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From the Departments of Neuropsychiatry*
and
Microbiology,
Sapporo Medical University,
Sapporo, the Second Department of Medicine,
Hokkaido University, Sapporo, the Kansai College of Oriental
Medicine,§ Osaka, Japan, and the Department
of Neuropsychiatry,¶ Tokyo University,
Tokyo, Japan, and Laboratory of Central Nervous System
Studies,||
National Institute of Neurological Disorders
and Stroke, National Institutes of Health, Bethesda, Maryland
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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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Alzheimer's disease (AD) is the most common cause of dementia in Western countries and Japan. Pathologically, AD is characterized by the presence of neurofibrillary tangles (NFTs) and senile plaques, the major constituents of which are tau protein and amyloid ß protein (Aß), respectively. Aß is deposited in a variety of plaques and in the cerebral vessels as cerebral amyloid angiopathy (CAA). The deposition of Aß peptides is thought to be an early and causative event in the pathogenesis of AD and increases markedly during progression of the disease, leading in turn to the generation of NFTs and finally neuronal death.5
It has recently been demonstrated that AGEs can be identified immunohistochemically in both senile plaques and NFTs from AD.6 Glycation of tau, in addition to hyperphosphorylation, appears to enhance the formation of paired helical filaments,7,8 and glycation of Aß enhances its aggregation in vitro.9 Plaques and NFTs are not found only in AD brains but also in the brains of patients with other neurodegenerative disorders.5 However, little is known about whether or not AGEs are involved in the pathogenesis of other neurodegenerative diseases.
To investigate the role of AGE modification in AD and other neurodegenerative disorders, we performed immunohistochemical studies using antibodies for AGEs, Aß, tau, ubiquitin, and apolipoprotein E (ApoE). Our data demonstrated that AGE modification was involved in pathological changes observed in both AD and other neurodegenerative disorders, implying that AGEs may be an important factor in the progression of various neurodegenerative disorders.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Brain tissue specimens were obtained from five pathologically
verified cases of AD, three of PSP, three of Pick's disease, three of
Guamanian Parkinsonism-dementia complex (PDC), three of Guamanian
amyotrophic lateral sclerosis (ALS), two of Guamanian ALS/PDC, three of
diabetes mellitus (DM), and three age-matched controls. Histological
sections were prepared from the cerebral cortex (temporal lobe and
parietal lobe) and the hippocampus. Except for the three patients with
DM, none of the subjects had diabetes. The clinical features of the
subjects are summarized in Table 1
.
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The rabbit anti-AGE-modified ribonuclease antibody used was described previously.10 The antibody detected AGE formed in vivo, such as AGE-collagen and AGE-hemoglobin, as well as glucose-derived AGE RNAse, glucose-derived AGE albumin, glucose-derived AGE low-density lipoprotein (LDL), and glucose-derived AGE collagen.10,11 However, the antibody did not recognize unmodified RNAse, albumin, hemoglobin, LDL, acetyl LDL, or collagen as well as previously reported AGE structures such as 2-furoyl-4(5)-[2-furanyl]-1-H-imidazole (FFI), 1-alkyl-2-formyl-3,4-diglycosyl-pyrroles (AFGP), pyrraline, pentosidine, or CML.10,11
Monoclonal antibodies against tau and Aß were established in our laboratory and were designated as GP144 (mouse IgM) and Aß90/12 (mouse IgG), respectively. These antibodies have been described elsewhere in detail.12,13 Rabbit anti-ubiquitin antibody and a polyclonal goat anti-ApoE antibody were purchased from Dako (Glostrup, Denmark) and Chemicon International (Temecula, CA), respectively.
Immunohistochemical Staining
Serial paraffin sections were immunostained according to the standard streptavidin-biotin peroxidase technique using a Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA). Autoclaving for 30 minutes was performed before tau and AGE immunocytochemistry to retrieve antigenicity.14 All sections were treated with 90% formic acid for 5 minutes, and the sections used for AGE staining were also treated with 0.05% proteinase K for 60 minutes. Endogenous peroxidase was inhibited with 0.3% hydrogen peroxidase (H2O2) in methanol for 30 minutes. These sections were also incubated with 10% horse serum (for tau, Aß, and ubiquitin), 10% rabbit serum (for ApoE), or 10% goat serum (for AGEs) to eliminate nonspecific binding. This was followed by incubation overnight at 4°C with the primary antibodies diluted 1:500 to 1:1000 in 10 mmol/L phosphate-buffered saline (PBS; pH 7.4). The sections were then sequentially incubated with the biotinylated secondary antibody for 60 minutes, with streptavidin-biotin-horseradish peroxidase for 60 minutes, and with 3,3'-diaminobenzidine/H2O2 until the reaction products were visualized (1 to 3 minutes). Then the sections were counterstained with hematoxylin. Specificity was confirmed by 1) applying PBS instead of the primary antibodies or 2) by replacing the primary antibodies with preimmune serum. As an additional control for anti-AGE immunoreactivity, positively stained tissues were subjected to absorption experiments in which anti-AGE antiserum was preincubated with AGE/bovine serum albumin (0.01 to 1 mg/ml) for 1 hour at 37°C, as described previously.15 For double immunostaining, a combination of the peroxidase-antiperoxidase and alkaline phosphatase techniques was used. The sections were visualized with the Histofine fast blue substrate for alkaline phosphatase staining or the Histofine ACE substrate (Nichirei, Tokyo, Japan) for peroxidase-antiperoxidase staining.
Double-stained sections were prepared sequentially, first with a cell-type-specific antibody, such as Aß90/12 and GP144, and after visualization the section was incubated with anti-AGE antibody. To confirm the specificity of anti-AGE antibody, additional double staining was performed by applying the primary antibody and secondary antibody in reverse order (first anti-AGE antibody and then the cell-type-specific antibody). These double-stained sections were not counterstained.
Semiquantification of AGE-Positive Structures
The number of senile plaques and cerebral blood vessels reacting with anti-Aß or AGE antibodies was counted on both serial tissue sections and double-stained sections in three AD patients. In each patient, immunoreactive plaques and vessels were counted in the hippocampus and the parahippocampal gyrus using three photomicrographs (1.3 mm2 each) at x100 magnification. NFTs and Pick bodies (in three patients with Pick's disease) that reacted with anti-tau or AGE antibodies were counted in the same way. Granulovacuolar degeneration granules, stained by hematoxylin and eosin (H&E) or reacted with anti-AGE antibody, were counted at x400 magnification.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
shows a representative AD brain
immunostained for Aß (Figure 1, A and D)
, AGE (Figure 1, B and E)
,
and ApoE (Figure 1, C and F)
. Double immunostaining of AD brains with
anti-Aß and AGE is also shown. In Figure 1G
, the section was
incubated with anti-AGE antibody before anti-Aß antibody, whereas
Figure 1H
shows a section incubated with anti-Aß antibody before
anti-AGE antibody. With double staining, Aß deposits were stained
blue and AGEs were stained red, whereas co-localization of AGE and Aß
was stained purple (Figure 1, G and H)
.
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.
Adjacent serial sections stained with the anti-AGE antibody also showed
various types of amyloid plaques (Figure 1, B and E)
. More than 90% of
the Aß-positive plaques were also stained by the anti-AGE antibody.
All types of plaques (diffuse plaques, primitive plaques, and classical
plaques) were recognized by anti-AGE (Figure 1, B and E)
. Diffuse
plaques are considered to represent an early stage of senile plaque,
whereas primitive plaques and classical plaques are thought to
represent mature senile plaque.17
Thus, AGE modification
was confirmed to occur in the early stage of plaque development, and
the most intense AGE accumulation was observed in the cores of senile
plaques (Figure 1, B and G)
. The extent of AGE accumulation paralleled
the development of senile plaques, indicating that a gradual and
constant increase of AGE accumulation, shown as an increase of AGE
immunoreactivity, occurred in AD along with the maturation of senile
plaques (Figure 1, B and E)
. The corona showed only weak AGE
immunoreactivity (Figure 1, B and G)
.
Most ApoE-positive plaques, including diffuse plaques, also showed AGE
immunoreactivity (Figure 1, C and F)
, but there was a subtle difference
in the staining patterns of AGE and ApoE in the same plaque. ApoE
stained both the corona and the core similarly, whereas AGE stained the
core much more strongly than the corona (Figure 1, B and C)
.
Double staining for AGE and Aß (Figure 1G)
showed that most senile
plaques were stained by both antibodies. However, occasional
AGE-positive plaques (approximately 5%) were recognized to be Aß
negative or very weakly Aß positive (Figure 1H)
.
Immunohistochemical studies of patients with various other neurodegenerative disorders revealed that all senile plaques, representing the aging process, observed in these disorders (three cases of Pick's disease, one case of PSP, one case of DM, and one age-matched control) also reacted with anti-Aß, anti-ApoE, and anti-AGE antibodies. The staining characteristics were similar to those of AD plaques.
Figure 2
shows representative staining
with anti-Aß (Figure 2A)
, anti-AGE (Figure 2B)
, and anti-ApoE (Figure 2C)
in serial sections of CAA from an AD brain. Most vessels with CAA
were positive for Aß, AGE, and ApoE. In these vessels, the
AGE-positive area corresponded with Aß deposits, or else the Aß
deposits lay within the AGE-positive region (Figure 2B)
. However, as
shown in Figure 2, D–F
, the vessels without amyloid deposits
(approximately 10%) were stained for AGE. Among these Aß-negative
and AGE-positive vessels, approximately 30% showed ApoE
immunoreactivity. Aß-negative and AGE-/ApoE-positive vessels were
also observed in other neurodegenerative diseases such as Guamanian PDC
as well as in DM and control cases. In this study, we did not observe
any Aß-positive, but AGE-negative vessels and AGE immunoreactivity
was present in some of the endothelial cells.
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shows typical immunohistochemical
findings with anti-tau (Figure 3, A, D, and G)
, anti-AGE (Figure 3, B, E, and H)
, and anti-ApoE (Figure 3, C, F, and I)
in serial sections
from subjects with AD (Figure 3, A–C)
, Guamanian PDC (Figure 3, D–F)
,
and a control (Figure 3, G–I)
. In these NFT-positive cases,
approximately 20% of I-NFTs were stained with anti-AGE antibody, but
E-NFTs were AGE negative or were only weakly stained. The AGE
immunoreactivity of E-NFTs was always enhanced by autoclaving, but that
of I-NFTs was obvious without autoclaving (Figure 3, B, E, and H)
. In
Guamanian cases, numerous I-NFTs and E-NFTs were positive for anti-tau
antibody. E-NFTs showed very strong ApoE immunoreactivity, but only AGE
was detected in I-NFTs (Figure 3, D–F)
. There were no remarkable
differences among Guamanian PDC, ALS, and ALS/PDC in the staining for
AGE and ApoE. Control subjects also had NFTs, which were thought to
represent the aging process, and these NFTs showed same staining
pattern for AGE and ApoE (Figure 3, G–I)
.
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. Some Pick cells were
very weakly AGE positive, but the reaction varied considerably
depending on the pretreatment conditions.
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.
Immunohistochemical comparison of AGE-positive intracellular granules
was performed in hippocampal pyramidal neurons of AD (Figure 4E)
, PSP,
Pick's disease, Guamanian PDC (Figure 4F)
, DM, and control subjects.
Numerous AGE-positive granules were present in all of these subjects
and showed a characteristic granular pattern.18
The number
and intensity of AGE-immunoreactive neurons in AD and other
neurodegenerative disease were similar to those in the control cases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our immunopathological studies using serial brain sections and double staining provided the following evidence supporting a role of AGE in the pathogenesis of AD: 1) all types of plaques (diffuse, mature, and classical) showed AGE immunoreactivity, 2) AGE was present from an early stage of Aß accumulation in diffuse plaques, and 3) approximately 5% of plaques stained by the anti-AGE antibody were very weakly stained or not stained by anti-Aß. This was the most interesting observation from the present study and may raise two possibilities. First, AGE modification could occur secondary to Aß deposition, but some Aß deposits are too small to detect. The second possibility is that another protein than Aß protein may be glycated in AGE-positive, Aß-negative plaques. In this case, which protein undergoes glycation instead? Amyloid plaques are composed of a variety of proteins that are designated as amyloid-associated proteins. It seems possible that the protein undergoing glycation may be one of these amyloid-associated proteins or else some unidentified protein. With respect to ApoE, we have already reported the presence of ApoE-positive but Aß-negative plaque-like formation in AD.21 In addition, Dickson et al reported that the staining patterns of AGE and ApoE were similar, suggesting a possible relationship between AGE and ApoE in AD.22 Tabaton et al, however, failed to detect glycated Aß and glycated ApoE using immunoprecipitation techniques and concluded that other amyloid-associated proteins may be candidates for glycation.23 As mentioned earlier, another possibility is that some novel or unidentified protein may be glycated. In fact, Schmidt et al very recently reported that there were novel plaque-like deposits stained by monoclonal antibodies to a 100-kd protein without Aß deposits in AD brains.24 Considering these reports, additional studies are needed to identify the protein undergoing glycation in amyloid plaques.
Regarding NFTs and AGEs, we found that our anti-AGE reacted mainly with I-NFTs in AD brains, whereas E-NFTs were weakly positive. In contrast, anti-ApoE reacted strongly with E-NFTs but weakly with I-NFTs. These findings were confirmed in the other neurodegenerative disorders studied and in our age-matched controls. Contrary to our observation, Dickson et al reported that AGE immunoreactivity is intense in E-NFTs but weak in I-NFTs.22 As AGEs are a heterogeneous group of structures,4 the difference between our findings and Dickson's may reflect differences in the antibodies used or differences in the method of tissue preparation. AGE determinants were localized in paired helical filaments by immunoelectron microscopy,8 and tau protein isolated from AD brains was shown to be glycated at its tubulin-binding domain.25 Furthermore, it has been shown that AGE tau induces neuronal oxidative stress that results in increased expression of cytokine gene and amyloid precursor protein as well as release of Aß peptide.26
AGEs are nonenzymatic products that are considered to be relatively indigestible. It has been reported that AGEs are degraded in lysosomes after being taken up via particular cell-surface binding sites, the receptors for AGE or RAGE, and macrophage scavenger receptor, whereas proteolytic degradation releases small AGE peptides or fragments likely to contain variable portions of AGE moieties.4 Considering these observations, our findings could also raise the possibility that weak AGE immunoreactivity in E-NFT compared with I-NFT may be related to receptor-mediated degradation. In this respect, we demonstrated that some of the granules in granulovacuolar degeneration were AGE positive in AD and other neurodegenerative diseases. Granulovacuolar degeneration is derived from autophasic mechanism involving lysosomes,27 and our findings may provide some evidence suggesting that receptor-mediated degradation takes place in AD and other neurodegenerative disorders.
AGEs are involved in pathological structures, Pick bodies, and ballooned neurons in Pick's disease,28 substantia nigra neurons in Parkinson's disease, and cortical Lewy bodies in diffuse Lewy body disease.29 Reactive oxygen intermediates can be generated during AGE modification,30 and the binding of AGEs to specific receptors can also generate oxidative stress31 as well as the production of pro-inflammatory cytokines.32 In various neurodegenerative diseases other than AD, AGEs were present in some neuropathological structures, including senile plaques, NFTs, Pick bodies, and granulovacuolar degeneration. Under these circumstances, oxidative stress may be induced by AGE generation and receptor-mediated reactions. Therefore, AGE may contribute to eventual neuronal dysfunction and death as an important factor in the progression of various neurodegenerative diseases as well as AD.
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Footnotes |
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Supported by grants-in-aid for scientific research 0761097 and 09470204 from the Ministry of Education, Science, and Culture of Japan.
Accepted for publication July 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein from Alzheimer's disease patients is glycated at its tubulin-binding domain. J Neurochem 1995, 65:1658-1664[Medline]
. Nature 1995, 374:647-650[Medline]
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