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An abnormality of plasma amyloid protein precursor in Alzheimer's disease.

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An Abnormahty of Plasma Amyloid
Protein Precursor in Alzheimer's Disease
Ashley I. Bush, FRANZCP,"? Scott Whyte, FRACP," Linda D. Thomas, BSc(Hons),"
Timothy G. Williamson, BSc(Hons)," Cees J. Van Tiggelen, MD,X Jon Currie, FRACP," David H . Small, PhD,"
Robert D. Moir, BSc(Hons),* Qiao-Xin Li, PhD," Baden Rumble, PhD," Ursula Monning, PhD,S
Konrad Beyreuther, PhDJ and Colin L. Masters, MD"
PA4 amyloid deposition in the brain, which is characteristic of Alzheimer's disease (AD), may result from either
overexpression of the amyloid protein precursor (APP) or failure of APP to be correctly processed. A blood marker
reflecting this abnormal metabolism would be of diagnostic value and would provide a means of monitoring the
efficacy of therapeutic interventions. We analyzed immunoblots of plasma APP enriched by heparin-Sepharosechromatography from patients with moderate to severe AD dementia (n = 34) and control subjects (n = 77) and found an
approximately 50% increase in the proportion of 130-kd APP species in patients with AD (p < 0.001), no difference
in the 110-kd form, a 15 to 30% decrease in the 65-kd form (p < 0.001), and a 20 to 35% decrease in the proportion
of 42-kd APP (p < 0.001). These species of APP were soluble, lacked the carboxyl terminus, and the 110- and 42-kd
species were shown to be consistent with degradation products derived from the 130-kd species. A comparison of levels
of 130-kd plasma APP from moderately to severely demented patients with AD and control subjects distinguished the
two groups with a specificity of 87.0% and a sensitivity of 79.4%.
Bush AI, Whyte S, Thomas LD, Williamson TG, Van Tiggelen CJ, Currie J, Small DH, Moir RD, Li Q-X,
Rumble B, Monning U, Beyreuther K, Masters CL. An abnormality of plasma amyloid
protein precursor in Alzheimer's disease. Ann Neurol 1992;32:57-65
Alzheimer's disease (AD) is a dementia characterized
by the deposition of PA4, a 4-kd protein that accumulates by polymerization into amyloid fibrils in the brain
11, 2). PA4 is released by the abnormal processing of
amyloid precursor protein (APP). The prototype of
APP is the form with 695 amino acids (APP&, which
has the structural domains of an integral transmembrane protein 131. Alternatively, spliced amyloidogenic
forms include APP751,with a Kunitz protease inhibitory (KPI) domain 14, 51; APP,14, with an OX-2 domain insert 161; and APP,,,, with both the KPI domain
and an OX-2 domain 171.
At least three mechanisms could lead to the generation of PA4. One possibility is that aberrant cleavage
of APP may be caused by a defect in the constitutive
proteolysis known to occur within the PA4 domain 18,
91. Although candidate enzymes for APP processing
have been described 110, 111, an association between
an APP-cleaving protease activity and clinical AD has
yet to be shown. Conversely, an increase in substrate
may overwhelm the constitutive pathway and provide
APP for an amyloidogenic pathway. Such a mechanism
is likely in Down syndrome (DS), in which an extra
copy of chromosome 21 is associated with increased
expression of APP and premature deposition of PA4
112). A third mechanism could involve a modification
to the APP molecule itself, which then results in an
alteration of the constitutive proteolysis. Such a mechanism is possible in the closely related disorder of Dutch
congophilic angiopathy, which is associated with a mutation within the PA4 domain of APP 113, 141, and in
forms of famhal AD in which there are mutations
close to the carboxyl terminus of the PA4 peptide
115- 191.
Circulating forms of APP are concentrated in platelets 120-221, whereas lower amounts occur in plasma
112, 20, 23) and resting T lymphocytes 1241. We report evidence illustrating that the levels and processing
of plasma APP may be altered in AD. Furthermore,
we show that the profile of plasma APP in AD can be
From the *Department of Pathology, the University of Melbourne,
the Neuropathology Laboratory, Mental Health Research Institute
Of victoria, and the tDepamnent Of
Hospital, Parkvdle, Victoria, Australia; and the $Center for Molecu.
lar Biology, University of Heidelberg, Heidelberg, Germany.
Received Sep 10, 1991, and in revised form Jan 2, 1992. Accepted
for publication Jan 2, 1992.
Address correspondence to Dr Masters, Depmment of pathology,
Universiryof Melbourne, Parhde, 3052, Australia,
Copyright 0 1992 by the American Neurological Association 57
changed to approach that of control profiles by the
action of a serine protease activity that copurifies with
APP during heparin-Sepharose chromatography.
tion. The protein A-Sepharose was pelleted by centrifugation
(10,000 g; 3 min), and antiserum was then added to the
separated supernatant.
Materials and Methods
Western Blotting
Patient Selection
Patients with AD met National Institute of Neurological Disease and Stroke/Altheimer’s Disease and Related Disorders
Association (NINDUADRDA) clinical criteria [25], had
Mini-mental state examination {26} scores of less than 17,
and were excluded if they had clinical findings consistent with
peripheral vascular disease or mixed vascular dementia and
AD. Patients with familial AD were excluded from this study,
as were patients with AD in whom clinical onset was before
the age of 55 years. Age-matched control subjects each underwent a Mini-mental state examination and were excluded
if they scored less than 29. The different neurological diagnoses used to determine control subjects with nondemented
neurological disease (n = 6 ) were epilepsy, multiple sclerosis, hydrocephalus, and three with cerebrovascular disease;
this group was included as a small initial survey for the specificity of the changes seen in the A D group and as a preliminary to a more exhaustive survey of acute and chronic neurological and non-neurological disease that is beyond the scope
of this initial report. Ail volunteers were ambulatory, in stable health, and not suffering any acute illness at the time of
the study. Subjects were excluded from the control group if
they had a family history of any heritable dementing disorder.
Western blotting procedures were as described by Bush and
associates [20), modified by transferring for 5 hours at 0.6 A
and blocking with 3% (wh) bovine serum albumin (Fraction V; Sigma, St Louis, MO) in Tris-buffered saline (2 hours,
room temperature (RT]). The efficiency of protein transfer
was estimated by blotting onto two stacked nitrocellulose
filters (0.2 pm pore diameter) and staining the gels for untransferred protein with Coomassie blue following the blotting step. This process indicated that no immunoreactive
APP passed through the first filter under these conditions,
but that the efficiency of protein transfer was approximately
90% below 46 kd and gradually decreased to approximately
50% with increasing molecular weight between 46 and 130
kd. Blots were probed with mouse monoclonal antibody
(mAb) 2 2 C l l (Boehringer-Mannheim, Munich, Germany),
which recognizes an epitope on the amino terminus of APP
1281; it was diluted to 170 ng/mL in blocking buffer. Plasma
samples in this series of experiments were separated on 8.5%
polyacrylamide gels. Using these conditions, the 110-kd APP
immunoreactive band previously reported {20] resolved into
a doublet. For the purpose of our analysis, however, we regarded the sum of the signals generated by the doublet as
belonging to the one 110-kd region.
Partial Purification of A P P from Plasma
Refectance Analysis of Blots
Blood (20-40 mL) was drawn from fasting individuals with
a 2 1-gauge needle into heparinized collection tubes and centrifuged at 1,500 g for 15 minutes. The plasma fraction was
separated from the blood cell pellet and centrifuged at
19,000 g for 25 minutes at 4°C to remove any debris. Processing and assay of all plasma specimens were performed
with the operator unaware of the diagnostic category of the
sample.
To detect APP by Western blotting, APP was partially
purified from plasma by heparin-Sepharose chromatography.
Plasma (2.5 mL) was loaded onto a 0.25-mL. bed volume
heparin-Sepharose (Pharmacia, Uppsala, Sweden) column (8
x 5 mm) pre-equilibrated with buffer 1 (175 mmollL NaCl,
50 mmoVL Tris-HC1 [pH, 7 . 4 3 at 4°C. The column was then
washed with 3.25 mL buffer 1, and the APP was eluted with
750 pL elution buffer (550 mmol/L NaC1, 50 mmoVL TrisHCl [pH, 7.41). Protein concentration was determined with a
bicinchoninic acid protein assay using bovine serum albumin
standards (271.
Reflectance analysis was used to quantitate amounts of APP
on Western blots. Reflectance was assayed by video-capture
with a Videk Megaplus camera (Kodak, Canandaigua, NY)
operated by PixelTools vl. 1 (Perceptics, Knoxville, TN).
Quantitation was then performed with Image v1.29 software
(W. Rasband, National Institutes of Health Research Services Branch, National Institute of Mental Health, Bethesda,
MD), which facilitated precise alignments of the individual
blot lanes with the reflectance profile and the setting of exclusion limits of individual peaks in the four regions of interest
at 130, 110, 65, and 42 kd. The integrated reflectance (area
under the curve) was thus computed for each of the four
peaks in every sample. The values obtained were linear with
concentration for each region over a range of heparin-sepharose eluate loads (20-90 pg protein).
To compare the relative amounts of the four APP derivatives, the relative proportion of band immunoreactivity to
total lane immunoreactivity was determined in each plasma
sample and then averaged to give the values presented in the
Table and in Figure 1. Independent samples Student’s t-tests
between pooled control and A D groups were performed for
the four immunoreactive bands at a significance level of p =
0.0125 (0.05 divided by the number of comparisons 2291).
Where these comparisons were significantly different, the
test of simple effects [29] followed by Scheffk [30] post hoc
comparisons between all pairs of diagnostic groups (i.e., patients with AD, other neurological disease control subjects,
normal younger adult control subjects, and nondemented,
age-matched control subjects) were then performed.
Immunoprecipitation
Heparin-Sepharose eiuates (650 pg protein) were diluted
with 50 mmol/L Tris-HC1 (pH 7.4) to reduce the concentration of NaCl to 175 mmol/L and were immunoprecipitated
with rabbit polyclonal antisera (5 pL) according to the
method described by Bush and associates {20}, modified by
preincubating the samples with 10 mg protein A-Sepharose
(Pharmacia) for 1 hour at 4°C to reduce nonspecific absorp-
58 Annals of Neurology
Vol 32 No 1 July 1992
Ratios of Plasma APP F o m Analyzed by Image Capture in A D and Control Subjects"
Percentage of Total APP
Mean Age +- SD
AD (n = 34)
Pooled control
subjects (n = 77)
Neurological control
subjects (n = 6 )
Age-matched control
subjects (n = 46)
Younger adult control
subjects (n = 25)
67.9
61.5
* 8.9
* 16.6
64.0
2
6.8
* 10.2
41.5 * 10.9
70.6
130 kd
110 kd
36.5 5 10.6
23.9 +- 6.9b
24.7
23.6
25.0
&
8.3'
24.0 +- 7.2'
23.4
* 6.3'
S
SD
65 kd
* 6.6
* 5.3 (NS)
22.7 * 5.8
25.5 * 5.8
20.2 * 3.6
42 kd
21.5
26.8
5
7.1
17.4 2 6.0
25.7 5 7.2b
31.0
5
3.2'
21.3
25.0
* 5.8 (NS)
30.0
L
* 6.2b
6.6'
* 10.6 (NS
25.4 * 6.4'
27.4
-C
7.5'
"Heparin-Sepharose eluates of AD and control plasma samples were analyzed by Western blotting with mAb 22C11, and the intensities of th
bands at 130, 110, 65, and 42 kd were measured by computer-assisted image capture analysis. The relative amounts of the four APP band:
as percentages of total lane signal, were determined in each plasma sample and averaged to give the values presented. Independent sample
t-tests between pooled control and AD groups were performed in the four regions at a significance level of p = 0.0125. Comparisons wer
significant for three regions: 130,65, and 42 kd (two-taiied, p < 0.001). Comparisons between all pairs of diagnostic groups (patients with All
other neurological disease control subjects, normal younger adult control subjects, and non-demented, age-matched control subjects) were the
performed for the values obtained for the 130-, 65-, and 42-kd bands using a Scheffk test at a significance level of p < 0.05.
bt-test, p < 0.001.
'Significant by Scheffk test, p < 0.05.
APP
=
amyloid protein precursor; AD = Alzheimer's disease; NS = not significant; mAb
=
monoclonal antibody.
A
B
Fig 1. Analysis of amyloid protein precursor (APP) in control
and Alzheimer's disease (AD) plasma by Western blotting.
Heparin-Sepharose eluates from plasma (65 pg protein) were
analyzed by 8.5% sodium dodecyl sulfate polyacrylamih gel
electrophoresis and Western blotting with monoclonal antibody
22CI 1. The relative molecular mass of standard protein markers (Rainbow Stanhrds, Amersham, UK) are shown on the left.
APP immunoreactive bands of 130, 110, 65, and 42 kd are
indicated by arrows on the right. Only the relative abundances
of the 130- and 42-kd APP forms, as in the samples illustrated, could be used t o visibly discriminate between patients
with A D compared with both (A) nondemented el&& control
subjects and (B) n o m l younger control populations.
Bush et al: Abnormal Processing of APP in Alzheimer's Disease 59
h s a y of APP-degrading Protease
Aliquots (500 pL) of eluates from each heparin-Sepharose
column were desalted using a 1.7-mL Sephadex G25 (Pharmacia) column (8 x 34 mm) equilibrated with 175 mmoVL
NaCI, 50 mmoVL Tris-HC1, 1 mmol/L CaCl,, and 1 mmoV
L MgCl, (pH, 7.4), in the presence or absence of 20 pmoV
L ZnC1, at 4"C, and the protein concentration was adjusted
to 0.80 mg/mL with the same buffer. The samples were then
incubated at 37°C for 2 hours; an aliquot (80 G)was then
removed, and the protein in each aliquot was precipitated
with chloroformlmethanol(1:4 vlv), boiled in sodium dodecyl sulfate (SDS) sample buffer, and analyzed by Western
blotting using mAb 22C11.
The effects of inhibitors of various classes of proteases
were assayed by adding them to these incubation mixtures
and observing their influence on the degradation of 130-kd
APP. A sample (500 pL) of a heparin-Sepharose eluate from
the plasma of a normal younger adult control subject was
desalted into protease assay buffer (175 mmoYL NaCI, 50
mmoYL Tris-HC1, 1 mmolfl. CaCI,, 1 mmoYL MgCI,, 20
pmoVL ZnC1, [pH, 7.4)). Aliquots (containing 65 pg protein) were diluted to 0.80 mdmL with the same buffercontaining protease inhibitor, then incubated for 2 hours at
37°C. Protein was precipitated in each sample by the addition of chlorofordmethanol and analyzed by Western blotting. The final concentrations of inhibitors in the incubation
mixtures were ethylene diamine tetraacetic acid (EDTA)
(1 mmoYL), diisopropyl fluorophosphate (DFP) (1 mmoYL),
aprotinin (10 pg/mL), N-ethylmaleimide (NEM) (1 mmoYL),
pepstatin A (10 pg/mL), a1-antichymotrypsin (0.4 mghL),
and soybean trypsin inhibitor (SBTI) (1 mg/mL). The effects
of AI3Cl(2Opmol/L) and heparin (20 UImL)on APP proteolysis were also analyzed.
Plasma Zinc Assay
Zn2+ assays were performed by atomic absorption spectrophotometry according to the method of Davies and colleagues E317.
Results
Multiple Molecular Weight F o m s of APP in Plasma
Are Recognized by mAb 22C11 on Western Blots
To examine forms of APP in plasma, blood was collected from younger adult control subjects and plasma
was prepared. The APP in plasma was then partially
purified by heparin-Sepharose chromatography and the
bound protein analyzed by Western blotting. MAb
22C 11 identified four major immunoreactive bands of
APP (130, 110, 65, and 42 kd) in Western blots of
human plasma 120). This antibody has been previously
used in the identification of APP from platelets 120).
The staining of bands by mAb 22C11 on Western blots
of heparin-Sepharose eluates could be abolished by
preincubating 3 mL. of diluted antibody with 6 pg purified human brain APP (full-length, possessing the intact carboxyl terminus) for 2 hours at RT.
The identity of the four major bands labeled by
mAb 2 2 C l l as different molecular weight forms of
60 Annals of Neurology Vol 32 No 1 July 1992
Fig 2. Analysis of immunoreactive amyloid protein precursor
(APP) in plasma by Western blot analysis. APP immunoreactiue proteins in heparin-sepbarose eluates fmm plasma were analyzed by 8.5% sodium dodeql sugate polyacrylamide gel electrophoresis and Western blotting with monoclonal antibody
22C1I. Lane I: heparin-sepharose eluate of plasma (65 pg
protein); lane 2: heparinSepharose eluate immunoprecipitated
by antiserum 9013 (raised against full-length human brain
APP); lane 3: heparin-Sepharose eluate immunoprecigitated by
the prebleed to antiserum 9013; lane 4: heparinSephurose eluate
immunoprecipitated by anti-Fd-APP (raised against APP fusion protein). The relative molecular masses of standard protein
markers (Rainbmu Standards, Amersham, UK) are shmun on
the left. APP immunoreactive bands previously reported (20) of
130, 110 (a doublet), 65, and 42 Rd, are indicated by arrows
on the right.
APP was also confirmed by cross-reactivity with antibodies raised against native human brain full-length
APP and against synthetic peptides representing APP
domains. The immunoprecipitated protein was analyzed by Western blotting with mAb 2 2 C l l . A polyclonal antiserum (90/3)raised against full-length APP
purified from human brain, and another polyclonal
(anti-Fd-APP) 128) raised against an APPGg5fusion protein, both immunoprecipitated 130-, 110-, and 42-kd
bands detected by mAb 2 2 C l l on Western blots. The
prebleed from the 90/3 rabbit antiserum did not immunoprecipitate these proteins (Fig 2). An approximately
50-kd broad band detected in Western blots of these
immunoprecipitates (Fig 2, lanes 2-4) was the immunoglobulin G ( 1 6 ) heavy chain from the rabbit sera
used for immunoprecipitation being weakly recognized
by the anti-IgG secondary antibody. Antibody 9013
and its prebleed were also used to directly probe Western blots of heparin-Sepharose eluate (65 pg protein).
Unlike its prebleed, 9013 ( I : 10,000 in blocking buffer,
4"C, overnight) detected the 65-kd band recognized
by mAb 22C 11 on Western blots of heparin-Sepharose
eluate (data not shown).
The forms of APP observed in plasma are unlikely
to be full-length APP, which is known to be released
into the blood by vesiculation of platelet-plasma membranes [20, 211, because the APP signal detected by
Western blotting was unaffected by ultracentrifugation
of plasma (100,000 g x 1 hr). Two rabbit polyclonal
antisera, one raised against a synthetic peptide consisting of the last 43 residues of APP6,, (anti-CT) 120)
and the other raised against residues 667 to 676 of
APPos (anti-CTII) 112, 201, did not immunoprecipitate any protein from plasma heparin-Sepharose eluates that could be detected by mAb 22C11 on Western
blotting, indicating that the APP species detected by
mAb 22Cl l on Western blots are unlikely to possess
an intact carboxyl terminus or intact PA4 domain.
The concentration of 130- and 110-kd APP species
in normal plasma was estimated by comparing reflectance levels of these species in Western blots of
heparin-Sepharose eluates with levels from a known
amount of the same molecular weight soluble APP
species purified from normal human brain. Assuming
100% extraction of these APP forms from plasma during chromatography, we estimate the concentration of
these species to be 31 to 65 picomolar (range, n = 4)
in whole plasma.
Abnormal Projile dPlasma APP in AD
We surveyed the relative abundance of APP-immunoreactive bands from A D and control plasma samples. The largest apparent changes were an increase in
the 130-kd APP band and a decrease in the 42-kd
plasma APP band in AD samples compared with samples from all control groups. The control groups consisted of nondemented, age-matched persons (see Fig
1A); normal younger adults (see Fig 1B); and patients
with other neurological disease. There was an apparent
decrease in the levels of the 65-kd band in patients
with AD compared with normal younger adult control
subjects, but not compared with nondemented, agematched individuals. There was no consistent difference in the levels of the 110-kd band between patients
with AD and control subjects. The total amount of
APP immunoreactivity did not differ between patients
with AD and control subjects; the ratio of the total
immunoreactivity of patients with AD to control subjects was 1.02 0.22 (mean ? standard deviation).
Quantitation of these findings by image capture analysis (see Table) showed that the 130-kd band was significantly (t145.831 = 6.34, p < 0.001) increased, and
that the 65- and 42-kd bands were significantly (t{109)
= - 3 . 9 7 , ~< 0.001; and tIlO91 = - 5.88,p < 0.001,
respectively) decreased in patients with AD compared
with pooled control subjects (averaged data from other
*
neurological disease control subjects, normal younger
adult control subjects, and nondemented, age-matched
control subjects). Because readings of the 130-kd APP
band gave heterogeneous variances according to an F
test, a separate variance estimate was undertaken,
which adjusted the degrees of freedom (df) to yield
fractional df for analysis. These findings were also supported by the results of a Mann-Whitney U test (a
nonparametric analogue of the t-test) {32), which
showed the differences in the 130- and 42-kd bands in
patients with AD compared with pooled control
groups to be significant at the p < 0.0001 level and
the difference in the 65-kd band to be significsant at
the p < 0.001 level.
In patients with AD, there was a 53% increase in
the proportion of the 130-kd form, a 20% decrease
in the 65-kd form, and a concomitant 32% decrease
in the 42-kd form. The immunoreactivity pattern was
more evenly distributed between the APP species in
the pooled control group. This trend was maintained
throughout the comparisons made of the patients with
AD with the three control subgroups, where analysis
of variance on simple effects indicated significant differences between patients with AD and the three control groups in the 130-kd (F13, 107) = 18.17, p <
O.OOl), 65-kd (FE3, 107) = 8.70, p < O.OOl), and
42-kd (Fr3, 107) = 13.09,p < 0.001) bands. Further
post hoc analysis confirmed the significance of the difference between the patients with AD and each control
group in the 130-kd region (Fig 3A) and between the
patients with AD and the younger adult and elderly
control groups in the 42-kd region (Fig 3B) according
to a Scheffk procedure conducted at the p < 0.05 level
of significance. The levels of the 65-kd band were
found to be significantly lowered in patients with AD
compared with younger adult and neurological disease
control groups, but not compared with age-matched
elderly control subjects, therefore limiting the clinical
usefulness of 65 kd plasma APP levels in discriminating patients with AD. There were no significant differences between the mean reflectance proportions of the
three control groups in either the 130- or the 42-kd
regions. The proportion of 110-kd APP was notably
constant between all diagnostic groups. Further statistical analysis is being reserved for studies of the clinical
predictive value of these observations.
h e r Molecakzr Weight Plasma APP Species
Can Be Generatedfrom the 230-kd Species
b~u Serine Protease
The existence of a different profile of lower molecular
weight forms of APP in AD plasma may be consistent
with a defect in the proteolysis of APP. To examine
whether such a defect in proteolytic activity is reflected
in plasma, we studied the ability of proteases copurifying with APP from heparin-Sepharose to cleave APP.
Bush et ak Abnormal Processing of APP in Alzheimer's Disease 61
t
.
i
.
i
i
!
I
I
0
Y-a
ddts
&xmtch?d
contrds
n-25
n-46
Neurdogical
contrds
n - 6
A0
n-34
A
Fag 3. Rejectance analysis of immunoblots comparing Alzheimer's disease (AD) and control plasma amyloid protein precursor
(APP). The distribution of plasma APP immunoreactivity was
analyzed & rejlectance as detailed in the Table. A significant
diffence between the A D and the normal age-matched control
groups was observed only in the Levels of 130- and 42-kd species
of APP. The test of significance was the Scheffe'procedure conducted at the p < 0.05 level. Solid lines indicate the means for
each group. Asterisks indicate a significant dgference between a
control group and the A D group means. (A)Proportions of
130-kd APP in the A D group and the individual control
groups. The mean proportion of 1.30-kdAPP species was significantly (approximately 50%) greater in the A D group compared with each control group. The dotted line indicates a suggested threshold to distinguish the A D group, with 87.0%
specifcity and 79.4% sensitivity within these sample groups.
Diagnostic pwer was 84.7%. (B) Proportions of 42-kd APP
concentrations in the A D group compared with the individual
control groups. The mean proportion of the 42-kd A P P species
was significantly h e r (range, approximately 20 to 35%) in
the A D group compared with the younger adult and the agematched control groups. The dotted line indicates a threshold
that distinguishes the A D group, with 80.5% specificity and
73.5% sensitivity within these sample groups. Diagnostic
power was 78.4%.
When plasma APP from heparin-Sepharose eluates
was incubated at 37°C for 18 hours, the APP was degraded slowly, with loss of the 130-kd band and accentuation of lower bands (110 and 42 kd). This finding
indicated that the lower molecular weight forms of
APP in plasma could be degradation products of the
130-kd form, and that proteolysis of the 130-kd form
in an AD preparation changes the plasma APP profile
toward that of control subjects. Proteolysis was accelerated in the presence of Zn*+;the reaction yielded the
same products within 2 hours (Fig 4). The Zn2+ concentration used to stimulate proteolysis was 20 pmoY
L, within the range of the normal human plasma con62 Annals of Neurology Vol 32 No 1 July 1992
Younger
adults
Agctnatckd
controis
Neurological
controls
AD
n-25
n-46
n-6
n-34
B
centration. There was no clear difference between patients with AD and control subjects in the ability of
Zn2+ to stimulate APP breakdown. Identical degradation profiles were found in both groups. Zn2+enhanced APP proteolysis of a younger adult control
preparation over 2 hours was completely inhibited by
EDTA, heparin, and the serine protease inhibitors
aprotinin, DFP, SBTI, and incompletely inhibited by
a,-antichymotrypsin. AI,Cl, the cysteine-protease inhibitor N-ethylmaleimide, and the acid-protease inhibitor pepstatin A did not influence the reaction (data not
shown).
To test the possibility that a zinc deficiency might
contribute to decreased APP proteolysis in AD, we
assayed total 2nz+ levels in plasma from fasting patients with AD (14.8 ? 2.8 pmoVL; n = 17) and found
no significant differences from levels in fasting healthy
elderly control subjects (14.8 & 2.8 pmoVL; n = 40).
To determine whether APP might be processed in
whole plasma, we incubated fresh plasma from a young
adult control subject at 37°C over a period of seven
days and assayed the plasma APP by heparin-Sepharose chromatography. No significant degradation of
the 130-kd APP form was observed over this period,
indicating that constitutive processing of APP does not
occur in plasma.
Discussion
This study shows that there is an increase in the 130-kd
form and a decrease in the 65- and 42-kd forms of
APP in the plasma of moderately to severely demented
patients with AD. These observations may form the
basis for a peripheral biochemical marker for AD. A
comparison of levels of 130-kd plasma APP between
these patients with AD and control subjects showed
that a threshold of 30% total APP immunoreactiviry
Fig 4. Idntification of an amyloid protein precursor (APP)degrading protease in plasma. APP purified by heparin-sepharose chromatography of plasma fmm patients with Alzheimer's
disease (AD) and control subjects was incubated at 3 7°C in
saline buffer in the presence or absence of Zd'. Samples (6s pg
protein)from each incubation were analyzed by electrophoresis on 8.5 % polyacrylamide gels and Western blotting with
monoclonal antibody 22C11. The relative molecular masses of
stanakrd protein markers (Rainbow Standzrds, Amersham,
UK)are shown on the kfi. APP immunoreactive bands of 130,
110, 65, and 42 Rd are indicated by arrows on the right. Illustrated are samples representative of 6 patients with A D and
G normal young adult control subjects.
could distinguish the AD group with a sensitivity of
79.4% and a specificity of 87.0%. A plasma APP profile of less than 20% total APP for the 42-kd species
could distinguish the AD group with a sensitivity of
73.5% and a specificity of 80.5%. The overlapping
values in the AD and control groups could be explained by incorrect clinical diagnosis of AD, cases of
subclinical AD being detected in control subjects, and
the possibility that subgroups of AD (e.g., early onset
AD) are not represented by the same biochemical
lesion detected in this study. The diagnostic power
of this test within these sample groups at the thresholds recommended is 84.7% for 130-kd plasma APP
and 78.4% for 42-kd APP. Prospective studies are
now in progress to determine the predictive value of
the plasma APP profile for clinical or pathological
outcome.
Our data are at variance with those of Podlisny and
colleagues [23}, who reported no qualitative or quantitative differences in a soluble APP band of similar molecular weight (125 kd) in AD plasma. Several reports
have now documented changes in cerebrospinal fluid
levels of APP in patients with AD, but a consensus
has yet to emerge on the direction of these changes
133-351. Palmert and associates 1333 describe alterations in the relative amounts of APP bands of 125,
105, and 25 kd, which could also be attributed to altered processing of APP. The possibility also remains
that the altered plasma APP profile seen in patients
with AD could reflect a change in APP heparin-binding
affinity. Further studies are currently being undertaken
to determine the validity of this hypothesis.
Our estimates of the concentration of 130- and 110kd plasma APP (approximately 50 picomolar) agree
with previous estimates of plasma KPI-containing APP
of similar molecular weight [36), suggesting, like others 1231, that KPI-containing APP may be the predominant form in plasma. Therefore, our observations are
consistent with an increased production of fully processed 130-kd KPI-containing APP in AD. Several
lines of evidence support the possibility that an overproduction of KPI-containing APP or an increase in
the ratio of KPI-containing APP to APP,, is associated with PA4 amyloidogenesis. In DS, both APP messenger RNA (mRNA) and protein levels are increased
and are associated with the invariable premature onset
of AD [5, 12). In sporadic AD, the proportion of
01-containing APP mRNA is increased 15, 37, 381.
Increased mRNA and expression of KPI-containing
APP released by lymphoblastoid cells in patients with
familial AD have also been shown to be accompanied
by aberrant intra-PA4 proteolysis [39]. Finally, it has
been shown that PA4 immunoreactive deposits develop in transgenic mice overexpressing APP,,I in the
brain 1401.
The major APP fragments in plasma could be derived from the 130-kd form of APP. The decrease in
the 42-kd APP species in A D plasma suggests that
inhibition of constitutive proteolysis of 130-kd APP
may occur in the disease condition. Abnormal APP
processing could result either from an alteration in
APP itself or from an alteration in the activity of a
protease that constitutively hydrolyzes APP. It is also
possible that an increase in KPI-containing APP inhibits its own constitutive catabolism, resulting in a reduction in the amount of the 42-kd APP fragment appearing in AD plasma. The precise mechanism and
location of this altered processing, as well as the site
of production of plasma APP, remain to be elucidated.
The physiological relevance of the proteolytic mechanism copurifying with APP on heparin-Sepharose
chromatography is s t d to be determined. Its identity
may yield clues to the role of APP in blood and the
Bush et al: Abnormal Processing of APP in Alzheimer's Disease 63
nature of APP processing in general. To the best of
our knowledge, no mammalian Zn2+-stimulated serine
protease has been previously described; however, the
possibility that this activity may represent more than
one enzyme or a Zn2+-stimulated cofactor must also
be considered. The difference in plasma APP levels in
AD could not be attributed to a change in total plasma
zinc concentration, because total Zn2+levels in plasma
from fasting patients with AD were not significantly
different from levels in fasting elderly control subjects.
Although plasma APP was seen to be degraded by a
protease when heparin-Sepharose eluates were incubated at 37”C, our data indicate that this mechanism is
not active in whole plasma.
This study was supported by funds from the National Health and
Medical Research Council, the Victorian Health Promotion Foundation, and the Aluminium Development Council. Prof Beyreuther is
supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium fiir Forschung und Technologie.
We thank Dr Michael Berndt, Department of Medicine, Westmead
Hospital, Westmead, New South Wales, for his helpful discussions.
We also thank Drs A. Wooton, D. Deam, and S. Ratnaike, Department of Clinical Biochemistry, Royal Melbourne Hospital for plasma
Zn2+analysis; Dr Kurt Naujoks, Boehringer-Mannheim, for providing the mAb 22C11; Mr Dean McKenzie, Department of Psychological Medicine, Monash Universiry, for statistical advice; Mr Allyn
Radford for assistance with image capture analysis and photography;
Ms Valcy Malone for coordination of volunteers; and Mr Stuart
Portbury for technical assistance.
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