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Amyloid protein levels in cerebrospinal fluid are elevated in early-onset Alzheimer's disease.

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Amyloid p Protein Levels in Cerebrospinal
Fluid-Are Elevated in Early-Onset
Alzheimer's Disease
Tamiko Nakamura, MD," Mikio Shoji, MD, PhD," Yasuo Harigaya, MD, PhD," Mitsunori Watanabe, MD,"
Kenji Hosoda, PhD,? Tobun T. Cheung, PhD,$ Lillian M. Shaffer, PhD,$ Todd E. Golde, PhDJ
Lnda H. Younkin, MD, PhD,$ Steven G. Younkin, MD, PhD,$ and Shunsaku Hirai, MD, PhD"
The 4-kd amyloid P protein (AP) deposited as amyloid in Alzheimer's disease (AD) is produced and released by normal
proteolytic processing of the amyloid P protein precursor (PAPP) and is readily detected in cerebrospinal fluid (CSF).
Here, we present the levels of AP in CSF from a total of 95 subjects, including 38 patients with AD, 14 with early-onset
AD and 24 with late-onset AD, 25 normal control subjects, and 32 patients with other neurological diseases. The level
of AP decreased with normal aging, and there was a significant elevation in the level of AP in the CSF of early-onset
AD patients (4.14 2 1.37 pmollml, p < 0.01). Neither Mini-Mental State nor Functional Assessment Staging were
correlated with the amount of AP in the CSF. The AP/secreted form of PAPP ratio was elevated, but the level of
al-antichymotrypsin in the CSF did not correlate with the level of CSF AP in early-onset AD patients. Thus, the level
of AP in the CSF is elevated in early-onset AD patients and is suggested to be correlated with the pathology in the
brain that characterizes AD.
Nakamura T, Shoji M, Harigaya Y, Watanabe M, Hosoda K, Cheung 'IT, Shaffer LM, Golde TE,
Younkin LH, Younkin SG, Hirai S. Amyloid p protein levels in cerebrospinal fluid are
elevated in early-onset Alzheimer's disease. Ann Neurol 1994;36:903-911
The amyloid deposited in the Alzheimer's disease
(AD) brain is composed of a 39 to 43 amino acid
amyloid P protein (AP) 11, 2 ) derived from a set of
695 to 770 amino acid precursor proteins collectively
referred to as the amyloid P protein precursor (P APP)
C3f. In each of the 695 to 7 7 0 residue PAPPs, AP is
an internal peptide that begins 99 residues from the
carboxy-terminus and extends from the extracellular
intraluminal region into the middle of the hydrophobic, membrane-spanning domain. The PAPP is normally processed in a constitutive secretory pathway in
which it is cleaved at A p l 6 to produce a large, secreted
derivative (secreted form of PAPP, sPAPP) E4-111.
An alternative pathway, which OCCU'J -t least in part
in the endosomal-lysosomal system, produces a complex set of carboxy-terminal derivatives including potentially amyloidogenic forms with the entire AP at or
near their amino-termini [12-141. Recently, we reported that normal pAPP processing produces and releases a soluble 4-kd A@{15-19}, which is essentially
identical to the AP deposited as amyloid fibrils in AD
{20). A similar fragment has also been detected in cerebrospinal fluid (CSF) from individuals both with and
without AD El>, 171. Moreover, the processing of a
mutant PAPP found in a Swedish family with hereditary AD [ 2 1 ) is altered in a way that releases increased
amounts of AP C22, 23). Thus, it is important to determine whether the processing of PAPP is altered,
whether the amount of AP produced is increased in
AD, and finally how these alterations contribute to AD
pathology. In the present study, we measured the levels of AP in CSF from a large population of patients
to clarify these questions, and studied the correlation
between AP and al-antichymotrypsin (ACT) as a possible biochemical marker for AD [24-27).
From the 'Department of Neurology, Gunma University School of
Medicine, Gunma, and t Diagnostic; and Medicals Department, Teijin Institute for Biomedical Research, Tokyo, Japan; and $Division
of Neuropathology, Institute of Pathology, Case Western Reserve
University School of Medicine, Cleveland, OH.
Received Dec 10. 1993. and in revised form Mar 10 and Tun 7,
1994. Accepted for publication Jun 9, 1994.
Materials and Methods
Subjects
CSF was collected from a total of 95 subjects consisting of
38 patients with sporadic AD, 25 normal control subjects,
and 32 with other neurological diseases. Of the 38 A D cases,
14 were early-onset A D patients in whom onset was before
65 years of age and 24 were [ate-onset A D patients in whom
onset was after 65 years of age. The normal control group
was subclassified into a younger group less than 40 years old
and an older group more than 40 years old. The early-onset
A D group and older normal control group were age matched.
Address correspondence to D r Shoji, Department of Neurology,
Gunma University School of Medicine, 3-39-15 Showamachi, Maebashi, Gunma 371, Japan.
Copyright 0 1994 by the American Neurological Association
903
oping system (Amersham, USA) was used to detect the A@.
Bands visualized on exposed films were accurately quantified
by using an image analyzer (Fast Scan, Molecular Dynamics,
USA) 1153. Synthetic A@l-40 (5, 10, 25, and 50 pmol)
added to 3 ml of RIPA was also analyzed by the same immunoprecipitation method in each blot to allow a standard curve
to be plotted by first-order regression analysis, and the level
of A@ in each sample was calculated (Fig 1).The intraassay
coefficient of variation was 14.3% and the interassay coefficient of variation was 22.1%.
Clinical diagnosis of A D was based on the National Institute
of Neurological and Communicative Disorders and Stroke/
Alzheimer’s Disease and Related Disorders Association
(NINCDSIADRDA) criteria 1281.
All subjects underwent medical, neurological, and psychiatric examinations and appropriate diagnostic studies including computed tomographic (CT) scanning and magnetic resonance imaging (MRI) to exclude other disorders that may
have been responsible for dementia. We especially selected
CSF from persons with no brain atrophy as detected by MRI
as normal controls to exclude A D subjects who had not yet
shown clinical dementia. Normal control and A D patients
suffering from acute inflammatory diseases or malignancy
were excluded from the present study. The patients with A D
underwent Mini-Mental State (MMS) testing [29} to evaluate
severity of dementia and were classified into seven clinical
stages of A D using the Functional Assessment Staging
(FAST) 130). Duration of illness was also evaluated. After
informed consent was given, CSF was obtained by lumbar
puncture. One milliliter of CSF was taken for routine CSF
studies, after which 0.5 ml for sPAPP, 0.5 ml for ACT, and
3 ml for A@ were collected and used for each assay. CSF
samples were frozen at - 80°C until assay. Bloody CSF samples or CSF samples with abnormally high levels of protein
or cell counts from both A D and normal control subjects
were excluded from the study. The cell number and protein
concentration in CSF were not significantly different between
A D and normal control groups (Table 1).
CSF (50 ~ 1 and
)
10 pl of cold trichloroacetic acid were
mixed and incubated for 30 minutes at 0”C, followed by
centrifugation at 15,000 rpm. After washing twice in cold
100% ethanol, the pellet was separated by 10% Tris-tricinePAGE and transferred electrophoretically onto Immobilon
P membranes at 100 V for 1 hour. The s@APPon the membrane was detected with 2 2 C l l ( 5 pgiml), a mouse monoclonal antibody raised against @ APP (Boehringer, Mannheim, Germany), using an ECL developing system. Pooled
normal CSF was also analyzed as a standard. The total s@APP
(105 kd, 125 kd) signal on the exposed film was estimated
by Fast Scan, and the integrated optical density of the ratio
of sample CSF to standard pooled CSF was calculated at the
sample s@APPlevel.
Determination of CSF Amyloid p Protein Level
Enzyme Immunoassay of CSF a1-Antichymotrypsin
CSF (3 ml), 20 pl of SGY2134 (polyclonal rabbit antiserum
raised against A@1-40), 20 pl of protein A-agarose beads,
and 0.75 ml of 5 x RIPA buffer (150 mM NaCI, 1% Triton
X-100, 0.5% cholic acid, 0.1% sodium dodecyl sulfate in 50
mM Tris buffer, p H 8.0) containing 5 x protease inhibitor
(1 mM phenylmethylsulfonyl fluoride [PMSF], 1 pg/ml leupeptin, 0.1 pgiml pepstatin A, 1 pgiml Na-p-tosyl-L-lysine
chloromethyi ketone [TLCK], 1 mM EDTA) were mixed
and incubated at 4°C for 20 hours. After centrifugation and
washing, the immunoprecipitated protein was separated by
10 to 16.5% Tris-tricine-polyacrylamide gel electrophoresis
(Tris-tricine-PAGE) and electrophoretically transferred
onto Immobilon P membrane (Millipore, USA) at 100 V
for 2 hours. The blotting membrane was labeled with 4G8
(1 :500), a mouse monoclonal antibody raised against
PAPI 7-24. An electrochemiluminescence (ECL) devel-
An anti-ACT antibody (MBL, Japan) was digested to F(ab‘)*
by pepsin and coupled to horseradish peroxidase (HRP)
(anti-ACT-HRP) using maleimidobenzoyl-N-hydroxysuccinimide ester. Polystyrene beads (6 mm) immobilized with antiACT antibody (20 pgiml) were also prepared. Two hundred
microliters of standard ACT (from 0 to 200 ng/L in 1%
bovine serum albumin-phosphate-buffered saline (BSAPBS), p H 7.2; Calbiochem Corp, Germany) or diluted CSF
samples (1: loo), 200 pl of anti-ACT-HRP (125 ng/ml in
1% BSA-PBS, p H 7.2), and one anti-ACT-fixed bead for
each sample was placed in a glass tube and incubated at 37°C
for 1 hour. After washing the beads, 400 IJ-I of substrate
0.0034%
solution (0.045% 3,3’,5,5’-tetramethylbenzidine,
H,O, in 0.1 M phosphateIcitric acid buffer, p H 4.3) was
added and incubated at 37°C for 30 minutes. Optical density
at 450 nm was estimated after adding l m l of 1 M H,SO,.
Determination of Level of CSF Secreted Form
of Amyloid p Protein Precursor
Tabje I . Summary of Subjects (Alzheimerner’sDisease and Normal Controlj)
Age
Sex (n)
No. of
Patients
Years
Range (yr)
Male
Female
Duration of
Disease (yr)
MMS
Score
CSF Cell
(countImm’)
CSF Protein
(mgidl)
Alzheimer’s disease
Early onset
Late onset
38
14
24
72
59
79
40-94
40-7 1
68-94
15
9
23
8
15
3.2
4.1
2.7
13
15
12
3
2
4
37
34
38
Normal control
Younger group
25
43
27
57
16-75
11
14
16-39
45-75
6
5
6
8
-
30
30
30
3
3
29
28
30
Older group
12
13
6
Data are mean values.
904 Annals of Neurology Vol 36 No 6 December 1994
3
in relation to severity of dementia and duration-dependent
changes.
15000
Results
Amyloid /3 Protein Level in Normal Control Subjects
The mean level of AP was 4.00 -+ 2.92 pmol/ml in
the CSF of the total normal control group, but there
was a significant difference between that in the younger
(5.15 -+ 2.75 pmol/ml) and older groups (2.27 k 1.44
pmol/ml; p < 0.01; Fig 2). The level of CSF AP was
significantly reduced with aging ( p < 0.05; Fig 3). No
significant differences were observed between males
and females in all control groups.
10000
At3
5000
pmole/mI
11
0
10
0
10
20
30
40
9
50
.
prnole
8
.
0
7
0
5P
lop
25p 50p
Fig 1. Standurd curve of synthetic amyloid p protein (Ap)
(top);5,000, 10,000, and I J , o O O are integrated optical densities determined by Fast Scan. Signal of synthetic AD by electrochemiluminescence (bottom);5p, lop, 25p, and Sop = 5 pmol,
10 pmol, 25 pmol, and 50 pmol o f synthetic Apl-40, yespectively.
5
8 .
4
Statistical Analysis
Values are shown as mean 2 standard deviation (SD). Means
were evaluated using Student's t test and/or the CochranCox test after Smirnoff's elimination test and Bartlett's test
for uniformity of variances. First-order regression was used
for analysis of age-related changes in normal control subjects
30
0..
0
i
O
0
S
.
.
.
0.
-
8.
-0
1
a
.
31
3
08
$!
.
0
2
The reliable assay range of this method was from 0.4 to
200 ng/ml, and the sensitivity was 0.4 ng/ml. Recovery rate
was 99.3%. The intraassay coefficient of variation was 6.4%.
The level of CSF ACT detected by enzyme immunoassay
(EIA) correlated with that detected by the established radioimmunoassay system (n = 42, r = 0.96, p < 0.01) [25].
..
6
0
0
0
00
eAD IAD
yN ON
Alzheimer's
disease
Normal
control
Fig 2. Levels o f amyloid p protein (AD) in cerebrospinalftuid
in normal control and AlzheimeJs disease ( A D ) groups. eAD
= early-onset AD; LAD = late-onset AD; yN = younger normal control group; ON = older nomal control group. White circles show yN and eAD, and black dots show ON and A D .
Nakamura et al: CSF A P Is Increased in Early-Onset A D
905
~
ADprnole/rnl
12
a
a
1
n = 25
9
R = -0.43
P c: 0.05
6
Age
F i g 3. Relationship between amyloid p protein ( A p ) and norm
a
l aging. The level o j A ,B uias signifirantly reduced with norm l a g i n g (n = 25. r = -0.431, p < 0.03).
Amyloid p Protein Level in Alzheimer's
Disease Patients
The mean level of AP was 3.53 5 1.31 pmol/ml in
the CSF of the total AD group, 4.14 2 1.37 pmol/ml
in the early-onset AD group, and 3.17 f 1.15 pmoll
ml in the late-onset AD group (see Fig 2). There was
significant elevation of AP level in the early-onset AD
( p < 0.01) compared with the older normal control
group. No significant differences were observed in
level of CSF AP between the early- and late-onset AD
groups; however, the mean CSF AP level in the lateonset AD group was slightly higher than that in the
older normal control group.
There was no correlation between CSF AP level and
MMS in total AD, early-onset AD, or late-onset AD
groups. No significant differences were observed in
the level of AP between FAST stages (stage 3, n =
11, 3.30 ? 1.32 pmol/ml; stage 4, n = 6, 4.05 t
1.05 pmol/ml; stage 5, n = 4, 3.35 ? 1.83 pmol/ml;
stage 6, n = 11, 3.28 & 1.38 pmol/ml; stage 7, n =
6, 4.79 2 1.86 pmol/ml) or in FAST stages between
the early-onset AD and late-onset AD groups. The AP
level showed a significant correlation with duration of
illness in the late-onset AD ( r = 0.56, p < 0.01) and
in the total AD ( r = 0.47,p < 0.01) groups. However,
in the early-onset AD group, there was no correlation.
Amyloid p Protein Level in Other Neurological
Disease Patients
There was considerable overlap in the levels of CSF
AP observed in the AD and other neurological disease
groups. High levels of AP were observed in patients
with acute cerebral infarction (Patient l), GuillainBarre syndrome (Patient 9), viral encephalitis (Patient
l l ) , multiple system atrophy (Patient 19), and Kennedy-Alter-Sung disease (Patient 30; Table 2).
Secreted Form of Amyloid p Protein Preczlrsor Level in
Normal Control Subjects
As shown in Figure 4, the mean level of sPAPP was
similar in the total normal control group (1.35 -t_ 0.38),
in the younger group (1.37 t 0.55), and in the older
group (1.43 k 0.35).
Secreted Form of Amyloid p Protein Precursor Level in
Alzheimer's Disease and Other Neurological Disease
Patients
There were no significant differences between the AD
groups and the total normal control group (Figure 4).
There was also no correlation between sPAPP level in
the total AD group and MMS or between sPAPP level
in the total AD group and duration of illness. No significant differences in sPAPP level in the total AD
group were found between FAST stages (stage 3, n =
11, 1.21 -+ 0.40; stage 4, n = 6, 1.41 -t 0.43; stage
5 , n = 3, 1.22 k 0.14; stage 6, n = 10, 1.28 0.23;
stage 7, n = 6, 2.14 k 1.60). There was no difference
in level of sPAPP between early-onset and late-onset
AD groups. Among the other neurological disease patients, the level of sPAPP was high in those with Guillain-Barre syndrome (Patients 9 and lo), glioma (Patient 16), olivopontocerebellar atrophy (Patient ls),
multiple system atrophy (Patient 19), Emery-Dreifusstype muscular dystrophy (Patient 25), and KennedyAlter-Sung disease (Patient 30; Table 2).
*
APIsPAPP Ratio in CSF
The ratio of AP to sPAPP was calculated as the relative
activity of both the A@-generating pathway and the
constitutive secretory pathway of pAPP in the brain.
1.68 in the
The ratio of AP to sPAPP was 2.73
total normal control group, 3.46 & 1.25 in the
younger normal control group, 1.64 ? 1.13 in the
older normal control group, 2.86 ? 1.88 in the total
AD group, 3.53 t 2.05 in the early-onset AD group,
and 2.45 k 1.68 in the late-onset AD group (Fig 5).
The AP/sPAPP ratio also decreased with aging (n =
24, r = -0.50, p < 0.05). There was a significant
increase in the ratio of AP to sPAPP in the early-onset
AD group compared with that in the older normal
control group ( p < 0.01). No correlation was found
between the APIsPAPP ratio and MMS, duration of illness, and FAST stages in any ADgroups. In other neurological disease patients, those with acute cerebral infarction (Patient 2), herpes simplex encephalitis (Patient
12),neuro-Behget disease (Patient 14), and amyloid angiopathy with recurrent cerebral hemorrhage (Patient
32; see Table 2) showed elevated AP/sPAPP ratios.
*
a1 -Antichymotrypsin hue1 in Normal Control
Subjects and Alzeimer's Disease Patients
The mean level of ACT was 2.27 k 1.40 Kglml in
the total normal control group, 2.33 L 1.29 pglml
906 Annals of Neurology Vol 36 No 6 December 1994
Table 2. Summary of Subjects (Other Neurologzcal Diseases)
Patient
No.
Diagnosis
1
2
3
4
5
6
7
8
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Cerebral infarction (A, S)
Cerebral infarction (A, S)
Cerebral infarction (A, S)
Cerebral infarction (A, S)
Cerebral infarction (C, L)
Cerebral infarction (C, S)
Cerebral infarction (C, S)
Cerebral infarction (C, S)
Guillain-Barre syndrome
Guillain-Barre syndrome
Viral encephalitis
Herpes simplex encephalitis
Herpes simplex encephalitis
Neuro-Behqet disease
Meningitis carcinomatosa
Brain tumor (glioma)
Olivopontocerebellar atrophy
Olivopontocerebellar atrophy
Multiple system atrophy
Multiple system atrophy
Parkinson's disease
Leukoencephalopathy
Hydrocephalus
Lissencephaly
Muscular dystrophyb
Muscular dystrophy'
ACTH deficiency
Hypoparathyroidism
Amyotrophic lateral sclerosis
Kennedy-Alter-Sung disease
Creutzfeldt-Jakob disease
Amyloid angiopathy
Age
(yr)
44
59
73
81
55
63
65
71
22
50
32
38
42
47
43
42
41
50
60
61
61
45
58
40
38
58
60
65
69
17
54
74
Sex
MMS
M
M
F
M
M
26
N
23
M
M
M
F
F
M
F
M
M
M
F
M
M
F
M
F
M
F
F
M
F
M
M
M
Cell
(count/mm3)
0.7
1.3
3.0
1.0
3.0
33
40
32
60
-
-
0.3
1.3
2.3
3.0
120.7
2.3
15.7
16.3
5.0
0.7
0.3
0.0
0.3
0.0
2.7
0.3
0.7
1.o
2.0
7.0
0.0
0.3
1.o
2.0
0.3
0.3
112
22
102
N
AP
AP
11
25
N
N
N
0
15
18
N
N
19
N
24
24
N
-
11
28
30
30
16
19
N
M
N
F
F
1
0
Protein
(mgidl)
79
60
59
28
72
45
108
39
60
33
31
31
44
37
39
23
13
17
23
39
42
48
7
18
AP
(pmoliml)
5.45
2.89
0.28
2.14
0.06
2.19
2.99
3.87
5.72
2.13
9.61
1.14
0.00
3.45
2.58
3.7
4.21
2.96
8.07
2.55
1.83
2.37
0.93
1.38
1.82
0.70
3.54
1.26
3.94
5.32
0.81
2.72
sPAPPa
APisPAPP
1.58
0.53
0.97
1.37
0.50
1.51
0.74
1.16
2.84
2.40
1.96
0.2 1
0.28
0.22
1.72
2.72
1.16
2.12
2.95
1.75
1.15
0.86
2.04
0.66
2.40
1.76
0.88
0.92
1.71
3.22
3.46
5.47
0.29
1.56
0.12
1.45
3.18
3.33
2.01
0.89
4.70
5.47
0.00
15.91
1.50
1.27
3.63
1.40
2.74
-
-
0.52
5.56
1.46
1.60
2.77
0.45
2.08
0.76
0.5 1
4.03
1.37
2.06
1.65
ACT
(pgiml)
-
2.50
1.80
5.75
20.75
9.60
2.80
10.80
7.60
7.70
5.10
13.35
11.95
10.70
12.30
3.30
4.80
2.50
4.60
6.85
3.20
7.60
8.00
2.30
0.40
4.55
0.60
9.40
3.20
3.70
7.60
20.05
~~
aMeasurement by integrated optical density.
bEmery-Dreifuss-type muscular dystrophy.
CFacioscapulohumoraltype muscular dystrophy.
MMS = Mini-Mental State; AP = amyloid P protein; sPAPP = secreted form of amyloid P protein precursor; ACT = el-antichymotrypsin;
A = acute; S = small; (-) = not examined; N = normal; C = chronic; L = large; Ap = aphasia; ACTH = corticotropin.
in the younger group, and 2.21 k 1.56 pg/ml in the
older group (Fig 6). There was no correlation between
ACT level and aging. The mean level of ACT was 4.98
t 4.35 pg/ml in the total AD group, 3.12 k 2.18
pg/ml in the early-onset AD group, and 5.92 k 4.88
pg/ml in the late-onset AD group (Fig 6). No significant differences in CSF ACT levels were observed between the early-onset AD and the older normal control
group. There were no significant differences in levels
of ACT between FAST stages, MMS, and duration of
illness in any groups of AD. In other neurological disease patients, high levels of ACT were found in the
CSF of those with chronic cerebral infarction (Patients
5 and 8), herpes simplex encephalitis (Patients 12 and
13), neuro-Behcet disease (Patient 14), meningitis carcinomatosa (Patient 15), and amyloid angiopathy (Patient 32).
Relationship Between A p, sBAPP, and ACT
There was no correlation between CSF AP and ACT in
the normal control group and those in any AD group.
No correlation was found between sPAPP and ACT in
the normal control group or in the total AD group.
Discussion
pAPP contains the AP sequence near its membranespanning domain and is proteolytically processed to
many fragments including sPAPP and AP [8, 11-19].
Nakamura e t al: CSF A P Is Increased in Early-Onset AD
907
sRAPP
Integrated O.D.
AB/s BA PP
I
11
0
5
0
l 90 I
4
0
a-
2
1
7-
0
- &:
0
O
0
6-
0
'8:
i i'
0
00
!:
9800
00
0
0
O:O
0
IAD
Alzheimer's
disease
yN
50
4-
0
0
fl
0
eAD
.
0
0
3
0
ON
Normal
control
Fig 4. Levels of secreted form of amyloid p protein precursor
isPAPP) in normal control and Alzhetmer/r disease i A D )
groups. eAD = early-onset AD; IAD = late-onset AD; yN =
younger normal control group; ON = older normal control
group. White circles show yN and eAD, and black dots show
ON and A D .
Since antibodies raised against AP generally cannot distinguish A P from these proteolytic fragments of
PAPP, we investigated AP in CSF by western blotting
using two different antibodies to avoid overestimation
of its level [l5]. Previously, we found no obvious correlation between A D and the level of AP in CSF because of considerable interindividual variations { 151. In
the present report, we have demonstrated changes in
levels of A P in CSF under both normal and pathological conditions using samples from a larger population
of carefully diagnosed subjects, and we observed that
A@ levels in CSF were elevated in early-onset AD.
Since it was difficult to obtain CSF samples from 80and 70-year-old subjects who were free of disease and
had completely normal intelligence, we could not examine enough CSF samples from such persons as age-
908 Annals of Neurology Vol 36 No 6 December 1994
3-
8
'1
O8
1
00"
80
.
..
:
-
8
0
L
0.
0
eAD IAD
yN ON
Alzheimer's
disease
Normal
control
Fig 5 . Ratio of amyloid /3 protein to secreted form of amyloid p
protezn precursor ( APIS/3APP) in Alzheimer's disease ( A D )
and normal control groups. eAD = early-onset A D ; ZAD =
late-onset A D ; y N = younger normal control group; ON =
older normal control group. White circles show yN and eAD,
and black dots show ONand A D .
matched controls for the late-onset A D group. However, the amount of A P in CSF decreased with normal
aging, suggesting that the level of AP in CSF from the
late-onset A D group may be elevated relative to that
from normal persons of more advanced age. Further
study concerning the CSF AP levels in normal subjects
in their 80s and 90s is necessary to clarify whether the
CSF A @is actually increased in late-onset AD.
The severity of dementia (MMS) and the level of
disability (FAST) did not correlate with the level of
AP in either the total or early-onset A D groups. These
ACT
PI II
25
20
15
10
0
a
5
0
8
P
0
eAD IAD
yN ON
Alzheimer's
disease
control
Normal
Fig 6. Levels of al-antichymotrypsin ( A C T ) in cerebrospinal
Juid in Alzheimer's disease ( A D ) and normal control groups.
eAD = early-onset AD; LAD = late-onset AD; yN = younger
normal control group; ON = older normal control group. White
circles show yN and eAD, and black dots show ON and LAD.
findings are in agreement with those of a previous
pathological study by Hyman and colleagues {3 11,
which showed no correlation between plaque numbers
and duration of illness. This suggests that AP may increase continuously in early-onset AD.
The diagnostic value of sPAPP in CSF has already
been studied. However, no definite conclusions have
yet been reached C32-401. We measured the relative
optical densities of both 105-kd and 125-kd bands to
compare the activity of the A P-generating pathway to
that of the secretory pathway of PAPP. In contrast to
AP, sPAPP levels showed no clear differences with
normal aging in any AD group or in the normal control
subjects in the present study. However, AP/sPAPP
ratio decreased with normal aging and was elevated in
the early-onset AD group. This finding suggests that
the AP-generating pathway is predominant in younger
persons and is activated in AD. This hypothesis that
high levels of AP may be produced in AD corresponds
to the nucleation/seeding theory of AP proposed by
Jarrett and associates [41), who showed that continuous production of large amounts of AP is necessary to
accelerate amyloid formation and deposition. Another
possibility is that amyloid formation depends on local
AP generation and clearance. The level of CSF AP
may represent the generatiodclearance equilibrium of
AP. Although some proteins capable of binding to AP
{42, 431,and AP clearance from the blood to the brain
parenchyma, i.e., the blood-brain barrier {44), have
recently been reported, it was not shown how AP is
cleared from the extracellular spaces in the brain in
contrast to mechanism of production of A@.Thus, the
level of CSF A@is thought to represent activity of AP
generation in the brain.
Although the massive accumulation of AP as amyloid fibers in the AD brain was established histologically {45-481, quantitative studies reached no consensus on the levels of PAPP, their proteolytic fragments,
or AP in AD 148-52). Recent reports have shown that
a mutant PAPP identified in a family with hereditary
A D exhibits altered processing in a way that elevates
the release of AP, and this increased level of AP was
suggested to be one of the causes of A D 122, 231.
These previous reports suggested that continuous increases in AP level from birth to the onset of clinical
manifestations may be necessary for the development
of AD. Although the increase in level of AP in CSF
of AD patients may not be directly related to that in
the brain, our present findings suggest that the processing of PAPP may be altered, the AP-generating
pathway may be predominant, and the level of AP may
be increased in the brain of AD patients.
We designed the present study to determine
whether an assay of the level of AP in CSF is of diagnostic value for AD. However, the diagnostic value of
this assay was limited because of overlaps in the levels
of AP in the CSF of AD patients, normal control subjects, and those with other neurological diseases. Elevated levels of AP and sPAPP were observed in some
acute neurological disease and neurodegenerative disease patients. However, correction of the level of AP
by the level of sPAPP (AP/spAPP) showed the highest AP/sPAPP ratios to occur mainly in diseases resulting from acute cerebral damage. Transient activation of the AP-generating pathway may occur with
acute cerebral damage. This finding also corresponds
to the observation that acute cerebral damage causes
rapid alterations in pAPP synthesis in some animal
Nakamura et al: CSF AD Is Increased in Early-Onset AD
909
model systems [ 5 31. Thus, careful clinical assessment
is necessary for the use of CSF AP level as a diagnostic
marker of AD.
Previously, we reported the level of ACT in CSF as
a possible biochemical marker for AD [251. However,
some observed overlap in ACT levels in CSF between
AD and normal control subjects reduced somewhat its
diagnostic value. It is important to exclude patients
suffering from systemic inflammatory diseases and malignancy, and in the present study, we also found that
patients with acute diseases of the nervous system
showed elevated levels of ACT in their CSF. Moreover, we found that the level of CSF ACT is not increased in early-onset AD in contrast to AP. This finding suggests the possibility that CSF ACT level may be
related to a limited subgroup of late-onset AD patients.
9. PasternackJM, Palmert MR, Usiak M, et al. Alzheimer’s disease
10.
11.
2.
3.
4.
Note Added During Review
During the review of this manuscript, Slunt and colleagues reported that 22C11 cross-reacts with sAPLP2
Biol Chem 1994;267:2637-2664). This finding suggests that the levels of sPAPP might be influenced to
some extent by those of sAPLP2.
15.
Supported by the Univers Foundation and the Primary Amyloidosis
Research Committee of the Ministry of Health and Welfare ofJapan.
8.
We thank K. S. K m and H. M. Wisniewski for 4G8, M. Wakayama,
M. Morita, T. Yoshida, T. Arai, H. Yamaguchi, and K. Okamoto
for providing CSF samples, T. Kawarabayashi, M. Ikeda, and T.
Iizuka for useful discussion, and T. Tashiro for Fast Scan analysis.
9.
20.
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