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Short Communication |
From the Center for Neurologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, abundant Aß-negative AMY plaques were described immunohistochemically in AD brain.15 A monoclonal antibody (MAb) used in that study, AMY 117, was raised against an as-yet-unidentified 100-kd protein present in paired-helical-filaments-tau-rich AD brain extracts. An accompanying commentary to this report raised the possibility that AMY plaques are a non-amyloid precursor to Aß-bearing senile plaques.16 In collaboration with the authors of the original report, we sought to determine the temporal sequence of deposition of the AMY 117 antigen relative to that of Aß. The immunoreactivity (IR) of the AMY 117 MAb was compared with that of each of three Aß antibodies in brains obtained from three temporal models of AD pathogenesis: DS patients (aged 12 to 73 years), monkeys (aged 17 to 34 years), and PD-APP transgenic mice (aged 8 to 20 months). The immunohistochemical protocols were first optimized for each antibody in AD brain sections for each of the fixation and embedding conditions used in these three temporal models of AD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Autopsied brains from 22 AD patients (aged 64 to 96 years; mean, 83 years) and 10 aged human controls (aged 60 to 87 years; mean, 78 years) were used to optimize immunostaining protocols for each tissue preparation and to characterize the spatial patterns of immunoreactivity (IR) of antibodies to Aß or AMY. A series of 29 brains from clinically diagnosed DS patients (aged 12 to 73 years; mean, 38 years), the neuropathology of most of which has been previously described,5 was examined to determine the temporal sequence of deposition of Aß and AMY relative to each other. Within this series, brain tissues from 10 young DS cases (aged 12 to 29 years) were generously provided by Dr. K. Wisniewski (Institute for Basic Research in Developmental Disabilities, Staten Island, NY). In addition, brain sections from three young DS patients lacking AD pathology were kindly provided by Dr. D. Anthony (Department of Pathology, Harvard Medical School, Boston, MA). Brain tissues from the remaining 16 DS patients (aged 36 to 73 years) were collected at autopsy by us at Brigham and Women's Hospital. Brain tissues from two animal models of AD pathogenesis were also examined: eight monkeys (aged 17 to 34 years) and 30 PD-APP transgenic mice17 aged 8 to 20 months (kindly provided by Athena Neurosciences, South San Francisco, CA).
Tissue Preparation
Blocks of human and monkey brain tissues from cerebral cortex, hippocampus, and cerebellum were fixed in 10% neutral buffered formalin for three time intervals ranging from 1) 1 to 2 hours (AD, aged human controls, older DS (>29 years), and monkey brains; brief fixation) to 2) an unknown period longer than 1 week (duplicate blocks from AD, older DS, and monkey brains; routine fixation) to 3) several years (12- to 29-year-old DS brains; long-term fixation). For several AD, DS, and aged control brains, additional blocks were fixed in 70% ethanol in 125 mmol/L NaCl at 4°C for 2 days. PD-APP transgenic mice were saline perfused and their hemibrains immersion fixed in 70% ethanol in 125 mmol/L NaCl at 4°C for 1 to 2 days. Fixed brain tissue was dehydrated and embedded in paraffin. Eight-micron sections were baked at 58°C for 1 hour. In addition, fresh-frozen 6-µm cryostat sections were prepared for several subjects in each group (except the monkeys). Frozen sections were fixed with cold acetone before immunolabeling.
Immunohistochemistry
Serial sections were immunostained with the avidin-biotin horseradish peroxidase/diaminobenzidine (DAB) method (rabbit or mouse ABC Elite kit, Vector Laboratories, Burlingame, CA), using the antibodies detailed below. Details of the immunostaining protocol have been previously described.5 Double labeling was accomplished using the horseradish peroxidase/DAB kit to detect the first primary antibody and the alkaline phosphatase ABC kit with Vector Red substrate (Vector Laboratories) to detect the second primary antibody. All antibodies were tested on long-term, routinely and briefly fixed AD and DS paraffin sections as well as on frozen sections to determine optimal staining conditions. Our sensitive general Aß antibody R1282 (1:1000),18 which detects multiple Aß forms, was used as a reference antibody for Aß plaque distribution. A highly sensitive Aß42-endspecific MAb, 21F12 (1:1000),19 was used to detect early, diffuse plaques as well as more mature plaques (gift of Athena Neurosciences, South San Francisco, CA). In long-term and routinely formalin-fixed tissues, both Aß antibodies required formic acid pretreatment (88% formic acid for 8 minutes at room temperature (RT)) to optimize visualization of Aß deposits. AMY 117 hybridoma supernatant MAb (AMY 117; gift of Lee and Trojanowski Laboratories, The Center for Neurodegenerative Research, University of Pennsylvania School of Medicine, Philadelphia, PA)15 was used neat on all tissues. AMY 117 ascites MAb (AMY 117asc; 1:5000; gift of Lee and Trojanowski Laboratories) was used in absorption experiments with Aß1–40 and Aß1–42 peptides (20 µg of Aß peptide/1 µl of antibody) as well as to confirm AMY 117 staining. In long-term and routinely formalin-fixed tissues, the AMY 117 staining required a double pretreatment: antigen retrieval by microwaving the sections for 10 minutes in citrate buffer solution (BioGenex, San Ramon, CA) followed by proteinase K digestion (Dako Corp., Carpinteria, CA) for 6 minutes at RT. No pretreatment was required for AMY 117 immunostaining of briefly formalin-fixed or ethanol-fixed paraffin sections or on cryosections. The HistoMouse-SP kit (Zymed Laboratories, South San Francisco, CA), an immunohistochemistry kit designed to use MAbs on mouse sections, was used in conjunction with MAb AMY 117 on the PD-APP transgenic mouse brain sections so as to avoid cross-reactivity with endogenous mouse IgG. Selected sections from all subject groups were stained for amyloid deposits with thioflavin S.
Microscopy
For light microscopy, photomicrographs were generated using an
Olympus BX50 microscope. For the confocal image shown in Figure 1
,
kindly provided by Dr. M. L. Schmidt (The Center for
Neurodegenerative Diseases, University of Pennsylvania School of
Medicine, Philadelphia, PA), a polyclonal antibody raised against
native Aß purified from AD brain and made in our laboratory, Angela
(1:250),20
was used for double-immunofluorescent labeling
of Aß and AMY 117 on a 40-µm ethanol-fixed, cryoprotected AD brain
section. Here, Aß IR was visualized using a Texas-Red-conjugated
donkey anti-rabbit secondary antibody (1:400; Jackson ImmunoResearch
Laboratories, West Grove, PA) whereas AMY 117 IR was visualized using a
fluorescein-isothiocyanate-conjugated donkey anti-mouse secondary
antibody (1:100; Jackson ImmunoResearch Laboratories). A series of
six confocal images were obtained through the section at 1-µm
intervals using a Leica confocal laser scanning microscope, as
previously described.15
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In both single-labeled adjacent sections and in double-labeled
sections, the vast majority of AMY 117 immunoreactive plaques were
co-localized to some degree with Aß immunoreactivity (IR) detected by
antibodies R1282 (a general Aß polyclonal antibody) and 21F12
(Aß42-specific MAb) in both AD and older (
29 years) DS brains. AMY
117 IR was always restricted to those cortical and hippocampal regions
that contained Aß deposits. Many, but not all, AMY IR plaques
overlapped with thioflavin-S-labeled amyloid plaques. As exemplified in
Figure 1
, AMY 117 IR frequently
co-localized (eg, overlapped) with Aß IR (Figure 1, a–c)
, was
interspersed with it (Figure 1c)
, or, in the more pathologically severe
brains, surrounded it (Figure 1, d–f)
within an individual plaque
lesion. In the latter case, the two antigens were found to partially
overlap or to segregate but abut each other. Subpial Aß deposits,
large diffuse Aß42 IR bands, cerebellar Aß deposits, and vessel
wall Aß were all devoid of any AMY 117 IR (see asterisks in Figure 1, d–f
). Occasionally, small punctate AMY 117 immunoreactive deposits
that did not appear to overlap with Aß IR were observed; such
deposits occurred only in brain regions bearing abundant Aß IR
plaques (see arrowheads in Figure 1, d and e
). However, the presence of
both antigens could often be detected in these same lesions in sections
just above or below the plane of the initially stained section (see
arrowhead in Figure 1f
). Absorption of antibodies R1282 (Aß) and AMY
117asc with synthetic Aß1–40 and Aß1–42 peptides
caused ablation of plaque staining by R1282 (absorption with Aß1–40
peptide shown in Figure 2, a and b
) but
did not diminish AMY 117 IR (Figure 2, c and d)
. Figure 2
further
illustrates the close co-occurrence of Aß and AMY 117 within plaques,
at both high and low magnification, in the brain of a 65-year-old DS
patient. Regions of compacted Aß42 IR plaques in hippocampus,
parahippocampal gyrus, and temporal cortex were also AMY 117 IR, as
shown at low magnification in Figure 2
(eg, small arrowheads in e and
f). However, AMY 117 IR was absent in Aß42 IR diffuse plaques in the
parahippocampal gyrus (arrows in Figure 2, e and f
), in Aß42 IR
plaques in the subpial layers of temporal cortex (asterisks in Figure 2, e and f
), and in Aß42 IR plaques in deep cortical layers and white
matter (large arrowheads in Figure 2, e and f
) as judged in immediately
adjacent sections. In AD and DS brains in general, Aß IR, especially
as detected with the Aß42 MAb 21F12, was more abundant than AMY 117
IR.
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Of the 10 nondemented aged control brains examined, 2 had no Aß or AMY 117 IR. In the remaining eight cases, varying amounts of Aß deposition were detected in the cortex in each brain. Only three of the eight Aß-bearing brains had any AMY 117 IR; Aß IR was always much more abundant than that of AMY 117. In general, AMY 117 IR was associated with spherical, compacted plaques and plaques apparently undergoing compaction, but not with diffuse, thioflavin-negative plaques in these aged control brains (data not shown). As in AD brains, no vessel wall AMY 117 IR was detected, even when Aß IR was present in the blood vessel.
Down Syndrome Brains
To determine the relative temporal sequence of deposition of Aß
and the AMY 117 antigen, we immunolabeled adjacent sections of DS
brains from patients ranging from 12 to 73 years old using the highly
sensitive Aß42 MAb 21F12 and the AMY 117 MAb. Frontal cortex sections
from 13 young DS patients (aged 12 to 29 years) were examined; in
addition, large sections containing both temporal cortex and
hippocampus were available for three of these young DS cases and were
immunostained with both antibodies. Because the young DS brains had
been subjected to long-term fixation in formalin, various pretreatments
were tested and then employed to allow visualization of the AMY 117
antigen. A combination pretreatment involving antigen retrieval by
microwaving the section in a citrate buffer solution followed by a
brief proteinase K digestion allowed the unmasking of the AMY 117
antigen in the long-term fixed tissues. Aß42 plaque IR was observed
in 7 of the 13 young DS brains (aged 12, 15, 16, 17, 21, 27, and 29
years) (exemplified in Figure 3, a, c, e, and g
). Quantitative analyses of Aß deposition and other
neuropathological characterization of these brains has been previously
reported.5
AMY 117 IR was detected in three of the seven
young DS brains that had Aß deposits (and in none of those that did
not). Specifically, the frontal and temporal cortices and hippocampus
of a 15-year-old DS patient having thioflavin-positive amyloid plaques
and shown previously to have compacted and cored Aß IR plaques,
gliosis, and some neuritic changes had some AMY 117 IR plaques (not
shown); the hippocampus of a 16-year-old DS patient that showed
compacted Aß IR plaques had some AMY 117 IR (Figure 3, e and f)
,
whereas the frontal and temporal cortices having only diffuse Aß42 IR
plaques did not (Figure 3, c and d)
; and the frontal cortex of a
29-year-old DS patient (Figure 3, g and h)
previously shown to have
compacted and cored Aß IR plaques, gliosis, and some neuritic changes
had many plaques positive for both Aß and AMY 117. In the brains of
the four young DS patients (aged 12, 17, 21, and 27 years) that had
almost exclusively diffuse Aß42 IR plaques, AMY 117 IR was not
detected (eg, Figure 3, a and b
). Brains from middle-aged and older DS
patients showed AMY 117 IR very similar to that described above for AD,
as demonstrated in the brain of a 65-year-old DS patient in Figure 2
.
As in AD cases, no vascular or cerebellar AMY 117 IR was detected in DS
brains at any age, even though abundant Aß deposition occurred in
each structure in these older DS brains.
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To further characterize the temporal accrual of AMY 117 in Aß plaque lesions, two animal models of AD pathogenesis were examined. Cortical sections bearing Aß (R1282 and 21F12) immunoreactive plaques from the brains of eight monkeys ranging in age from 17 to 34 years were immunostained with the AMY 117 MAb. In addition, hemibrain sections from 30 Aß (R1282) immunoreactive plaque-bearing PD-APP transgenic mice, aged 8 to 20 months, were examined for AMY 117 IR (using the HistoMouse kit (Zymed Laboratories, South San Francisco, CA) to avoid mouse IgG cross-reactivity). No AMY 117 IR was detected in any of these monkey or transgenic mouse brains, regardless of Aß plaque deposition (data not shown). Even in the 18- and 20-month-old PD-APP transgenic mice that have very abundant compacted and cored Aß IR and thioflavin-positive plaques, AMY 117 IR was not observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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indicate
that AMY 117 and Aß can both co-mingle and specifically co-localize
(eg, overlap) within an individual plaque. In a subset of more mature
plaques, AMY 117 IR surrounds that of Aß; in such cases, the two
antigens may partially overlap or may segregate but abut each other.
AMY 117 IR was not diminished by absorption of the antibody with Aß
peptide, suggesting that AMY 117 is a non-Aß antigen. In general, the
Aß antigen was more abundant than the AMY 117 antigen in AD, aged
human, and DS brains. The discrepancy between our results and those reported earlier,15 in which limited or sometimes no co-localization was described between AMY 117 and Aß IR in AD brain sections, is probably due, in part, to differences in staining conditions. First, the pretreatments used in our study enhanced the Aß IR, sometimes even in the ethanol-fixed sections, implying that some Aß deposits may have been missed in the study by Schmidt et al.15 Second, the Aß42 MAb 21F12 and the Aß polyclonal antibody R1282 are extremely sensitive at detecting multiple forms of Aß deposits and, as such, allowed greatly increased detection of Aß deposits relative to those detected by the polyclonal Aß antibody 2332 used in the previous study.15 Indeed, we have performed side-by-side comparisons of the three antibodies under the optimal staining conditions for each and have consistently found Aß42 MAb 21F12 to detect the greatest number of Aß deposits (C. A. Lemere and T. J. Grenfell, unpublished data). Our Aß polyclonal antibody R1282 also detected more Aß deposits than polyclonal antibody 2332 under optimal conditions but, in some cases, labeled fewer Aß deposits relative to those stained by Aß42 MAb 21F12. Third, in our study, we always single-labeled sections adjacent to the double-labeled sections so as to characterize the staining pattern for each antibody on its own to avoid the potential for steric competition between the antibodies for their respective antigens within lesions. Both in our hands, and recently in those of M. L. Schmidt (personal communication), a competition between Aß and AMY 117 antibodies for their respective antigens in individual plaque lesions was observed and may explain why some AMY-positive plaques appeared to be Aß negative in their study.
In full agreement with the earlier report, no AMY 117 IR was observed
in Aß-bearing blood vessels or in cerebellum, implying that there is
a regional and cellular specificity for this protein to associate with
Aß. In our study, AMY 117 IR occurred only in those brain areas
having Aß immunoreactive plaques and never in regions devoid of Aß.
In contrast, low-magnification photomicrographs previously published by
Schmidt and colleagues15
(as shown in Figure 3
of their
paper) illustrate an example of an AD case in which AMY 117 IR was
detected in a region of parahippocampal gyrus devoid of Aß using
polyclonal antibody 2332. Upon request, the authors kindly provided us
with several adjacent sections from the same block of tissue depicted
in the figure. In our hands, the region previously described as being
devoid of Aß was Aß42 plaque-rich using a different Aß antibody
(Aß42 MAb 21F12) and pretreatment of the tissue with formic acid.
Pretreatment of tissue and improved Aß antibody sensitivity may
account for the increased detection of Aß deposits and, as such, the
visualization of a much greater co-occurrence of Aß with AMY 117 in
the current study. Our conclusion is that regions of AMY IR are
Aß-rich. Careful inspection of Table 1 in the aforementioned paper
confirms that in AD brain regions where both antigens were examined
(positive staining listed as present (Y) or absent (N)), Aß IR alone
or Aß and AMY 117 IR together were described, but not AMY 117 IR
alone.15
In the current study, small, punctate AMY 117
immunoreactive deposits that were not directly associated with Aß IR
were occasionally observed, but only in areas of abundant Aß
immunoreactive plaques. In general, these small, punctate AMY 117
deposits were located between and close to plaques and, as such, may
represent the outer edge of a plaque that exists above or below the
plane of the AMY-117-stained section, so that Aß may be surrounded by
the AMY 117 antigen (as we indeed observed in mature plaques (Figure 1, d–f
, arrows)) or the two antigens partially overlap (exemplified by
Figure 1, d–f
, arrowheads).
By performing immunohistochemical studies of plaque development in a
unique temporal series of 29 DS brains from patients between the ages
of 12 and 73 years, we were able to determine that Aß deposition
clearly precedes that of the AMY 117 antigen. Early, diffuse Aß42
plaques in young DS brains were AMY 117 negative; it was only after the
appearance of more mature, compacted Aß plaques that AMY 117 IR was
observed. This point was best exemplified by the immunostaining of
adjacent sections from a single block of brain tissue containing both
temporal cortex and hippocampus from a 16-year-old DS patient (see
Figure 3, c–f
). Diffuse Aß42 IR was detected throughout the temporal
cortex, whereas more compacted Aß42 IR plaques were seen in the
hippocampus in the same section. In the adjacent section, AMY 117 IR
was observed only in the more compacted plaques in the hippocampus; the
temporal cortex in the same section was entirely AMY 117 negative. We
cannot exclude the possibility that the AMY 117 antigen within diffuse
plaques (but not compacted plaques) was destroyed by the harsh
(long-term) fixation conditions or by the double pretreatment to expose
antigens in the young DS brains. However, the lack of AMY 117 IR in the
long-term-fixed young DS brains bearing exclusively diffuse Aß42 IR
is consistent with the lack of AMY 117 IR in diffuse Aß42 IR deposits
observed in briefly fixed tissues from middle-age and older DS patients
(see Figure 2, e and f
) and in AD cases. Furthermore, the staining
protocol for each antibody under each of the fixation conditions was
optimized before use. Therefore, we believe it is very unlikely that
technical factors could explain the lack of AMY 117 IR in the
aforementioned young DS brains.
The pattern of AMY 117 IR seen in the young DS brains was also observed in briefly fixed aged human control brains in which only three of eight Aß-bearing brains showed any AMY 117 IR, and then only in a small portion of all Aß plaques. Again, it was the Aß plaques that appeared to be in the process of compaction that showed AMY 117 IR. Neither AMY 117 nor Aß IR were observed in two aged human control brains and in six young DS brains, lending further support to the conclusion that the AMY 117 antigen is not detectable in brain lesions before the appearance of Aß.
Brains from two animal models of AD pathogenesis, aged monkey (17 to 24 years) and PD-APP transgenic mice (aged 8 to 20 months), were examined for Aß and AMY 117 IR. Aß IR deposits were observed in all of the monkey and mouse brains, and the number of Aß deposits increased strikingly with age. AMY 117 IR was not detected in either species, regardless of Aß plaque burden. The lack of AMY 117 IR in monkey and PD-APP transgenic mouse brain suggests two possibilities. First, a species difference in the AMY 117 antigen may make it unrecognizable by the human MAb. Second, insufficient maturation of the plaques in monkeys and transgenic mice, compared with that in AD brain, may be responsible for the lack of AMY 117 detection in the plaques of these animals.
In summary, we conclude that the AMY 117 antigen is a non-Aß amyloid-associated protein that accrues in AD plaques after Aß deposition, rather than existing as the subunit of a novel, Aß-negative lesion in Alzheimer's disease. Because AMY 117 appears at the time of compaction of Aß plaques, it may turn out to play a significant role in the evolution of the plaque. This new information is critical to the interpretation of AMY IR plaques in AD brain and to the further search for the AMY antigen.
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Acknowledgements |
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and to Drs. Schmidt and John Trojanowski for many
interesting and helpful discussions. In addition, we are grateful to
Drs. Krystyna Wisniewski (Institute for Basic Research in Developmental
Disabilities, Staten Island, NY) and Douglas Anthony (Department of
Pathology, Harvard Medical School, Boston, MA) for generously providing
brain tissues from young DS patients. Lastly, we thank Dr. Dora Games
and Karen Khan for kindly providing fixed PD-APP transgenic mouse
brains. |
Footnotes |
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Supported by NIH grant (AG06173).
Accepted for publication March 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease. Cell 1988, 52:487-501[Medline]
1-antichymotrypsin and apolipoprotein E promote the assembly of the Alzheimer ß-protein into filaments. Nature 1994, 372:92-94[Medline]
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