N . Sasaki et al. Brain Research 888 2001 256 –262
257
human brain [3,11], while its expression by cortical glucose-derived AGE collagen [14,15]. This antibody does
neurons increases and becomes more widespread in AD not recognize unmodified RNase, albumin, hemoglobin,
[27]. Since it was reported that RAGE may be the nerve LDL, acetyl LDL, or collagen, as well as previously
cell receptor for amyloid b protein Ab, the role of RAGE reported AGE structures such as 2-furoyl-4 [5]-[2-furanyl]-
in the pathogenesis of AD has attracted considerable 1-H-imidazole
FFI, 1-alkyl-2-formyl-3,4-diglycosyl-
attention [6,11,28]. pyrroles AFGP, pyrraline, pentosidine, or CML [14,15].
We have previous studied the distribution of AGE in AD An anti-Ab antibody 1–42, C-terminal was purchased
and several neurodegenerative diseases, and have sug- from Bachem Feinchemikaline AG Swiss, and a mouse
gested that AGE may be an important factor in the anti-glial fibrillary acidi protein GFAP antibody was
progression of various neurodegenerative disorders [20]. purchased from Dakopatts Denmark.
However, little is known about the detailed role of RAGE A polyclonal antibody against RAGE were raised in
in AD [28]. rabbits. The peptides were synthesized according to the
To investigate the role of RAGE in AD and DM, we amino acid sequences of RAGE, residues 167–180 [6].
established an anti-RAGE antibody and performed im- Synthesized peptide was coupled to keyhole limpet
munohistochemical studies. Our data demonstrated that hemocyanin, and mixed with an equal volume of Freud’s
RAGE was present in astrocytes from AD brains along complete adjuvant. The conjugated peptide were injected
with AGE and Ab, suggesting that RAGE-mediated degra- into rabbits 33 at 2-week intervals. Serum was obtained 2
dation of Ab occurs in astrocytes. weeks after last injection, and antibody titer was assessed
by ELISA. When antibody levels plateaued, the serum was collected and stored at 2808C until required.
2. Materials and methods
2.1. Subjects and specimens 2.3. Tissue preparation and Western blot analysis
Brain tissue
specimens were
obtained from
five Western blot analysis was performed on samples of
pathologically verified cases of AD, three cases of DM, bovine lung tissue and human brains obtained at post-
and three age-matched controls. Histological sections were mortem from subjects with no history of any neurological
prepared from the cerebral cortex temporal and parietal or psychiatric disorders. Bovine lung powder Sigma, 3 g
lobes and the hippocampus. None of AD patients or was incubated with Tris 20 mM, NaCl 0.1 M, PMSF 1
controls had diabetes. The clinical features of the subjects mM, trasylol 0.1, and octyl-b-glucoside 1, pH 7.4,
are summarized in Table 1. total 30 ml for 16 h at 48C with constant mixing.
Insoluble material
was removed
by centrifugation
2.2. Antibodies 11,0003g for 30 min at 48C, and the supernatant was
saved lung extract. Human brain cortex was homogen- A rabbit anti-AGE-modified ribonuclease antibody was
ized in buffer containing 20 mM HEPES, 0.25 M sucrose, used, which has been described previously [14]. This
0.3 mM phenylmethlsulfonyl fluoride PMSF, 1 mM antibody detects AGE formed in vivo, such as AGE-
dithiothreitol DTT, 1 mM EGTA, and 1 mM MgCl .
2
collagen and AGE-hemoglobin, as well as AGE formed in Samples containing 50 mg lung extract or 10 mg
vivo, such as glucose-derived AGE RNase, glucose-de- human brain of protein were loaded onto 10 acrylamide
rived AGE albumin, glucose-derived AGE LDL, and gels. Proteins were separated by sodium dodecyl sulfate
SDS–polyacrylamide gel electrophoresis PAGE and electrophoretically transferred to nitrocellulose filters, as
Table 1
a
described by Towbin et al. [24]. After transfer, the filters
Characteristics of subjects
were blocked by incubation with 5 nonfat dry milk in
Case Diagnosis
Sex Age at
Durations of disease
PBS-T buffer phosphate-buffered saline-tween; 140 mM
autopsy years
NaCl, 27 mM KCl, 81 mM NaHPO , 15 mM KH PO ,
4 2
4
1 AD
F 64
7
0.1 Tween 20, pH 7.4 overnight at room temperature.
2 AD
M 48
5
Then the filters were incubated for 1 h with PBS-T
3 AD
F 68
8 4
AD M
45 5
containing 0.1 nonfat dry milk and anti-RAGE antibody
5 AD
F 52
6
at 1:2500 dilution. Next, the filters were washed three
6 DM
F 57
times in PBS-T and incubated with PBS-T containing
7 DM
F 68
0.1 nonfat dry milk and horseradish peroxidase-linked
8 DM
F 75
anti-rabbit Ig [Fab9 ] Amersham-Life Science diluted
9 Control
F 73
2
10 Control
M 66
1:5000 for 1 h at room temperature. Filters were washed
11 Control
F 61
three more times in PBS-T, and immunoreactivity was
a
AD: Alzheimer’s disease. DM: Diabetes mellitus.
detected using an enhanced chemiluminescence Western
258 N
blot detection system Amersham, followed by exposure to ECL HYPER film Amersham.
2.4. Immunohistochemical staining Serial paraffin sections were immunostained according
to the standard streptavidin–biotin peroxidase technique using a Vectastain ABC elite kit Vector Lab., CA. All
sections were treated with 99 formic acid for 3 min, and the sections used for AGE staining were also treated with
0.05 proteinase-K for 30 min. Endogenous peroxidase was inhibited with 0.3 hydrogen peroxidase H O in
Fig. 1. Characterization of the anti-RAGE antibody by Western blotting
2 2
of human brain and bovine lung extracts. Lane 1, anti-RAGE antibody
methanol for 30 min. These sections were also incubated
and immobilized human brain 10 mg lane. Lane 4, anti-RAGE antibody
with 10 horse serum for GFAP, or 10 goat serum
and immobilized bovine lung extract 50 mg lane. Bands are observed at
for Ab, AGE, and RAGE to eliminate nonspecific
approximately 35 kd, 50 kd, and 60 kd in lanes 1 and 4. Lane 2 human
binding. This was followed by incubation overnight at 48C
brain and lane 5 bovine lung show that the immunoreaction was
with the primary antibodies diluted to 1:500|1:1000 in 10
diminished after absorption by the synthesized RAGE peptide. Lanes 3 and 6 show non-immune rabbit IgG and immobilized human brain or
mM PBS pH 7.4. The sections were then sequentially
bovine lung extract, respectively, with no bands being observed. Migra-
incubated with the biotinylated secondary antibody for 1 h,
tion of the protein standards is indicated to the left of lane 1.
with streptavidin–biotin-horseradish peroxidase for 1 h, and with 3,39-diaminobenzidine H O
until the reaction
2 2
products were visualized 1–3 min. Then the sections Western blotting and immunohistochemistry in the same
were counterstained with hematoxylin. Specificity was procedure as described above.
confirmed by applying PBS instead of the primary anti- bodies, or by replacing the primary antibodies with non-
2.6. Semiquantification of RAGE-positive astrocytes immune serum.
The combinations of antibodies used for double staining The number of astrocytes reacting with anti-Ab, AGE or
are summarized in Table 2. We used the anti-GFAP RAGE antibodies was counted in both serial tissue sections
antibody as a marker of reactive astrocytes. It was diluted and double stained sections. In each subject, immuno-
extensively 1:2000–4000, so that observation of im- reactive astrocytes were counted in the hippocampus using
munopositive granules was not prevented.
2
three photomicrographs 1.3 mm each obtained at 3100 magnification.
2.5. Absorption test Specificity of the antibody was verified by absorption
3. Results
