y s i n s y n t h e s i s in t h e cell o r recycling o f t h e s y n a p t o p h y s i n f r o m t h e n e r v e t e r m i•n a l s . -~ 4 R e g i o n s o f t h e w h i t e m a t t e r were v i r t u a l l y u n s t a i n e d • Tables 2 and 3 summarize the data from s y n a p t o p h y s i n - l i k e i m m u n o r e a c t i v i t y in t h e h i p p o c a m p u s a n d t h e e n t o r h i n a l cortex. In c o n t r o l s , s y n a p t o p h y s i n - l i k e i m m u n o s t a i n i n g in t h e i n n e r a n d the outer molecular layer of the dentate gyrus showed n o d i s t i n c t difference (Fig. 2A, B). T h e p a t i e n t s with
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Table 4. CD-1 and/~/A4 immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients and controls
Hilus CDI + neurons DSP NSP CA3 Neurons CD1 + neurons DSP NSP CA1 Neurons CD + neurons DSP NSP Subiculum CD + neurons DSP NSP EC layer II CD + neurons DSP NSP EC layer III CD + neurons DSP NSP EC layer IV V CD + neurons DSP NSP
Control n= 3
MID n= 3
Possible AD n= 3
AD n= 8
Kruskall Wallis P
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.7 + 1.2 0.5 ± 0.7 0.0 ± 0.0
2.7 + 2.3 0.0 ± 0.0 0.0 ± 0.0
34.0±20.0 13.0±15.7 2.9±4.6
<0.01 <0.05 N.S.
MannWhitney U-test 0,3;2,3;3,1 0,3;2,3
290.0 ± 0.0 ± 0.0 ± 0.0 +
90.5 0.0 0.0 0.0
173.5 ± 0.0 ± 0.0 ± 0.0 +
57.3 0.0 0.0 0.0
299.0 ± 2.3 ± 0.0 ± 0.3 ±
33.8 4.0 0.0 0.6
249.0 __+54.7 51.0 ± 42.5 9.7 ± 7.9 1.3 ± 2.2
N.S. <0.05 N.S. N.S.
235.7 ± 12.7 + 0.0 + 0.0 ±
53.0 21.9 0.0 0.0
299.3 + 22.0 ± 23.5 ± 0.0 ±
55.4 30.6 33.2 0.0
299.3 ± 53.7 + 11.0+ 0.7_+
5.5 86.1 19.1 1.1
259.7 ± 48.9 128.2 _+ 49.6 10.5 + 8.6 4.7 ± 7.7
N.S. <0.05 N.S. N.S.
0,3; 3.1 0,3 0,3; 2,3
0,3;3,1
0.0 + 0.0 3.7 __+6.4 0.0 + 0.0
8.0+ 11.3 3.5 ± 4.9 0.5 ± 0.7
13.3 ± 11.9 8.7 ± 11.5 3.0 + 1.7
161.4_+ 117.0
<0.05
15.6 ± 28.1
N.S.
5.8 ± 9.2
N.S.
0.0 + 0.0 0.3 ± 0.6 0.0 ± 0.0
0.0 __+0.0 14.5 __+20.5 0.0 ± 0.0
95.0 ± 82.7 4.3 ± 7.5 4.3 ± 7.5
245.0± 171.3 27.0__+ 14.2 8.0 __+9.0
<0.01 <0.05 N.S.
0,3; 3,1 0,3; 2,3
0.0 + 0.0 0.3+0.6 0.0 + 0.0
13.7 ± 22.0 10.0±14.1 2.0 ± 2.8
39.3 + 42.3 17.3±16.2 1.3 ___2.3
99.7 ± 74.0 53.6___42.5 3.0 ± 3.9
<0.05 <0.05 N.S.
0,3 0,3; 3,1
32.3 ± 30.3 25.7±22.9 1.3 ± 2.3
128.1 ± 51.4 17.3±11.2 0.4__+ 1.1
<0.01 N.S. N.S.
0,3; 2,3; 3,1 0,3
0.0 ± 0.0 0.3±0.6 0.0 ± 0.0
1.3 ± 1.5 9.0± 1 2 . 7 3.5 ± 4.9
0,2
Neurons, CDI + neurons, diffuse (DSP) and neuritic (NSP) fl/A4 immunopositive plaques were counted per 1 mm 2. Results are expressed as mean _ S.D. EC, entorhinal cortex; N.S., not significant. The differences across the study groups were tested using the Kruskall-Wallis one-way analysis of variance. The numbers in the last column indicate which two groups differ significantly in the Mann-Whitney U-test. 0, control; 1, multi-infarct dementia; 2, possible Alzheimer's disease; 3, Alzheimer's disease.
A D showed a visible line o f d e m a r c a t i o n in the molecular layer separating a zone of light staining in the outer h a l f from one o f denser staining in the inner h a l f (Fig. 2C, D). The possible a n d definite A D patients showed a 2 0 % decrease of control values in synaptophysin-like i m m u n o s t a i n i n g in the outer molecular layer, whereas the inner molecular layer was n o t affected. In the hilus, the possible a n d definite A D patients h a d a loss o f 3 6 % a n d 27% in synaptophysin-like immunoreactivity. In the CA3 area the synaptophysin-like i m m u n o r e a c t i v i t y was decreased 32% in patients with possible A D a n d 37% in patients with definite AD. In the CA1 area the loss in i m m u n o s t a i n i n g was 2 4 % for possible A D patients a n d 4 3 % for definite A D patients. The analysis of the subiculum revealed a 34% a n d a 4 1 % loss in synaptophysin-like i m m u n o r e a c t i v i t y in patients with possible a n d definite A D reaching a statistical significance c o m p a r e d to controls ( M a n n W h i t n e y U-test, P < 0.05). In the e n t o r h i n a l cortex the patients with possible a n d definite A D showed a significant loss o f g r a n u l a r staining in layers II, III a n d I V - V varying from a loss of 35% to 53% in possible A D patients a n d 33% to 4 2 % in definite A D patients in different laminae.
In M I D patients, the synaptophysin-like staining was c o m p a r a b l e with the staining in controls in the hilus, CA1, CA3, a n d subiculum. Synaptophysin-like staining was even slightly m o r e intense in the molecular layer of the dentate gyrus in M I D patients t h a n in controls. The e n t o r h i n a l cortex o f M I D patients showed a ~ 13% decrease in synaptophysinlike staining c o m p a r e d to controls. Synaptophysin-like i m m u n o p o s i t i v i t y was not related to age in the whole study g r o u p or in the A D group. However, the subjects in the study represented a relatively n a r r o w age range from 60 to 91 years.
Arnyloid-~-protein immunoreactivity Table 4 presents the n u m b e r of /~/A4 i m m u n o reactive diffuse and neuritic p l a q u e s / m m 2 in the h i p p o c a m p u s a n d the e n t o r h i n a l cortex of d e m e n t i a patients a n d controls. As expected, the patients with definite A D h a d the highest n u m b e r o f / ~ / A 4 positive senile plaques. In patients with definite A D , diffuse plaques were most frequent in layer III of the e n t o r h i n a l cortex, whereas most neuritic plaques were f o u n d in layer II of the e n t o r h i n a l cortex. Diffuse senile plaques o u t n u m b e r e d the neuritic forms in all areas studied. The patients with possible A D showed
Synapse loss and fl-amyloidosis in Alzheimer's disease the highest number of diffuse plaques in layers IV-V of the entorhinal cortex, and neuritic plaques were most frequently seen in layer II of the entorhinal cortex. The hilus and the CA3 area were almost free of plaques in this group. Patients with definite AD showed a moderate to high degree of senile plaques in the molecular layer of the dentate gyrus. Plaques were seen both in the inner and outer half of the molecular layer. Two patients with possible AD (nos 8 and 9) had few diffuse plaques in the molecular layer and one possible AD subject (no. 7) was free of senile plaques in this area. None of the controls had plaques in the molecular layer of the dentate gyrus. The MID patients had a considerable amount of ///A4 positive diffuse plaques in the CAI region. Furthermore, senile plaques were found in the entorhinal cortex. The hilus and the CA3 area showed only few or no plaques.
C D -1 irnmunopositivity The CD-1 antibody recognized neurofibrillary tangles, dystrophic neurites in and around senile plaques, as well as neuropil threads. Positive immunoreactivity was also found as granular staining in a proportion of neurons possibly representing an early cytological change that precedes the formation of tangles and plaques. Table 4 shows CD-1 immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients. Patients with definite AD showed the most frequent immunostaining in every area. Layer II of the entorhinal cortex had the highest number of immunopositive neurons. This was also the case in patients with possible AD. In MID patients the highest number of immunopositive neurons was in the CA1 area and a considerable number of stained neurons were seen also in layer lII of the entorhinal cortex. The analysis
381
of the molecular layer of the dentate gyrus of patients with definite AD showed only a few or moderate number of CD-1 immunopositive neurons. The immunopositive neurons distributed both to the inner and outer molecular layer. The possible AD cases or controls had no immunopositive neurons in this area.
Neuron count The total number of neurons in the CA1 and CA3 areas was calculated and it did not differ significantly between study groups.
Spatial relationship between synaptophysin-like immunoreactivity, amyloid-~3-protein and CD-1 immunoreactivity The spatial relationship of /~/A4, CD-I and synaptophysin-like immunoreactivities was studied locally in each area as well as considering the intrinsic circuits in the hippocampal formation. We did not find any strict local correlation between the degree of synaptophysin-like immunoreactivity and /3/A4 positive plaques (Fig. 3) or CD-I immunoreactive neurons. No local correlation between the number of neurons and the degree of synaptophysin-like immunostaining in the CAI and the CA3 regions was detected either. Instead, the number of CD-1immunoreactive neurons in layer II of the entorhinal cortex correlated almost significantly with the synaptophysin-like immunostaining of the outer molecular layer of the dentate gyrus in the whole study group (r = - 0 . 5 2 , n = 14, P =0.056). This correlation did not reach the statistical significance if only definite and possible AD cases were included in the analysis ( r = - 0 . 5 8 , n = 9 , P=0.101). The synaptophysin-like immunoreactivity of the outer molecular layer did not correlate with the number of diffuse or neuritic plaques of layer II of the entorhinal cortex.
60 1
50
L
m
~
I ~3E 40 ~ 0
I
-----J
A
00
m
O~
I. . . . .
0.8 0
l___l
0.8
30
I
~. 20
i
lO
I . . . . . . . . .
........
; ........
Hilus
o.2
~
o
~
.......
."
CA3
----
,°,
~0
- ........
CA1 Subiculum EC II
EC III
,
EC IV-V
Synaptophysin-like immunoreactivity Diffuse plaques
.....
Neuritic plaques
Fig. 3. A local relationship of synaptophysin-likeimmunoreactivity (absolute OD) and numbers of senile plaques/mm2in the hippocampus and the entorhinal cortex. There is no local correlation between synaptic density and plaques.
O. Heinonen et al.
382
In general, we found no distinct correlation between /~/A4 and CD-1 immunopositivities in a particular area. All definite AD patients showed a number of CD-l-immunopositive neurons in layer II of the entorhinal cortex, and all of them had a high amount of/3/A4 immunolabelling in the molecular layer of the dentate gyrus. One patient with possible AD (7) had numerous CD-1-positive neurons in layer If of the entorhinal cortex but no /~/A4 positive plaques in the molecular layer.
DISCUSSION
Synaptic loss is centrally involved in the pathogenesis of Alzheimer's disease We report here a loss in synaptophysin-like immunoreactivity in the hippocampus and the entorhinal cortex of patients with possible and definitive AD. Our findings are in agreement with previous data demonstrating synaptic loss in the neocortex and the hippocampus by synaptophysin immunohistochemistry as well as by electronmicroscopy. 6'14'21 In their recent study, Honer and coworkers ~4 reported a 77% reduction in synaptophysin-like immunoreactivity (EPI0 antibody) in the hippocampus and a 54% reduction in the temporal cortex in AD. Masliah and coworkers 21 demonstrated a 45% loss in synaptophysin immunoreactivity in the frontal and parietal cortex, and to a lesser extent in the hippocampus and entorhinal cortex. In the present study, the loss varied from 19% to 53% depending on the specific area, the most prominent loss of staining being in the entorhinal cortex. In contrast to our study, Scheffand coworkers 29 recently reported that there were no changes in synaptic density between control and AD subjects in the entorhinal cortex. Differences in methods, AD patient populations, and severity in cognitive decline of the patients may account for divergent results. It has been proposed that, when neuropathological alterations proceed beyond an individual threshold, the brain fails to compensate such changes. Thus, the loss of synaptophysin-like immunoreactivity in the present study may reflect the overdraft of the individual capability of a plasticity response to replace lost synaptic contacts. Alternatively, the alteration in the pattern of synaptophysin staining in the present study do not represent, at least as a whole, the actual loss of synapses but the insufficient amount of nerve terminal protein density and malfunction of synapses. In accordance with earlier reports, 5'12'14the present study demonstrates a striking decrease in immunoreactivity for synaptophysin in the outer molecular layer of the dentate gyrus in the brains of AD patients, compared with controls. Interestingly, patients with possible and definite AD did not differ from each other in the degree of synaptophysin-like immunoreactivity supporting the hypothesis that the
loss of presynaptic terminals is a relatively early phenomenon in AD. 21 In contrast to AD cases, we found no synaptic loss in the hippocampus of MID patients. Thus, our results suggest that synaptic loss is centrally involved in the pathogenesis of AD. The unaltered synaptophysin-like immunoreactivity in AD in the primary visual cortex, 4 which is a relatively spared area in AD, further supports this hypothesis.
Lack of positive spatial relationship between amyloid deposits and synaptic degeneration Recent studies indicate that the synaptic loss in AD might be largely independent of the [~/A4 deposition. 3"~'28"29Our study contributes to this by specifying plaque analysis by brain region and plaque subtypes. In the present study there was a lack of strict spatial correlation between the loss of synaptophysin-like staining and a high amount of []/A4 in a certain area (Fig. 3). Especially, diffuse plaques seem clearly to have no effect on synapse count. The lack of correlation between [:1/A4 deposition and synaptophysin-like immunoreactivity was clearer in the entorhinal cortex compared to the hippocampus (Fig. 3). One could not ignore the possibility of the regional susceptibility of the amyloid-induced injury. Such a scenario of regional susceptibility is postulated, e.g. for the cerebellum, where amyloid deposits occur without neuronal injury. However, our results, in agreement with previous results, do not unambiguously support the amyloid cascade hypothesis ~3 of AD that [~/A4 accumulation leads to the loss of synapses. Given the small number of cases, we should, however, be cautious in the interpretation of the correlation analysis. Furthermore, the absolute numbers of [4/A4 immunopositive plaques do not necessarily demonstrate the extent of fl/A4 load in a certain area. [4/A4 or fragments thereof were reported to be toxic to differentiated neurons ~'37 The neurodegenerative effect of [4/A4 in these studies suggests that neuronal death in AD is a direct consequence of [¢/A4. However, several other studies have found no direct toxicity of /~/A4. 9"26'3t If [I/A4 exerts a widespread toxic effect, there should be a positive spatial relationship between amyloid deposition and degeneration of synapses; the more [~/A4 the fewer synapses at a certain region. We did not see such a relationship. Thus, alternative models to the amyloid cascade hypothesis will be encouraged by these data. There may be other determinants of synaptic vulnerability and/or integrity, which may, in turn, affect cognitive decline through mechanisms independent of amyloid deposition. Additional study into the relationship between pathologic changes characteristic to AD and intrinsic anatomico-functional circuits should contribute to the understanding of the pathogenesis of AD. Recent evidence suggests that in the very early stages of AD, damage to the entorhinal cortex and/or subiculum efficiently isolates the hippocampal formation by
Synapse loss and/:~-amyloidosis in Alzheimer's disease disrupting its connections to cerebral association areas. This is followed by progression of the disease in a stepwise fashion along cortico-cortical connections. 7 The synaptic change in the outer molecular layer of the dentate gyrus might be explained as a secondary result of the disruption of the perforant pathway 1~ described by Van Hoesen and coworkers 34 and H y m a n and coworkers ~5'~6in the brains of A D patients. This pathway originates in large neurons clustered in layer II of the entorhinal cortex, 3°32'35 and serves as a major source of synaptic terminals in the outer portion of the dentate molecular layer. The entorhinal fibers also terminate in the subiculum, CA1, and CA3.1"3°'323435 In the present study, the number of CD-l-immunopositive neurons in layer lI of the entorhinal cortex correlated almost significantly with the synaptophysin-like immunolabelling of the outer molecular layer of the dentate gyrus in the whole study group. This inverse correlation did not, however, reach the statistical significance if only definite and possible A D patients were included in the analysis. The synaptophysin-like immunostaining of the outer molecular layer did not correlate wih the number of diffuse or neuritic plaques of layer II of the entorhinal cortex. In definite AD, a significant number of senile plaques were found in every area studied. In the present study the laminar and regional distribution was not the same for diffuse and neuritic plaques. The distribution of plaques between definite and possible A D cases was not identical. The number of patients in the present study is not high enough, however, to make any strict conclusions concerning differences in regional or laminar distribution o f / ~ / A 4 deposition between early and late stages of AD. Moreover, certain populations of synapses may be more susceptible than others to degeneration in AD. Thus, studies with other synaptic markers, such as chromo-
383
granins, might refine the relationship between amyloid accumulation and synapse integrity. Finally, studies investigating the inflammatory component of the plaques (e.g. complement proteins) might show differences in the distribution of actively inflammatory and inert plaques. This, in turn, might help elucidate the relationship between plaques with different characteristics and synaptic pathology. There may be injurious plaque types in which this association is obscured by the analysis of all plaques. CONCLUSION Synaptophysin-like immunoreactivity is decreased in the hippocampus and the entorhinal cortex in patients with possible and definite A D but not in M I D patients compared to nondemented controls. Equal synaptic loss in possible A D cases who are considered to represent early A D and definite A D cases supports the idea that synaptic loss is an early marker of AD. Moreover, the preservation of synapses in patients with M I D suggests that synapse loss is centrally involved in the pathogenesis of AD. The loss of synaptophysin-like immunoreactivity does not correlate with a high amount of [~/A4 in a particular region. This does not support the amyloid cascade hypothesis of A D that [~/A4 accumulation leads to the synapse loss. Moreover, we did not find a strict spatial relationship between senile plaques and degenerating neurons. Acknowledgements We thank Abbott Laboratories, IL, for the generous gift of the CD-I antibody. We also thank Ms Tarja Karjalainen and Ms Anna-Lisa Gidlund for their skilful technical assistance. The present work was supported by The Medical Research Council of the Academy of Finland, University of Kuopio, Orion Farmos Corporation, Finland, and The North Savo Regional Fund of the Finnish Cultural Foundation.
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LOSS OF S Y N A P T O P H Y S I N - L I K E I M M U N O R E A C T I V I T Y IN THE H I P P O C A M P A L F O R M A T I O N IS A N E A R L Y P H E N O M E N O N IN A L Z H E I M E R ' S DISEASE O. H E I N O N E N , * H. S O I N I N E N , * H. S O R V A R I , * O. KOSUNEN,~" L. PALJ,~,RVI,'~ E. K O I V I S T O * and P. J. R I E K K I N E N Sr*++§ *Departments of Neurology and tPathology and ++A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, SF-70211 Kuopio, Finland
Abstract--We
studied a synaptophysin-like immunoreactivity in the hippocampal formation of patients with definite Alzheimer's disease, multi-infarct dementia, patients with no evidence of clinical dementia with neuropathological findings fulfilling the criteria of possible Alzheimer's disease, and age-matched nondemented controls. Possible Alzheimer's disease cases were of special interest because they were considered to represent early Alzheimer's disease. We also studied the spatial relationship of synaptophysin-like immunopositivity with amyloid-/~-protein immunopositive senile plaques and anti-paired helical filament immunopositive degenerating neurons locally as well as considering the intrinsic circuits in the hippocampal formation. The synaptophysin-like immunoreactivity was decreased in the hippocampus and the entorhinal cortex in patients with definite and possible Alzheimer's disease but not in multi-infarct dementia patients compared to controls. Equal loss of synapses in possible and definite Alzheimer's disease patients supports the hypothesis that synaptic loss is an early phenomenon in Alzheimefs disease. Unchanged synaptophysin-like immunopositivity in patients with multi-infarct dementia suggests that the loss of synapses is centrally involved in the pathogenesis of Alzheimer's disease and not dementia per se. There was no spatial correlation between loss of synapses and amyloid-,g-protein positive senile plaques, Moreover, we could not find a strict spatial relationship between senile plaques and degenerating neurons. Our results do not support the amyloid cascade hypothesis of Alzheimer's disease that local accumulation of amyloid-/~-protein leads to the loss of synapses.
Neuronal death in patients with Alzheimer's disease (AD) has been suggested to be closely associated with the extensive synapse loss in the neocortex. Although two earlier studies described no change in synaptic number in A D cortex, 1°'25 more recent studies indicate that loss of synapses occur in the neocortex and the hippocampus of A D brain. 5"6"14'2°'21"2s The synapse loss in A D has been proposed to be a relatively early phenomenon, possibly preceding the loss of neurons. Thus, synapse damage would be a primary phenomenon rather than a secondary reflection of degeneration occurring in the perikaryon. :~ Moreover, the relationship of synaptic pathology to amyloid-fl-protein (3/A4) accumulation and cytoskeletal pathology in A D is of particular interest. Several studies suggest that the neocortical synapse loss, rather than the number of senile plaques or neurofibrillary tangles, correlates with the cognitive impairment in AD. This supports the view that the loss of synapses rather than ~/A4 deposition should be considered as a fundamental lesion in AD. 6'2~'~3
§To whom correspondence should be addressed. Abbret~iations: AD, Alzheimer's disease; []/A4, amyloid-/~'protein; DAB, 3,3'-diaminobenzidine; MID, multi-infarct dementia: MMS, Mini-Mental State; OD, optical density: PBS, phosphate-buffered saline. 375
Synaptophysin (p38) is a 38,000 mol. wt calcium binding glycoprotein :7 specifically located in the membrane of presynaptic vesicles in most and probably all neocortical axonal endings. ~7 Quantification of synaptophysin-like immunoreactivity is a valuable method for studying presynaptic terminals in normal and pathologic conditions. 2° Previous studies employing this method showed a significant synaptic loss in the neocortex and in the molecular layer of the hippocampus in patients with A D . 21"22'12 The decrement in synaptic connections in critical brain areas most probably cause impairment in memory, cognitive and behavioural functions. 12 We report here quantitative immunohistochemical data on synaptophysin-like immunoreactivity in the hippocampus and the entorhinal cortex of patients with definite AD, patients with multi-infarct dementia (MID), patients with no evidence of dementia but whose neuropathological findings fulfilled the criteria of possible AD, and age-matched nondemented controls. Especially, subjects without clinical evidence of dementia, but who had a sufficient amount of senile plaques for the diagnosis of possible AD, are of interest in studying the synapse pathology, since they probably represent the very early stage of AD. In addition, we studied the spatial relationship of synaptophysin-like immunoreactivity with senile
376
O. Heinonen et al. Table 1. Clinical characteristics of patients and controls Case no. 1 2 3 4 5 6 7 8 9 l0 11 12 13 14 15 16 17
Age/Sex
Duration of the disease (years)
88 M 60 F 65 M 77 F 75 F 86 M 90 F 77 F 72 M 91 F 77 F 80 F 84 F 87 F 81 M 84 F 75 F
---1 5 7 ---18 4 18 9 3 6 12 9
MMS
-12.00 6.00 6.00 ---0.00 8.00 0.00 0.00 9.00 1.00 0.00 0.00
Neuropathologic diagnosis Control Control Control MID MID MID Possible A D Possible A D Possible A D AD AD AD AD AD AD AD AD
Cause o f death Pneumonia Myocardial infarction Aortic valve stenosis Pneumonia Pneumonia Pneumonia Pulmonar embolism Myocardial infarction Myocardial infarction Pneumonia Pneumonia Pneumonia Pneumonia Chronic pyelonephritis Pneumonia Pneumonia Pneumonia
F, female; M, male; - - , not determined. p l a q u e s a n d d e g e n e r a t i n g n e u r o n s . Senile p l a q u e s were d e t e c t e d b y e m p l o y i n g /~/A4 i m m u n o h i s t o c h e m i s t r y . C y t o s k e l e t a l a l t e r a t i o n s were d e m o n s t r a t e d b y u s i n g a n t i - p a i r e d helical f i l a m e n t a n t i b o d y C D - 1 u w h i c h is a v a r i a n t o f t h e A l z - 5 0 a n t i b o d y 36 reacting against AD associated proteins including A68.
EXPERIMENTAL PROCEDURES
Patients and controls We used brain tissue from 17 subjects whose clinical characteristics are shown in Table 1. The mean post-mortem delay ( + S.D.) was 4.5 _ 1.9 h and it did not differ across the study groups. The histopathological diagnosis of A D was based on the C E R A D guidelines and criteria described by Mirra and coworkers, z3 We used modified Bielschowsky's silver impregnation staining to detect plaques and tangles. The Congo Red staining was also used for rating cerebral amyloid, particularly in the cerebral blood vessel walls. Eight patients had definite AD. Their mean age was 82.4 _ 5.2 years, duration of the disease was 9.9 _+ 5.8 years, and the mean score of Mini-Mental Status examination (MMS) 8 was 2.3 _+ 3.9. Three patients had had recurrent strokes with dementia and they fulfilled the D S M lII criteria of MID. z The patients with M I D did not show neocortical plaques in Bielschowsky's silver staining, and thus they did not fulfil the neuropathologic criteria o f AD. Their mean age was 79.3 + 5.9 years, mean duration of dementia was 4.3 + 3.1 years, and the mean M M S score was 8.0__+ 3.6. The patients with definitive A D and M I D went through lifetime examinations including neuropsychological testing, electroencephalogram, and cerebrospinal fluid analysis. The remaining six cases derived from an autopsy series of Kuopio University Hospital. None of them had clinical evidence of dementia. However, three of these cases (mean age 79.7 ___9.3 years) showed plaques and tangles by
Bielschowsky's silver staining allowing a diagnosis of possible A D according to the C E R A D criteria. The other three subjects (mean age 71.0 + 14.9 years) without clinical dementia and without plaques and tangles in Bielschowsky's staining served as controls. Immunohistochemistry We fixed samples from the hippocampal formation (including the dentate gyrus, the hippocampus proper, the subicular complex, and the entorhinal cortex) overnight in 4% buffered formalin, sectioned at 5 # m and collected onto 3-aminopropyltriethoxysilane (Sigma, St Louis, MO) pretreated slides. After the deparaffinization, we incubated sections for /~/A4 immunohistochemistry in 90% formic acid for 10min to intensify the staining. We blocked the endogenous peroxidase activity by using 5% hydrogen peroxide in distilled water for 5 min. Before the primary serum, we incubated the sections in normal horse serum (1:67, Vector, Burlingame, CA) for 1 h to avoid nonspecific staining. We stained the first set of sections using a monoclonal anti-synaptophysin antibody (SY 38, Dako, l/~g/ml), the second set of sections with a monoclonal mouse antibody to h u m a n fl-amyloid (anti-fl/A4) (6F/30, Dako, A/S Denmark, 0.13/zg/ml), and the third set of sections employing a monoclonal anti-paired helical filament (CD-I) antibody (Abbott Laboratories, IL, 1 ~g/ml), at + 4 ° C overnight. After the primary serum, we incubated the sections in biotinylated anti-mouse serum (made in horse, 1:200, Vector) for 30 min followed by the incubation in avidin-biotin-peroxidase complex (Vectastain ABC-standard kit, Vector) for 40 min. lmmunoperoxidase reaction was developed using 0.05% 3,3'-diaminobenzidine (DAB, Sigma) and 0.03% hydrogen peroxide for 5 min. Between different steps we thoroughly washed the sections in phosphate-buffered saline (PBS), p H 7.4. Finally, we counterstained the sections with anti-fl/A4 and anti-CD-I in Meyer's haematoxylin-eosin, dehydrated, mounted with DePex (BDH, Broom Rd, Poole, U.K.) and covered by coverslips. The immunolabelling protocol for antisynaptophysin was repeated twice in adjacent sections to
Fig. 1. Synaptophysin-like immunoreactivity in the entorhinal cortex of a control case. Immunopositivity is seen as a granular staining pattern in the neuropil. Neuronal cytoplasma remain unstained or show a low granular immunostaining. The grain size and the intensity of the staining of different granules vary. Layer II of the entorhinal cortex shows a denser staining compared to layer I and layer III. Asterix shows the site of magnification. I, II and III represent entorhinal layers. Scale bars = 150 # m (A) and 45 # m (B).
Fig. 2. (A, B) Synaptophysin-like immunoreactivity in the hippocampus of a control case. The outer molecular layer shows a slighly denser staining compared to the inner molecular layer. (C, D) A patient with AD shows a visible line of demarcation in the molecular layer separating a zone of light staining in the outer half from one of denser staining in the inner half. Around plaques there are large synaptophysin-like immunopositive grains representing abnormal dilated terminals (open arrows). Asterix shows the site of magnification. H, hilus; GL, granular cell layer; IML, inner molecular layer; OML, outer molecular layer. Scale bars = 150#m (A, C) and 45/~m (B, D). 378
Synapse loss a n d / L a m y l o i d o s i s in Alzheimer's disease
379
Table 2. Synaptophysin immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients and controls
Dentate gyrus Outer molecular layer Inner molecular layer Hilus CA3 CA I Subiculum Entorhinal cortex layer II Entorhinal cortex layer Ill Entorhinal cortex layer IV V
Control n=3
MID n =3
Possible A D n=3
AD n =8
Kruskall Waltis P
0.71 _+ 0.19 1.02 ± 0.25 1.07 ± 0.34 1.57 ± 0.25 1.57 £ 0.37 1.28 _+ 0•08 1.40 ± 0.17 1.40 ± 0.08 1.16 ± 0.23
1.2 ± 0.76 1.45 -+ 0.42 .19 _+ 0•40 .56 _ 0.71 .58 _+ 0.85 .28 _+ 1.02 .25 _+ 0.42 .26 _+ 0.29 0.98 _+ 0.43
0.57_+0.23 0.93 ± 0.35 0.69_+0.11 1.07 ± 0.08 1.19 ± 0.42 0.84+0.17 0.80_+0.12 0.79 ± 0.09 0.66+0.10
0.55_+ 0.17 1.03 + 0.31 0.78_+0.31 0.99 ± 0.47 0.90 _+ 0.32 0.75_+0.27 0.90_+0.31 0.81 ± 0.34 0.75-+0.31
0.4186 0•4081 0.2179 0.1904 0•2304 0.1795 0.0786 0.0330 0.2463
MannWhitney U-test
0,2 0,3:0,2 0,3;0,2 0,3;0,2 0,2
Data are background corrected absolute O D s of the neuropil. Results are expressed as mean + S.D. The differences across the study groups were tested using Kruskall-Wallis one-way analysis of variance• The numbers in the last column indicate which two groups differ significantly in the M a n n Whitney U-test. 0, control; 1, multi-infarct dementia: 2. possible Alzheimer's disease; 3, Alzheimer's disease. assure reproducibility of results. For control staining of each case we omitted the primary serum, otherwise the procedure was similar.
Quant(fication o[' optical density We measured optical densities (OD) of synaptophysinlike immunoreactivity by using a Quantimet 570 Image Analysis System (Leica Cambridge Ltd, Cambridge, England). The image was acquired using a Sony 3CCD video camera DXC-750P. The camera was mounted in an Olympus Vanox-T Research Microscope with a x 2 0 SPlan objective, x'2.5 intermediate lens, and x I0 oculars (Olympus Optical Co., Ltd, Tokyo, Japan)• We calibrated the system for O D measurements as follows: the section was aligned in the field of view and a level of light intensity for video camera performance was adjusted under computer control; an electronic shading correction was applied to the image to compensate for possible unevenness present in the illumination; a calibration curve was established with three filters with known ODs (Kodak Wratten Gelatin Filter, 75 m m x 75 mm, Eastman Kodak Company, Rochester, N.Y.). The O D was obtained by manually positioning the area of interest of the immunostained section in the camera field of view. The neuronal perikarya, blood vessels, and senile plaques were excluded• Ten fields of each area of interest were measured and the O D measurements were averaged. The O D of the white matter on the same section served as the value of the background. The absolute OD values were calculated according to the calibration curve. The background corrected absolute OD of the neuropil was obtained
Table 3. Synaptophysin-like immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients compared to controls. The results are presented as percentage of control values
Dentate gyrus Outer molecular layer Inner molecular layer Hilus CA3 CAI Subiculum Entorhinal cortex Layer II Layer III Layer IV V
MID n =3
Possible A D n =3
AD n =8
169% 142% 111% 99% 100% 100%
80% 91% 64% 68% 76% 66%
77% 100% 73% 63% 57% 59%
89% 90% 84%
57% 56% 57%
64% 58% 65%
by subtracting the OD of the background from the OD of the neuropil.
Quantification of" amyloM-~-protein positit,e plaques and CD-I positive neurons The density of diffuse and neuritic senile plaques were counted separately per square millimeter (mm 2) in the hilus, in the CA3 and the CA1 areas, in the presubiculum and in layers I, II, Ill, IV V, and VI of the entorhinal cortex. The number of CD-I positive neurons per m m 2 were counted (including both tangle bearing and granularly stained neurons) in the same areas. The total number of neurons per m m 2 in the CA3 and the CAI areas were measured and the density of plaques and CD-1 positive neurons in the outer and the inner molecular layers of the dentate gyrus were described using the terms "~none", "moderate" and " m a n y " . The measurements were executed using Nikon Optiphot-2 microscope, x 10 or x 20 Plan objectives, x 10 oculars, and an ocular craticule (Nikon Corporation, Tokyo, Japan).
Statistical analysis Data were analysed using SPSS/PC V.3.2 software. Kruskal Wallis one-way analysis of variance was used, followed by M a n n Whitney U-test to detect differences between the study groups. Correlations were calculated by Pearson's correlation test. The level of significance was P < 0.05. RESULTS
Synaptophysin-like immunoreactivity S y n a p t o p h y s i n - l i k e i m m u n o r e a c t i v i t y w a s seen as g r a n u l a r s t a i n i n g p a t t e r n in t h e n e u r o p i l (Fig. IA, B). T h e g r a i n size a n d t h e i n t e n s i t y o f t h e s t a i n i n g o f different g r a n u l e s varied. A r o u n d senile p l a q u e s t h e r e were l a r g e s y n a p t o p h y s i n - l i k e i m m u n o p o s i t i v e g r a i n s r e p r e s e n t i n g a b n o r m a l dilated t e r m i n a l s (Fig. 2C, D). N e u r o n a l c y t o p l a s m a were n o t s t a i n e d or s h o w e d a low g r a n u l a r i m m u n o s t a i n i n g p r o b a b l y r e p r e s e n t i n g s y n a p t o p h y s i n s y n t h e s i s in t h e cell o r recycling o f t h e s y n a p t o p h y s i n f r o m t h e n e r v e t e r m i•n a l s . -~ 4 R e g i o n s o f t h e w h i t e m a t t e r were v i r t u a l l y u n s t a i n e d • Tables 2 and 3 summarize the data from s y n a p t o p h y s i n - l i k e i m m u n o r e a c t i v i t y in t h e h i p p o c a m p u s a n d t h e e n t o r h i n a l cortex. In c o n t r o l s , s y n a p t o p h y s i n - l i k e i m m u n o s t a i n i n g in t h e i n n e r a n d the outer molecular layer of the dentate gyrus showed n o d i s t i n c t difference (Fig. 2A, B). T h e p a t i e n t s with
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Table 4. CD-1 and/~/A4 immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients and controls
Hilus CDI + neurons DSP NSP CA3 Neurons CD1 + neurons DSP NSP CA1 Neurons CD + neurons DSP NSP Subiculum CD + neurons DSP NSP EC layer II CD + neurons DSP NSP EC layer III CD + neurons DSP NSP EC layer IV V CD + neurons DSP NSP
Control n= 3
MID n= 3
Possible AD n= 3
AD n= 8
Kruskall Wallis P
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.7 + 1.2 0.5 ± 0.7 0.0 ± 0.0
2.7 + 2.3 0.0 ± 0.0 0.0 ± 0.0
34.0±20.0 13.0±15.7 2.9±4.6
<0.01 <0.05 N.S.
MannWhitney U-test 0,3;2,3;3,1 0,3;2,3
290.0 ± 0.0 ± 0.0 ± 0.0 +
90.5 0.0 0.0 0.0
173.5 ± 0.0 ± 0.0 ± 0.0 +
57.3 0.0 0.0 0.0
299.0 ± 2.3 ± 0.0 ± 0.3 ±
33.8 4.0 0.0 0.6
249.0 __+54.7 51.0 ± 42.5 9.7 ± 7.9 1.3 ± 2.2
N.S. <0.05 N.S. N.S.
235.7 ± 12.7 + 0.0 + 0.0 ±
53.0 21.9 0.0 0.0
299.3 + 22.0 ± 23.5 ± 0.0 ±
55.4 30.6 33.2 0.0
299.3 ± 53.7 + 11.0+ 0.7_+
5.5 86.1 19.1 1.1
259.7 ± 48.9 128.2 _+ 49.6 10.5 + 8.6 4.7 ± 7.7
N.S. <0.05 N.S. N.S.
0,3; 3.1 0,3 0,3; 2,3
0,3;3,1
0.0 + 0.0 3.7 __+6.4 0.0 + 0.0
8.0+ 11.3 3.5 ± 4.9 0.5 ± 0.7
13.3 ± 11.9 8.7 ± 11.5 3.0 + 1.7
161.4_+ 117.0
<0.05
15.6 ± 28.1
N.S.
5.8 ± 9.2
N.S.
0.0 + 0.0 0.3 ± 0.6 0.0 ± 0.0
0.0 __+0.0 14.5 __+20.5 0.0 ± 0.0
95.0 ± 82.7 4.3 ± 7.5 4.3 ± 7.5
245.0± 171.3 27.0__+ 14.2 8.0 __+9.0
<0.01 <0.05 N.S.
0,3; 3,1 0,3; 2,3
0.0 + 0.0 0.3+0.6 0.0 + 0.0
13.7 ± 22.0 10.0±14.1 2.0 ± 2.8
39.3 + 42.3 17.3±16.2 1.3 ___2.3
99.7 ± 74.0 53.6___42.5 3.0 ± 3.9
<0.05 <0.05 N.S.
0,3 0,3; 3,1
32.3 ± 30.3 25.7±22.9 1.3 ± 2.3
128.1 ± 51.4 17.3±11.2 0.4__+ 1.1
<0.01 N.S. N.S.
0,3; 2,3; 3,1 0,3
0.0 ± 0.0 0.3±0.6 0.0 ± 0.0
1.3 ± 1.5 9.0± 1 2 . 7 3.5 ± 4.9
0,2
Neurons, CDI + neurons, diffuse (DSP) and neuritic (NSP) fl/A4 immunopositive plaques were counted per 1 mm 2. Results are expressed as mean _ S.D. EC, entorhinal cortex; N.S., not significant. The differences across the study groups were tested using the Kruskall-Wallis one-way analysis of variance. The numbers in the last column indicate which two groups differ significantly in the Mann-Whitney U-test. 0, control; 1, multi-infarct dementia; 2, possible Alzheimer's disease; 3, Alzheimer's disease.
A D showed a visible line o f d e m a r c a t i o n in the molecular layer separating a zone of light staining in the outer h a l f from one o f denser staining in the inner h a l f (Fig. 2C, D). The possible a n d definite A D patients showed a 2 0 % decrease of control values in synaptophysin-like i m m u n o s t a i n i n g in the outer molecular layer, whereas the inner molecular layer was n o t affected. In the hilus, the possible a n d definite A D patients h a d a loss o f 3 6 % a n d 27% in synaptophysin-like immunoreactivity. In the CA3 area the synaptophysin-like i m m u n o r e a c t i v i t y was decreased 32% in patients with possible A D a n d 37% in patients with definite AD. In the CA1 area the loss in i m m u n o s t a i n i n g was 2 4 % for possible A D patients a n d 4 3 % for definite A D patients. The analysis of the subiculum revealed a 34% a n d a 4 1 % loss in synaptophysin-like i m m u n o r e a c t i v i t y in patients with possible a n d definite A D reaching a statistical significance c o m p a r e d to controls ( M a n n W h i t n e y U-test, P < 0.05). In the e n t o r h i n a l cortex the patients with possible a n d definite A D showed a significant loss o f g r a n u l a r staining in layers II, III a n d I V - V varying from a loss of 35% to 53% in possible A D patients a n d 33% to 4 2 % in definite A D patients in different laminae.
In M I D patients, the synaptophysin-like staining was c o m p a r a b l e with the staining in controls in the hilus, CA1, CA3, a n d subiculum. Synaptophysin-like staining was even slightly m o r e intense in the molecular layer of the dentate gyrus in M I D patients t h a n in controls. The e n t o r h i n a l cortex o f M I D patients showed a ~ 13% decrease in synaptophysinlike staining c o m p a r e d to controls. Synaptophysin-like i m m u n o p o s i t i v i t y was not related to age in the whole study g r o u p or in the A D group. However, the subjects in the study represented a relatively n a r r o w age range from 60 to 91 years.
Arnyloid-~-protein immunoreactivity Table 4 presents the n u m b e r of /~/A4 i m m u n o reactive diffuse and neuritic p l a q u e s / m m 2 in the h i p p o c a m p u s a n d the e n t o r h i n a l cortex of d e m e n t i a patients a n d controls. As expected, the patients with definite A D h a d the highest n u m b e r o f / ~ / A 4 positive senile plaques. In patients with definite A D , diffuse plaques were most frequent in layer III of the e n t o r h i n a l cortex, whereas most neuritic plaques were f o u n d in layer II of the e n t o r h i n a l cortex. Diffuse senile plaques o u t n u m b e r e d the neuritic forms in all areas studied. The patients with possible A D showed
Synapse loss and fl-amyloidosis in Alzheimer's disease the highest number of diffuse plaques in layers IV-V of the entorhinal cortex, and neuritic plaques were most frequently seen in layer II of the entorhinal cortex. The hilus and the CA3 area were almost free of plaques in this group. Patients with definite AD showed a moderate to high degree of senile plaques in the molecular layer of the dentate gyrus. Plaques were seen both in the inner and outer half of the molecular layer. Two patients with possible AD (nos 8 and 9) had few diffuse plaques in the molecular layer and one possible AD subject (no. 7) was free of senile plaques in this area. None of the controls had plaques in the molecular layer of the dentate gyrus. The MID patients had a considerable amount of ///A4 positive diffuse plaques in the CAI region. Furthermore, senile plaques were found in the entorhinal cortex. The hilus and the CA3 area showed only few or no plaques.
C D -1 irnmunopositivity The CD-1 antibody recognized neurofibrillary tangles, dystrophic neurites in and around senile plaques, as well as neuropil threads. Positive immunoreactivity was also found as granular staining in a proportion of neurons possibly representing an early cytological change that precedes the formation of tangles and plaques. Table 4 shows CD-1 immunoreactivity in the hippocampus and the entorhinal cortex of dementia patients. Patients with definite AD showed the most frequent immunostaining in every area. Layer II of the entorhinal cortex had the highest number of immunopositive neurons. This was also the case in patients with possible AD. In MID patients the highest number of immunopositive neurons was in the CA1 area and a considerable number of stained neurons were seen also in layer lII of the entorhinal cortex. The analysis
381
of the molecular layer of the dentate gyrus of patients with definite AD showed only a few or moderate number of CD-1 immunopositive neurons. The immunopositive neurons distributed both to the inner and outer molecular layer. The possible AD cases or controls had no immunopositive neurons in this area.
Neuron count The total number of neurons in the CA1 and CA3 areas was calculated and it did not differ significantly between study groups.
Spatial relationship between synaptophysin-like immunoreactivity, amyloid-~3-protein and CD-1 immunoreactivity The spatial relationship of /~/A4, CD-I and synaptophysin-like immunoreactivities was studied locally in each area as well as considering the intrinsic circuits in the hippocampal formation. We did not find any strict local correlation between the degree of synaptophysin-like immunoreactivity and /3/A4 positive plaques (Fig. 3) or CD-I immunoreactive neurons. No local correlation between the number of neurons and the degree of synaptophysin-like immunostaining in the CAI and the CA3 regions was detected either. Instead, the number of CD-1immunoreactive neurons in layer II of the entorhinal cortex correlated almost significantly with the synaptophysin-like immunostaining of the outer molecular layer of the dentate gyrus in the whole study group (r = - 0 . 5 2 , n = 14, P =0.056). This correlation did not reach the statistical significance if only definite and possible AD cases were included in the analysis ( r = - 0 . 5 8 , n = 9 , P=0.101). The synaptophysin-like immunoreactivity of the outer molecular layer did not correlate with the number of diffuse or neuritic plaques of layer II of the entorhinal cortex.
60 1
50
L
m
~
I ~3E 40 ~ 0
I
-----J
A
00
m
O~
I. . . . .
0.8 0
l___l
0.8
30
I
~. 20
i
lO
I . . . . . . . . .
........
; ........
Hilus
o.2
~
o
~
.......
."
CA3
----
,°,
~0
- ........
CA1 Subiculum EC II
EC III
,
EC IV-V
Synaptophysin-like immunoreactivity Diffuse plaques
.....
Neuritic plaques
Fig. 3. A local relationship of synaptophysin-likeimmunoreactivity (absolute OD) and numbers of senile plaques/mm2in the hippocampus and the entorhinal cortex. There is no local correlation between synaptic density and plaques.
O. Heinonen et al.
382
In general, we found no distinct correlation between /~/A4 and CD-1 immunopositivities in a particular area. All definite AD patients showed a number of CD-l-immunopositive neurons in layer II of the entorhinal cortex, and all of them had a high amount of/3/A4 immunolabelling in the molecular layer of the dentate gyrus. One patient with possible AD (7) had numerous CD-1-positive neurons in layer If of the entorhinal cortex but no /~/A4 positive plaques in the molecular layer.
DISCUSSION
Synaptic loss is centrally involved in the pathogenesis of Alzheimer's disease We report here a loss in synaptophysin-like immunoreactivity in the hippocampus and the entorhinal cortex of patients with possible and definitive AD. Our findings are in agreement with previous data demonstrating synaptic loss in the neocortex and the hippocampus by synaptophysin immunohistochemistry as well as by electronmicroscopy. 6'14'21 In their recent study, Honer and coworkers ~4 reported a 77% reduction in synaptophysin-like immunoreactivity (EPI0 antibody) in the hippocampus and a 54% reduction in the temporal cortex in AD. Masliah and coworkers 21 demonstrated a 45% loss in synaptophysin immunoreactivity in the frontal and parietal cortex, and to a lesser extent in the hippocampus and entorhinal cortex. In the present study, the loss varied from 19% to 53% depending on the specific area, the most prominent loss of staining being in the entorhinal cortex. In contrast to our study, Scheffand coworkers 29 recently reported that there were no changes in synaptic density between control and AD subjects in the entorhinal cortex. Differences in methods, AD patient populations, and severity in cognitive decline of the patients may account for divergent results. It has been proposed that, when neuropathological alterations proceed beyond an individual threshold, the brain fails to compensate such changes. Thus, the loss of synaptophysin-like immunoreactivity in the present study may reflect the overdraft of the individual capability of a plasticity response to replace lost synaptic contacts. Alternatively, the alteration in the pattern of synaptophysin staining in the present study do not represent, at least as a whole, the actual loss of synapses but the insufficient amount of nerve terminal protein density and malfunction of synapses. In accordance with earlier reports, 5'12'14the present study demonstrates a striking decrease in immunoreactivity for synaptophysin in the outer molecular layer of the dentate gyrus in the brains of AD patients, compared with controls. Interestingly, patients with possible and definite AD did not differ from each other in the degree of synaptophysin-like immunoreactivity supporting the hypothesis that the
loss of presynaptic terminals is a relatively early phenomenon in AD. 21 In contrast to AD cases, we found no synaptic loss in the hippocampus of MID patients. Thus, our results suggest that synaptic loss is centrally involved in the pathogenesis of AD. The unaltered synaptophysin-like immunoreactivity in AD in the primary visual cortex, 4 which is a relatively spared area in AD, further supports this hypothesis.
Lack of positive spatial relationship between amyloid deposits and synaptic degeneration Recent studies indicate that the synaptic loss in AD might be largely independent of the [~/A4 deposition. 3"~'28"29Our study contributes to this by specifying plaque analysis by brain region and plaque subtypes. In the present study there was a lack of strict spatial correlation between the loss of synaptophysin-like staining and a high amount of []/A4 in a certain area (Fig. 3). Especially, diffuse plaques seem clearly to have no effect on synapse count. The lack of correlation between [:1/A4 deposition and synaptophysin-like immunoreactivity was clearer in the entorhinal cortex compared to the hippocampus (Fig. 3). One could not ignore the possibility of the regional susceptibility of the amyloid-induced injury. Such a scenario of regional susceptibility is postulated, e.g. for the cerebellum, where amyloid deposits occur without neuronal injury. However, our results, in agreement with previous results, do not unambiguously support the amyloid cascade hypothesis ~3 of AD that [~/A4 accumulation leads to the loss of synapses. Given the small number of cases, we should, however, be cautious in the interpretation of the correlation analysis. Furthermore, the absolute numbers of [4/A4 immunopositive plaques do not necessarily demonstrate the extent of fl/A4 load in a certain area. [4/A4 or fragments thereof were reported to be toxic to differentiated neurons ~'37 The neurodegenerative effect of [4/A4 in these studies suggests that neuronal death in AD is a direct consequence of [¢/A4. However, several other studies have found no direct toxicity of /~/A4. 9"26'3t If [I/A4 exerts a widespread toxic effect, there should be a positive spatial relationship between amyloid deposition and degeneration of synapses; the more [~/A4 the fewer synapses at a certain region. We did not see such a relationship. Thus, alternative models to the amyloid cascade hypothesis will be encouraged by these data. There may be other determinants of synaptic vulnerability and/or integrity, which may, in turn, affect cognitive decline through mechanisms independent of amyloid deposition. Additional study into the relationship between pathologic changes characteristic to AD and intrinsic anatomico-functional circuits should contribute to the understanding of the pathogenesis of AD. Recent evidence suggests that in the very early stages of AD, damage to the entorhinal cortex and/or subiculum efficiently isolates the hippocampal formation by
Synapse loss and/:~-amyloidosis in Alzheimer's disease disrupting its connections to cerebral association areas. This is followed by progression of the disease in a stepwise fashion along cortico-cortical connections. 7 The synaptic change in the outer molecular layer of the dentate gyrus might be explained as a secondary result of the disruption of the perforant pathway 1~ described by Van Hoesen and coworkers 34 and H y m a n and coworkers ~5'~6in the brains of A D patients. This pathway originates in large neurons clustered in layer II of the entorhinal cortex, 3°32'35 and serves as a major source of synaptic terminals in the outer portion of the dentate molecular layer. The entorhinal fibers also terminate in the subiculum, CA1, and CA3.1"3°'323435 In the present study, the number of CD-l-immunopositive neurons in layer lI of the entorhinal cortex correlated almost significantly with the synaptophysin-like immunolabelling of the outer molecular layer of the dentate gyrus in the whole study group. This inverse correlation did not, however, reach the statistical significance if only definite and possible A D patients were included in the analysis. The synaptophysin-like immunostaining of the outer molecular layer did not correlate wih the number of diffuse or neuritic plaques of layer II of the entorhinal cortex. In definite AD, a significant number of senile plaques were found in every area studied. In the present study the laminar and regional distribution was not the same for diffuse and neuritic plaques. The distribution of plaques between definite and possible A D cases was not identical. The number of patients in the present study is not high enough, however, to make any strict conclusions concerning differences in regional or laminar distribution o f / ~ / A 4 deposition between early and late stages of AD. Moreover, certain populations of synapses may be more susceptible than others to degeneration in AD. Thus, studies with other synaptic markers, such as chromo-
383
granins, might refine the relationship between amyloid accumulation and synapse integrity. Finally, studies investigating the inflammatory component of the plaques (e.g. complement proteins) might show differences in the distribution of actively inflammatory and inert plaques. This, in turn, might help elucidate the relationship between plaques with different characteristics and synaptic pathology. There may be injurious plaque types in which this association is obscured by the analysis of all plaques. CONCLUSION Synaptophysin-like immunoreactivity is decreased in the hippocampus and the entorhinal cortex in patients with possible and definite A D but not in M I D patients compared to nondemented controls. Equal synaptic loss in possible A D cases who are considered to represent early A D and definite A D cases supports the idea that synaptic loss is an early marker of AD. Moreover, the preservation of synapses in patients with M I D suggests that synapse loss is centrally involved in the pathogenesis of AD. The loss of synaptophysin-like immunoreactivity does not correlate with a high amount of [~/A4 in a particular region. This does not support the amyloid cascade hypothesis of A D that [~/A4 accumulation leads to the synapse loss. Moreover, we did not find a strict spatial relationship between senile plaques and degenerating neurons. Acknowledgements We thank Abbott Laboratories, IL, for the generous gift of the CD-I antibody. We also thank Ms Tarja Karjalainen and Ms Anna-Lisa Gidlund for their skilful technical assistance. The present work was supported by The Medical Research Council of the Academy of Finland, University of Kuopio, Orion Farmos Corporation, Finland, and The North Savo Regional Fund of the Finnish Cultural Foundation.
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