Alterations in Alzheimer's Disease\p=m-\AssociatedProtein
in Alzheimer's Disease Frontal and Temporal Cortex
Garth Bissette,
PhD; Wayne
H. Smith; Kenneth C. Dole; Barbara Crain, MD, PhD;
Barney Miller, PhD; Charles B. Nemeroff, MD, PhD
\s=b\ Alzheimer's disease
(AD)\p=m-\associatedprotein is present in
brain and cerebrospinal fluid of patients with AD but not in
adult, nondemented, normal controls. This protein may
represent an abnormal epitope of the "tau" microtubuleassociated protein and has been detected before the appearance of senile plaques and neurofibrillary tangles. The
amount of AD\p=m-\associatedprotein in the frontal and temporal cortices in 93 cases of neuropathologically confirmed
AD was compared with the amount that was present in 20
cases without AD. The amount of AD\p=m-\associatedprotein
was significantly increased in the cases of AD for both brain
regions compared with that in the cases without AD. The
presence of high levels of this protein is a useful adjunct,
postmortem marker of the presence of AD and may eventually lead to tests that allow early detection of individuals
at risk for this disease.
(Arch Gen Psychiatry. 1991;48:1009-1012)
Wolozin and colleagues1 reported the presence
In of1986,
novel antigen in brain with Alzheimer's disease
that
monoclonal
detected
a
(AD)
(ALZ-50)
by raising a
antibody
homogenates of ventral forebrain from
four patients with AD. Partial purification and Western
blot techniques showed that this antigen was a single
protein with a molecular weight of 68 000 d, and it was
subsequently named A68.2 The A68 protein was recently
reported to be one of three ALZ-50 immunoreactive ele¬
ments of a larger protein termed Alzheimer's diseaseassociated protein (ADAP),3 with a total aggregate weight of
200000 d as measured by size-exclusion column elution.
The presence of ALZ-50 immunoreactivity in brain tis¬
sue from normal human infants aged younger than
2 years,4 its subsequent disappearance in normal adults,
and its apparent reexpression in AD have generated con¬
siderable interest in this protein as the basis of a potential
was
to the
Accepted
for publication February 14, 1991.
From the Departments of Psychiatry (Drs Bissette and Nemeroff
and Messrs Smith and Dole), Pharmacology (Dr Nemeroff), and
Pathology (Dr Crain), and the Joseph and Kathleen Bryan Alzheimer's Disease Research Center (Drs Crain and Bissette), Duke University Medical Center, Durham, NC, and the Abbott Diagnostics
Divisions, Abbott Laboratories, Abbott Park, III (Drs Ghanbari and
Miller).
Reprint requests
to Duke
University Medical Center,
3859, Durham, NC 27710 (Dr Bissette).
PO Box
Hossein
Ghanbari, PhD;
diagnostic marker for AD. While there have been several
confirmatory reports of ALZ-50 immunoreactivity in
AD,5,6 neurofibrillary tangles in non-AD dementia and
Pick bodies also react with the ALZ-50 antibody.7,8 In ad¬
dition, microtubule-associated "tau" protein may be rec¬
ognized by ALZ-50,9,10 even though tauprotein is different
from the A68 protein in that tau-associated ALZ-50
immunoreactivity is not destroyed by trypsin. Thus, a
more specific means of detecting ADAP was necessary
before such measurements could be developed into a
useful clinical test. For this purpose, we have constructed
two-antibody sandwich, enzyme-linked immunoassay
was designed to avoid cross-reactivity
with such nonspecific epitopes as tau protein. This goal
has been successfully reached with the development of
the ALZ-EIA, which was used in this study, and it has
been reported in detail elsewhere.11 In the present study,
we evaluated the potential diagnostic utility of this labo¬
ratory test by measuring ADAP immunoreactivity in
temporal and frontal cortical punch samples from post¬
mortem brains with and without neuropathological diag¬
a
(ALZ-EIA) that
noses
of AD.
SUBJECTS AND METHODS
Brain tissue was obtained from the Kathleen Price Bryan Brain
Bank of the Joseph and Kathleen Bryan Alzheimer's Disease Re¬
search Center at Duke Medical Center, Durham, NC. Tissues
that were sampled represented 93 patients with a confirmed
neuropathological diagnosis of AD1213 and 20 patients without
AD. The patients in the group with AD included 11 cases with
additional diagnoses (six with Parkinson's disease, four with
diffuse Lewy body disease, and one with progressive supranuclear palsy). Diagnoses were reviewed by a single neuropathol¬
ogist (B.C.) who provided counts of senile plaques and neu¬
rofibrillary tangles on representative silver-stained14 sections of
solution-fixed temporal and frontal cortex.
Counts were made on the most severely affected area in each
section. The control group without AD included four neurologically normal, nondemented controls, three nondemented pa¬
tients with neurological disease (one each with amyotrophic lat¬
eral sclerosis, idiopathic Parkinson's disease, and Huntington's
disease), and 13 patients with non-AD dementias. The neuro¬
pathological diagnoses that were assigned to these demented
patients without AD included two with Binswanger's disease,
two with unclassified neurodegenerative disease, two with Par¬
kinson's disease, two with Pick's disease, and one each with
chronic subdural hematoma, chronic meningoencephalitis,
formaldehyde
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Mean ± SEM
No. of
Patients
Group
Nondemented
Normal
Neurological disease
Demented
Without Alzheimer's disease
With Alzheimer's disease
Sex
4
3
3
2
13
93
F, 1 M
F, 1 M
F, 9 M
50 F, 43 M
4
multi-infarct dementia, anoxie encephalopathy, and no discern¬
ible anatomical cause for dementia.
Micropunch (2-mm diameter) plugs of the middle temporal
gyrus (middle of Brodmann's area 21) and the superior frontal
gyrus (middle of Brodmann's area 11) that contained from 15 to
20 mg of brain protein were taken from the frozen brain hemi¬
sphere of each case and placed in 1.5-mL plastic microcentrifuge
tubes. All samples were coded, and personnel were blind to the
diagnosis of individual samples. Tissues were homogenized
with a motor-driven Teflon pestle in 4 µ of buffer (0.1 mmol of
TRIS, 150 mL of sodium chloride, and 0.5% ethylene glycol bis, , ', '-tetra acetic acid [EGTA], gentamicin sulfate, and so¬
dium alkyl paraben [Nipaset, Nipa Labs Ine, Wilmington, Del;
pH 7.5]) per milligram of wet tissue weight. This homogenate was
centrifuged in a fluorescence polarization immunoassay (TDx,
Abbott Laboratories, Abbott Park, 111) centrifuge for 1 minute,
and duplicate samples that contained 50-µ aliquote of the su¬
pernatant were placed in the assay wells. As described in detail
elsewhere,11 the ALZ-EIA uses a rabbit poly clonai antiserum to
ADAP (a complex with three ALZ-50 immunoreactive subunits,
one of which is the A68 protein) that was developed by Abbott
Laboratories. The IgG fraction of this poly clonai antiserum is
bound to polystyrene beads and binds to and separates effec¬
tively the ADAP epitope from the other nonspecific, supernatant
ALZ-50 immunoreactive species. This bead-antigen complex is
next reacted with ALZ-50 antibody, followed by incubation with
a horseradish peroxidase-conjugated goat anti-mouse IgM. Af¬
ter incubation with an o-phenylenediamine hydrochloric acid
solution, the resulting yellow liquor is read at a 492-nm wave¬
length with the use of an (Quantum) analyzer (Abbott Labo¬
ratories) on 60-well plastic plates. This photometric assay
measures the amount of ALZ-50 bound to the bead. Absorbance is calculated as units per milligram of protein. Protein
was measured by a modification of the Folin-phenol method
of Lowry et al.15
The ADAP-positive standards were manufactured from two di¬
lutions of brain extract that were obtained from Abbott Laborato¬
ries and one dilution of brain extract that was obtained from the
Duke University Medical Center; these extracts were derived from
tissues that previously were determined to have high levels of
ADAP. All samples were measured in duplicate, and infra-assay
and interassay variation was 9% and 15%, respectively. Data were
analyzed by analysis of variance and Student's f test. Correlations
among regional concentrations of ADAP, numbers of senile
plaques and neurofibrillary tangles, age, sex, postmortem delay,
freezer storage time, and length of disease were sought with the
Spearman Rank Correlation Coefficient test.
RESULTS
The group means and SEMs (mean ± SEM) for age, postmortem
delay time, and frozen tissue storage time are shown in the Table
for the group with AD (n 93), the demented controls without
AD (n 13), and the nondemented controls (n 7, including
three nondemented controls with neurologic disease and four
nondemented, neurologically normal controls). For the group
with AD, the average length of illness of 70 patients was
=
=
=
(Range)
Postmortem
Interval, h
Age, y
Freezer
Storage Time, mo
6.3 ±4.6 (0.8-20)
2.0 ±1.1 (1.0-3.2)
20.5 ±10.4 (4-49)
61.7 ±8.7 (52-79)
72.2 ± 3.4 (47-90)
6.5 ±2.5
(0.7-26.0)
7.1 ±1.0 (0.3-36)
23.6 ±4.2
66.2 ±5.5
(51-78)
77.0 ±1.0 (63-92)
42.0 ±3.1 (40-48)
(2-45)
24.5 ±1.5 (2-63)
111.3±9.5 months (mean±SEM), while for 12 of the demented
patients without AD, the average length of illness was 65.8± 14.5
months (mean±SEM). One of the neurologically normal, nonde¬
mented controls exhibited moderate plaque density in the frontal
and temporal cortices, as did three of the 13 demented controls
without AD. However, only one of these 13 demented patients
without AD had a single neurofibrillary tangle per 20 x field in
these cortical regions. In contrast, all 93 of the patients with AD
had demonstrable plaques, and only 21 did not exhibit neu¬
rofibrillary tangles in either the frontal or temporal cortex.
As illustrated in the Figure, the patients with AD exhibited
markedly higher ADAP concentrations in both the frontal and
temporal cortices (0.398+0.029 and 0.594±0.43 absorbance U/mg of
protein, respectively) compared with that in the group without AD
(0.175±0.028 and 0.281 ±0.054 absorbance U/mg of protein, respec¬
tively). The difference was significant to the P<.001 level for both
brain regions. None of the four neurologically normal, nonde¬
mented controls had ADAP concentrations above 0.200 absorbance
U/mg of protein in either the temporal or frontal cortex. However,
two of the three nondemented patients with neurological disease
(Huntington's disease and Parkinson's disease) had ADAP con¬
centrations well above 0.200 absorbance U/mg of protein in the
temporal but not the frontal cortex. Only two of the 13 demented
controls without AD had ADAP levels above 0.200 absorbance
U/mg of protein in the frontal cortex, while four of the 13 demented
patients without AD had ADAP concentrations greater than 0.200
absorbance U/mg of protein in the temporal cortex. In contrast, only
10 of the 93 patients with AD had ADAP concentrations below
0.200 absorbance U/mg of protein in both the frontal and temporal
cortices. The samples of patients without AD but with substantial
concentrations of ADAP were examined by Western blot analysis
and did indeed have authentic ADAP present, while all the sam¬
ples with AD but without ADAP, as measured by the ALZ-EIA,
were also negative by Western blot techniques (data not shown).
of freezer
Postmortem delay, length of disease, and
storage time were not strongly correlated to any of the variables
that were examined. The ADAP concentrations in the frontal and
temporal cortices were positively correlated (P<.001) for both
groups with and without AD. Temporal cortex concentrations of
ADAP were significantly correlated with senile plaque numbers
in both the temporal and frontal cortices of the patients with AD,
but the concentration of frontal cortex ADAP was not similarly
correlated. In the group with AD, ADAP concentrations in the
frontal cortex were negatively correlated (P<.001) with patient
age; temporal cortical ADAP was not. In addition, and not sur¬
prisingly, a significant positive correlation between numbers of
senile plaques in the frontal cortex and either numbers of tem¬
poral cortex plaques (P<.001) or numbers of frontal (P<.007) or
temporal (P<.04) cortex neurofibrillary tangles was present in
the group with AD.
iength
COMMENT
goal identifying definitive premortem or post¬
mortem diagnostic markers in AD has not yet been real¬
ized. Clinicians remain rather poor at diagnosing AD,16
The
of
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2.0
v*0
1.5
0
2 2
'5
o-
S-I
3
y
u
>
.
g
<-8
Q
<
O
J3
advanced Down's
< 0.5
syndrome,4,21
and the dense ALZ-50
immunoreactivity that is found in plaques and tangles of
AD tissue all mandate further investigation of the phys¬
iological and pathological roles of the ALZ-50 antigen.
n
0.0L
stained for ALZ-50 may eventually have developed tan¬
gles. Similarly, Mattiace et al21 have reported preliminary
findings of ALZ-50 immunoreactivity in Down's syn¬
drome that precedes development of the characteristic
changes of AD that are seen in patients with Down's syn¬
drome aged older than 30 years, such as neuritic plaque
amyloid and paired helical filament immunoreactivity.
The presence of the A68 protein in developing normal
human brain until about the age of 2 years,4 the reported
presence of ALZ-50 immunoreactivity in senescent mon¬
keys,22 the presence of ALZ-50 immunoreactivity in
Temporal
Cortex
Frontal
Cortex
Alzheimer's disease-associated protein (ADAP) concentration in
absorbance units per milligram of protein for temporal and frontal
cortex samples from 93 patients with AD (diamonds) and20 controls
without AD (open circles), including four nondemented, neuro¬
logically normal patients (X symbols), three nondemented patients
with neurological disease (N symbols), and 13 demented patients
without AD (O symbols). Bars represent the group mean with SEM
brackets. The concentration of ADAP in the group with AD was sig¬
nificantly (?<. 01) increased in both the frontal and temporal cortex
compared with the demented controls without AD alone, the non¬
demented controls alone, or both control groups together.
postmortem diagnosis still requires quantitative or
semiquantitative evaluation of neurofibrillary tangles and
senile plaques in stained tissue. This histological diagno¬
sis is time consuming and relatively subjective. Because
precise diagnoses, both premortem and postmortem, is a
necessary prerequisite for the elucidation of the etiology
and pathogenesis of AD, investigators have sought neuand
rochemical alterations in AD on which to base novel di¬
agnostic tests. The discovery of the A68 protein raises the
question of whether measurement of this protein will
provide the basis for a diagnostic test for AD in postmor¬
tem tissue or a premortem test in brain biopsy samples or
cerebrospinal fluid. The data presented here confirm
findings from previous reports17,18 of increased brain
ADAP concentrations in patients with a neuropathologically confirmed diagnosis of AD. The demented controls
without AD but with high ADAP concentrations were
found to have true ADAP immunoreactivity as shown by
Western blot analysis and may represent patients at risk
for AD who contracted another dementing disease that
led to death before classical neuropathological changes
associated with the diagnosis of AD could develop.
The regional distribution of ADAP within the brain of
patients with AD is similar to the regional distribution of
other neuropathological and neurochemical alterations
that are seen in AD. The affected brain regions with
increased levels of ADAP in AD include the hippocam¬
pus, subiculum, amygdala, temporal, frontal, and pari¬
etal cortices, and nucleus basalis of Meynert.17 However,
a recent
report19 of ALZ-50 immunoreactivity in the
somatosensory cortex in a neuropathologically confirmed
case of AD with only moderate cholinergic deficiency and
normal somatostatin immunoreactivity emphasized the
wide variability among individual cases of AD. Hyman et
al20 have reported ALZ-50 staining of both normalappearing and tangle-bearing neurons in AD, and they
have postulated that those neurons without tangles that
While much remains unknown, at present, the A68
to be one of three ALZ-50 immunoreac¬
tive subunits of a complex termed ADAP.3 The ALZ-50
antibody has also been shown to recognize a nonphosphatase-sensitive epitope of the microtubuleassociated protein known as tau.9-23 The A68 protein im¬
munoreactivity is distinct from tau-1, however, as the
ALZ-50 immunoreactive epitope on the A68 protein is
destroyed by trypsin and the ALZ-50 immunoreactive
component of tau is not.9 Tau protein has also recently
been reported to be decreased, not increased as ADAP is,
in AD.10 The ALZ-50 immunoreactivity has been shown
to be present in neurons of the neonatal rat and to
decrease with ensuing development.24 This rodent
ALZ-50 immunoreactivity codistributes with microtubule-associated protein 2. The ALZ-50 immunoreactiv¬
ity has also been reported to be present in neocortical
subplate neurons that are marked for naturally occurring
cell death.25 Recent evidence has shown that ALZ-50 im¬
munoreactivity is degraded by the cyclic adenosine
monophosphate-mediated action of ubiquitin.26
Until the abnormal epitope represented in ADAP can be
purified and sequenced and the gene cloned, the func¬
tional relationship between the presence of this antigen
and the dementia and neuropathological degeneration
associated with AD will likely remain obscure. The pres¬
ence of the ADAP antigen in an accessible physiological
compartment, such as cerebrospinal fluid, might allow
early identification of patients at risk for AD. Intensive
study of such individuals is required for development of
rational and novel pharmacotherapies for this common
dementing illness.
protein appears
This study was supported by grant MH-40524 from the National
Institute of Mental Health and by grant AG 05128 from the National
Institute on Aging, Bethesda, Md.
We are grateful to Sharon Rhoden for preparation of the manu¬
script.
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