288 Y
ubiquitin immunostaining. Therefore, immuno-histoch- pathological criteria for AD [23] as follows: none, sparse,
emistry using anti-a-synuclein antibodies is considered the moderate and frequent. We examined the areas in which
preferred method for detecting LBs and LNs rather than SPs existed in maximum density. NFTs were identified by
ubiquitin immuno-histochemistry. immuno-histochemistry using AT8. The distribution pat-
Recent studies indicated that in the amygdala of early- tern of NFTs and neuropil threads NTs was evaluated
onset familial AD patients [19], and Down’s syndrome based on Braak and Braak staging of neurofibrillary
brains with AD [20], numerous a-synuclein-positive LBs changes of AD [5], and was classified into six stages as
and neurites were noted. These observations led us to follows: transentorhinal stage, stages I–II; limbic stage,
determine the relationship between AD pathology and stages III–IV; and isocortical stage, stages V–VI.
a-synuclein-associated pathology. We examined a-synuclein immunoreactivity in brains of
2.4. Immno-histochemical analysis using anti-a-synuclein demented patients who were clinically diagnosed as having
antibodies sporadic AD. This study had the following aims: 1 to
investigate the relationship between AD pathology focus- We examined immunoreactivities of a-synuclein against
ing on the distribution and frequency of senile plaques two antibodies. One is a monoclonal antibody LB509,
SPs and NFTs and a-synuclein-positive structures; and raised against purified LBs specifically from DLB brains
2 to describe the morphology of a-synuclein-positive [4], and recognizes amino acids 115–122 [14], the other is
structures. a polyclonal antibody [17 [7], raised against a synthetic
peptide corresponding to amino acids 1–10 of human a-synuclein. For immuno-histochemistry using anti-a-
2. Materials and methods synuclein antibodies, 6-mm thick paraffin sections were
obtained from four selected regions of the brain of each 2.1. Cases
case: the hippocampus entorhinal cortex, amygdala, mid- brain, and cingulate gyrus. The sections were deparaffin-
In the present study, we examined 27 consecutive brains ized and pretreated with hydrolytic autoclaving in citrate
obtained at autopsy from demented patients, who were buffer for 15 min at 1218C to enhance the immuno-
clinically diagnosed as having probable AD based on the reactivity. Thereafter, they were incubated in 0.01 H O
2 2
NINCDS-ADRDA criteria [22] in the Hatsuishi Hospital. in methanol at room temperature for 30 min to block
Twenty-seven patients with probable AD were divided into endogenous peroxidase activity and pretreated to block
seven men and 20 women. The average age of death was nonspecific immunoreactivity prior to the addition of
82.0 years range 60–94 years. The average duration of LB509 dilution of 1:50 or [17 dilution of 1:1000 with
illness was 10.2 years range 2–22 years. The average normal rabbit serum, or normal goat serum respectively.
brain weight was 1022.2 g range 740–1240 g. This was followed by one-overnight incubation at 48C.
Staining was performed using the avidin–biotin complex 2.2. Tissue sampling and routine staining
technique with 3,39-diaminobenzidine DAB as the chromogen and followed by counterstaining with hemato-
All brains examined in the present study were obtained xylin. Sections of the midbrain of PD patients, in which
at autopsy within 24 h after death. The entire brain was typical brainstem-type LBs were detected on HE staining,
fixed in 10 formaldehyde for about 2 weeks and then cut were used as positive controls.
in coronal slices. For overall neuropathologic evaluation, All four sections were immunolabeled with anti-a-synu-
tissue blocks were obtained from selected regions: middle clein antibodies, and were then examined for the presence
frontal, inferior parietal and superior temporal neocortex, of a-synuclein-positive structures in each case. In cases in
hippocampus entorhinal cortex, basal ganglia, cerebellum, which a-synuclein-positive structures could be found in at
midbrain, pons, medulla oblongata. These tissue blocks least one region, five additional regions were selected.
were embedded in paraffin and 4-mm thick sections were These additional regions were the frontal, parietal, tempo-
¨ cut and stained with HE, Kluver-Barrera and Bodian
ral neocortex, pons and medulla oblongata, which were staining. Immuno-histochemical staining of 6-mm thick
recommended in the guidelines for brain sampling by the sections was performed using the anti-phosphorylated tau
consortium on DLB international workshop [21], and were monoclonal antibody mAb AT8 Innogenetics, dilution of
immunolabeled in the same manner. 1:3000, anti-human beta-amyloid mAb 6F 3D Dako,
dilution of 1:100 and rabbit anti-ubiquitin antibody 2.5. Quantitation of a-synuclein-positive structures
DAKO, dilution of 1:100. Initially, each section was scanned at a magnification of
2.3. Evaluation of neocortical SPs and NFTs 3100 to check whether or not there is any a-synuclein-
positive structures and identify the regions in which the Neocortical SPs were identified by Bodian staining, and
most abundant a-synuclein-positive structures were ob- their distribution was assessed based on CERAD neuro-
served. a-Synuclein-positive structures were divided into
Y . Arai et al. Brain Research 888 2001 287 –296
289
a-synuclein-positive intra-cytoplasmic inclusions and a- synuclein-positive neurites. To semiquantitatively evaluate
them, a score ranging from 0 to 3 was assigned according to the number of intra-cytoplasmic inclusions and the
density of neurites in an area of maximum density for each region. Intra-cytoplasmic inclusions were defined as a-
synuclein-positive structures including the nucleus in neurons in the plane of sections. Their number was
counted in three nonoverlapping fields at a magnification of 3200 for each section, and the final score was the
average of the three fields as follows: score 0, 0; score 1, 1–5; score 2, 6–10; score 3, .10. Similarly, the density
Fig. 1. Comparison of frequency and density of SPs between two groups.
of a-synuclein-positive neurites was scored as follows:
Neuritic plaques were identified in Bodian-stained tissue preparations according to CERAD criteria for AD. There were no significant differ-
score 0, none; score 1, a few neurites; score 2, some
ences between a-synuclein-positive and-negative groups. P; a-synuclein-
neurites; score 3, many neurites.
positive group, N; a-synuclein-negative group.
Additionally, a-synuclein-positive intra-cytoplasmic in- clusions were morphologically subdivided into two groups
according to whether or not they were of the LBs-type, as the positive group. Nine cases in the positive group had
which are revealed as spherical, ellipsoidal, angular and some a-synuclein-positive structures in all four regions,
reniform by staining. The regional rate of the a-synuclein- and the four cases in the positive groups had a-synuclein-
positive intra-cytoplasmic inclusions, which were impos- positive structures in only two or three regions. The
sible to identify as LBs morphologically at a magnification remaining 14 cases did not have a-synuclein-positive
of 3400 5non-LB type, was expressed as the percentage structures in all four regions, and were defined as the
relative to the total a-synuclein-positive intra-cytoplasmic negative group. We compared the positive group with the
inclusions. The quantitation was performed for all cases negative group with respect to age of onset, age of death,
and sections by the same examiner. duration of illness, and brain weight. There was a tendency
that cases of the positive group were slightly older at the 2.6. Confocal microscopy
time of death, and had shorter duration of illness, and lighter brain weight than those of the negative group. The
To assure that a-synuclein-positive intra-cytoplasmic two groups were not found to be significantly different by
inclusions were colocalized with NFTs, sections were the Mann–Whitney U-test.
double-labeled with the anti-a-synuclein [17 antibody and anti-tau AT8 antibody. Then, [17 and AT8 were
3.2. The frequency of SPs and NFTs in two groups fluorescence-labeled with Alexa 488 Anti-Rabbit IgG
H1L, detected as green, and with Alexa 594 Anti- The SP density determined based on CERAD neuro-
Mouse IgG H1L, detected as red, respectively. Sections pathological criteria for AD [23] did not differ significantly
were observed under a confocal microscopy system TCS- among the neocortical three regions between the two
SP, Leica, Heidelberg, Germany. groups. The average SP density was the highest in the
temporal cortex, followed by the parietal cortex and by the frontal cortex Fig. 1. The distribution pattern of NFTs
3. Results and NTs based on Braak and Braak staging [5] showed
