Amyloid Load and Neural Elements in Alzheimer's Disease and Nondemented Individuals with High Amyloid Plaque Density

EXPERIMENTAL NEUROLOGY ARTICLE NO.

142, 89–102 (1996)

0181

Amyloid Load and Neural Elements in Alzheimer’s Disease and Nondemented Individuals with High Amyloid Plaque Density AKIHIDE MOCHIZUKI,* JOHN W. PETERSON,* ELLIOTT J. MUFSON,†

AND

BRUCE D. TRAPP*

*Department of Neurosciences, The Cleveland Clinic Foundation, Cleveland, Ohio 44195; and †Department of Neurological Sciences, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois 60612

particularly the hippocampal complex and regions of the cerebral cortex, both of which are involved in memory and higher association functions. Due to the extensive distribution of plaques within the brains of individuals with AD, these lesions have been the focus of intense research aimed at defining their role in the etiology of neural degeneration in this disease. In 1984, Glenner and Wong (22) partially sequenced a peptide that is a major component of the amyloid protein associated with senile plaques. Since these initial seminal observations, it has been established that amyloid consists of a 39- to 43-amino-acid peptide, termed bA4, that is derived from one or more larger integral membrane glycoproteins called the amyloid precursor protein (APP) (33, 45, 60, 62, 76). At least four forms of APP are generated by alternative splicing of a single gene located on chromosome 21. These APP isoforms contain 695, 714, 751, and 770 amino acids and undergo posttranslational modifications including glycosylation and phosphorylation. The 695 form is the most abundant in normal mammalian brain and APP 751 and 770 are expressed in a variety of tissues and share sequence homology with the KUNITZ-type of protease inhibitors (37, 60, 76). The amino acid sequence of APP deduced from cDNA clones predicts a large extracellular domain, a single transmembrane domain, and a small cytoplasmic domain (33, 37, 60, 76). bA4 comprises the first 28 amino acids of the extracellular domain of APP and 11 to 15 amino acids of the transmembrane domain. Proteolytic processing of APP appears to be a normal cellular event and C-terminal truncated forms of APP 695, 751, and 770 are released by a number of cell lines maintained in vitro and can be detected in brain and cerebral spinal fluid (58, 65, 68, 83). Normal processing and secretion of APP in vitro result in proteolytic cleavage within the bA4 domain of APP (17, 68). Generation of bA4, therefore, results from abnormal processing of APP which may occur in endocytic pathways (63). Several lines of evidence support a causative role for APP production and bA4 deposition in the pathogenesis of AD including: (i) consistent AD pathology in adult Down’s syndrome patients (42, 56) who have an

The amyloid burden and relationship between amyloid deposits and neural elements were investigated in sections of prefrontal neocortex from eight Alzheimer’s disease (AD) patients and four age-matched nondemented controls with high amyloid plaque density (HPND). Computer-based image analysis revealed that the total area occupied by bA4 immunoreactivity was significantly greater (P F 0.031) in AD (27.1%) than in HPND (14.5%) sections. The total bA4-positive area occupied by nondiffuse plaques was significantly greater (P F 0.05) in AD (13.6%) than in HPND (5.2%) sections. The percentage of diffuse (DPs) and nondiffuse plaques (NDPs) which contained neurons, astrocytes, microglia, dystrophic neurites, and amyloid precursor protein (APP) was also determined. The frequency of association between bA4 and these neural elements was similar between AD and HPND cases in both diffuse and nondiffuse plaques. Forty percent of DPs in AD and HPND sections contained neuronal perikarya. Microglia, dystrophic neurites, and APP were detected in most nondiffuse plaques in both AD and HPND sections. While astrocyte cell bodies were not present in either diffuse or nondiffuse plaques, their processes were detected in most. These findings indicate that amyloid deposition and nondiffuse plaques are greater in AD than in HPND sections. The association between microglia and nondiffuse plaques supports the hypothesis that these resident immune cells participate in aggregation and redistribution of amyloid deposits and possibly formation of dystrophic neurites. r 1996 Academic Press, Inc.

INTRODUCTION

Alzheimer’s disease (AD) is the most common human neurodegenerative disease. Senile or amyloid plaques, neurofibrillary tangles, and cerebrovascular amyloid are the most prominent characteristics of this disease and form the diagnostic features of postmortem AD brain (7, 45). The density of senile plaques and neurofibrillary tangles generally correlate with the severity of dementia and loss of neurons in AD brains. These lesions are most pronounced in select brain regions, 89

0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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additional copy of the APP gene and in whom the first recognizable neuropathological change is diffuse bA4 deposition (51); (ii) several AD pedigrees display APP gene mutations (23, 55, 80); and (iii) evidence that bA4 may be neurotoxic under certain circumstances (38, 87). Since most bA4 is likely to be derived from APP which is produced within the central nervous system (CNS), the distribution of APP and bA4 can provide insights into mechanisms of amyloid deposition. Interestingly, abundant APP immunoreactivity has been detected in radial glia (78) or neurites ( 43) in fetal rodent brain, suggesting a role for this protein in CNS development. In adult mammalian brain, most APP has been detected in neuronal perikarya and their proximal dendrites (78) and in dystrophic neurites associated with senile plaques (66). Not all neurons contain detectable APP in adult brain and activated astrocytes may express APP in some circumstances (67). Based on morphological appearance, biochemical composition, and association of other CNS components, extracellular bA4 deposits can be classified into two general types: diffuse and nondiffuse (44, 85). Diffuse bA4 deposits vary in size, have poorly defined boundaries, and have bA4 concentrations which are less than those found in nondiffuse plaque. Most bA4 peptides in diffuse plaques contain amino acids 1–42 (30, 31). No consistent association between diffuse plaques and other CNS elements have been described. Nondiffuse or senile plaques consist of dense spherical bA4 deposits with well-demarcated edges and an average size of 40 µm. Many nondiffuse plaques contain irregularly shaped bA4-negative cavities which often surround a dense spherical amyloid core (approximately 10 µm in diameter) which is located at the center of the plaque. In addition to bA4 (1–42), nondiffuse plaques also contain bA4 (1–40) (30, 31). Two other CNS elements, dystrophic neurites and microglia, are also consistently associated with nondiffuse plaques (77). These may be responsible, in part, for the bA4-negative cavities. In this regard, it has been proposed that diffuse plaques evolve into nondiffuse plaques (40, 42, 51, 86). Such a process may involve aggregating properties of certain bA4 peptides, the presence of chaperon proteins, including APOE4 (54, 73), or the presence of microglia. Microglia have been proposed to be involved in APP and/or bA4 processing (29) and in cytokine production (26), which may increase the production of nondiffuse plaques and dystrophic neurites. Activated astrocytes have also been implicated in the generation of nondiffuse plaques and dystrophic neurites (16, 41, 52). The discovery of bA4 and APP has focused much of AD research on in vitro studies of APP processing, genetic linkage studies, and development of appropriate animal models. However, the source of APP from which bA4 is derived, the normal function of APP, and

how bA4 induces neuronal dysfunction still remain unknown. Neuropathological analysis of AD brains has an essential role in answering these and other questions which are fundamental to our understanding of AD pathogenesis. This report correlates neuropathological changes in AD brains with those in a unique group of age-matched control subjects which exhibit extensive plaque pathology without evidence of frank dementia (2–6). This group of individuals has been evaluated previously and the individuals are termed high-plaque nondemented controls (HPND) (2–6). These brains exhibit pathological changes which are pre-AD in density of tangles and development of neuritic plaques. We have determined the density of diffuse plaques and nondiffuse plaques in sections of prefrontal cortex and have determined the frequency of association between bA4 deposit and neurons, astrocytes, microglia, dystrophic neurites, and APP. MATERIALS AND METHODS

Subjects. A total of eight AD cases and four agedmatched controls were analyzed (Table 1). The average age of the AD and control groups was 79.3 (range 71–89 years) and 73.3 years (range 63–81 years), respectively. Postmortem interval averaged 5.6 h for the AD group and 3.9 h for the control group. The four aged nondemented individuals had no clinical history of AD. However, upon neuropathological evaluation, they displayed sufficient numbers of senile plaques in their neocortex to meet both the aged adjusted NIA/ADRDA (36) as well as the CERAD (50) criteria of AD. We have previously described significant pathological differences between this subgroup of patients and AD individuals (2–6) and have termed this group high plaque nondemented (HPND). These cases are similar to other populations which have been reported to be pathologically intermediate between normal aged and AD (9, 14, 35, 79). All cases were evaluated for AD type pathology (i.e., neuritic plaques and neurofibrillary tangles) using thioflavine-S and Bielschowsky silver staining procedures on paraffin embedded tissue sections taken from the entorhinal cortex, hippocampus, amygdala and temporal, frontal, parietal and occipital neocortices. None of the subjects had other confounding neurologic or neuropathologic disorders. Clinical evaluation of the HPND cases was based upon retrospective analysis of medical records. Furthermore, interviews with some of the physicians and immediate family members of many of the HPND cases provided further evidence that these individuals had not displayed evidence of frank memory impairment or dementia. However, it is important to keep in mind that in the absence of neuropsychological evaluation, it cannot be completely ruled out that these patients were exhibiting mild memory deficits that

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TABLE 1 Percentage of DP containing

Percentage of NDP containing

Amyloid Age Sex load

DP load

Clustered Clustered NDP MicroAstro- dystrophic MicroAstro- dystrophic load glia Neuron cyte neurites APP glia Neuron cyte neurites

AD patients 1 2 3 4 5 6 7 8

72 89 83 81 76 89 71 73

39.7 28.4 16.1 43.2 24.4 32.3 12.1 20.5

34.7 24.2 11.1 40.6 23.6 28.9 10.1 17.6

5.0 4.0 5.0 2.6 0.8 3.4 2.0 2.9

8.5 13.0 10.0 5.0 16.0 17.0 7.5 5.0

38.0 41.5 38.5 35.0 37.5 48.0 46.0 31.0

1.5 0.5 0.0 1.5 1.0 0.0 1.0 2.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

62.5 71.5 77.0 67.0 62.5 72.0 54.5 30.0

13.5 7.0 4.0 0.0 7.0 6.0 4.0 5.0

3.0 2.0 3.0 0.5 2.0 1.0 1.0 2.5

98.5 98.0 98.5 99.5 97.5 99.5 96.5 99.5

Mean SEM

79.3 7.3

27.1 a 11.0

23.9 a 10.8

3.2 a 1.5

10.3 4.7

39.4 d 5.6

0.9 0.7

0.0 0.0

0.0 0.0

62.1 d 14.7

5.8 3.8

1.9 b 1.0

98.4 d 1.1

95.4 d 3.0

HPND controls 1 2 3 4

63 64 85 81

8.5 11.6 15.6 22.5

7.5 11.3 14.9 21.9

1.0 0.3 0.7 0.6

7.5 5.5 5.0 7.0

34.0 47.5 40.0 38.0

1.5 0.5 0.0 1.0

0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0

27.0 52.0 35.0 57.0

2.5 4.5 4.0 2.0

2.5 3.5 1.5 2.5

95.5 95.0 95.5 96.0

91.0 91.0 90.5 92.0

Mean SEM

73.3 11.4

14.5 6.0

13.9 6.1

0.6 0.3

6.3 1.2

39.9 d 5.7

0.8 0.6

0.0 0.0

0.0 0.0

42.8 c 14.1

3.3 1.2

2.5 b 0.8

95.5 d 0.4

91.1 d 0.6

M M F F M F M M

F M M M

APP

98.0 93.5 97.0 100.0 92.0 96.5 91.5 94.5

Note. AD vs HPND: a P , 0.05. DP vs NDP: b P , 0.05, c P , 0.01, d P , 0.001.

were undetected by family members or by their physician. Tissue preparation. At autopsy, brains were removed from the calvarium and processed as described previously (2–6, 53). Briefly, each brain was cut into 1-cm-thick coronal slabs using a Plexiglas brain slice apparatus and immersion fixed in 4% paraformaldehyde for 24–48 h. Slabs containing the superior frontal gyrus were cut into 18 adjacent series of 30-µm-thick sections on a freezing sliding microtome and stored in phosphate buffer or cryoprotectant solution prior to processing. Immunohistochemistry. Sections were immunostained by the avidin–biotin complex procedure as described previously (61). Sections were pretreated with microwaving in a citrate buffer, pH 6.0, 2 3 5 min, placed in 44% formic acid for 2 min, placed in 1% H2O2 in 10% Triton X-100 (Sigma; St. Louis, MO) for 30 min, and then incubated in the following solutions: 3% normal goat serum for 30 min at 22°C; primary antibodies overnight at 4°C; biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) diluted 1:500 for 30 min at 22°C; enzyme (HRP)-linked biotin (1:1000) and avidin (1:1000) (Vector Laboratories) in phosphatebuffered saline for 1 h; diaminobenzidine/hydrogen peroxide for 5 min; and 0.1% osmium tetroxide for 30 s. Sections were mounted on glass slides and photographed with a Zeiss axiophot microscope. Sections for double immunofluorescent staining were pretreated as described above, placed in 0.1% osmium

tetroxide for 30 s to reduce autofluorescence and placed in the following solutions: 3% normal goat serum for 30 min at 22°C; primary antibodies overnight at 4°C; biotinylated secondary antibody (diluted 1:500) and Texas red-conjugated secondary antibodies (diluted 1:100; Jackson Laboratories, West Grove, PA) for 1 h; and fluoresceine-labeled avidin diluted 1:500 for 1 h. Sections were mounted in a Mowiol-based (Calbiochem, San Diego, CA) mounting medium containing 0.1% paraphenylenediamine hydrochloride. The primary antibodies used are characterized and included: mouse or rabbit anti-bA4 (1–42); mouse anti-APP (22C11, Boehringer Mannheim, Indianapolis, IN); mouse anti-nonphosphorylated neurofilament (SMI 32, Sternberger, Baltimore, MD); rabbit anti-glial fibrillary acidic protein (GFAP, Z334; Dako Corp., Carpinteria, CA); rabbit anti-ferritin antigen (Sigma); rabbit anti-ubiquitin (Z458; Dako Corp.). Confocal microscopy. Sections were analyzed on a Leica Aristoplan confocal laser scanning microscope (Leitz Wetzlar, Heidelberg, Germany) as described previously (8). Confocal images represented optical sections of approximately 0.35 µm axial resolution. In some cases, 8–10 consecutive optical sections were combined to form a ‘‘through-focus image.’’ Fluorescence in the red (Texas red) and green (fluorescein) channels was collected simultanously. In red/green merged images, areas of colocalization are yellow. Quantification. Amyloid plaques were classified into diffuse plaques and nondiffuse plaques using the crite-

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ria of Yamaguchi et al. (85). We determined the area occupied by bA4 immunoreactivity. Two hundred twelve square millimeters of cortex from each case was photographed and the negatives were scanned with a Nikon film scanner for computer analysis. The area occupied by amyloid deposits was determined with the assistance of NIH image 1.57 software at a resolution of 1 µm 5 68 pixels. The threshold signals for image analysis was set to eliminate autofluorescence produced by lipofuscin. To quantitate the association of neural elements with amyloid plaques, 200 DPs and 200 NDPs (.30 µm in diameter) were identified in double-labeled sections and the percentage containing neurons, astrocytes, microglia, dystrophic neurities, and APP was determined. Cells were considered plaque-associated when their nucleus was partly or totally contained within the plaque. Statistical analysis was performed using Student’s t test. RESULTS

Although amyloid deposition is a hallmark of AD pathology, its role in the etiology of dementia is unclear. To address this issue, we have quantitated the total amyloid load and the types of amyloid deposits in AD brains and compared these data with those obtained from HPND brains. All of our analysis was restricted to the cerebral cortex of the superior frontal gyrus. In addition, we have quantitated the association of neural elements with DPs and NDPs. Table 1 summarizes the features examined and presents the quantitative results for each.

tions. DPs contained trigonal, round, or linear bA4-free spaces which were occupied by neuronal perikarya, axons, dendrites, or vessels. Some larger DPs were surrounded by very small bA4 deposits (Fig. 3B). These deposits were filamentous in appearance and most contained branches (Fig. 3C). While they were detected throughout the cortex in both AD and HPND sections, they were most prevalent around DPs. NDPs contain thick and densely packed bA4-positive amyloid fibrils (Figs. 3D–3F). They are spherical with well-demarcated edges and average 40 µm in size. Irregularly shaped bA4-negative cavities were frequent within NDPs. Some contain small cavities which appear to be randomly distributed (Fig. 3D). Many have larger more continuous bA4-negative regions (Fig. 3E). Other NDPs contain large bA4-negative cavities which surround a spherical amyloid core of approximately 10 µm in diameter (Fig. 3F). Amyloid cores often have bA4-negative centers with jagged bA4-positive outer edges made up of thicker amyloid fibrils. bA4-negative spaces can totally surround the amyloid core and have been termed the ‘‘wreath’’ of core plaques. The amyloid core and the outer wreath wall are sometimes connected to each other by thick amyloid fibrils. In AD sections, NDPs were distributed throughout all layers of the cortex. In HPND sections, NDPs were concentrated in the deeper layers of the cortex. The percentage of total cortical area occupied by NDP and DP was also determined. In AD sections, approximately 14% of the total bA4-positive area was occupied by NDP (Fig. 2, Table 1). In HPND sections, NDPs represent approximately 5.5% of the total amyloid load.

Quantification and Characterization of Amyloid Deposits in AD and HPND Brains

Amyloid Deposits and Neural Elements

Using a computer-based quantification program we estimated the area of cortex occupied by bA4 immunoreactivity in AD (Fig. 1A) and HPND (Fig. 1B) sections. The total cortical area occupied by bA4 immunoreactivity was significantly greater (P , 0.031, Student t test) in AD (27.1%) than in HPND (14.5%) sections (Fig. 2, Table 1). Based on morphology, bA4 immunoreactivity can be classified into diffuse (DPs) and nondiffuse (NDPs) plaques. DPs consist of fibrillar amyloid deposits which are less dense than NDPs. DPs vary in size and have poorly defined boundaries (Fig. 3A). Many DPs measure no more than 10 µm in diameter while others can be over 100 µm in diameter. Many larger DPs have irregular shapes which may result from fusion of smaller plaques. Smaller DPs were evenly distributed throughout all layers of the cortex in AD and HPND sections. Larger DPs were distributed in all layers of the cortex in AD sections and were most abundant in the superficial and intermediate layers in HPND sec-

bA4 deposition requires the production and processing of APP within the CNS. To determine the possible source of APP or sites of bA4 formation, we determined the frequency of association between bA4 deposits and neurons, astrocytes, microglia, dystrophic neurites, and APP by double-labeling immunocytochemistry. bA4 immunoreactivity was divided into diffuse and nondiffuse plaques. A total of 200 diffuse and 200 nondiffuse plaques were identified and the percentage containing neuronal, astrocytic, or microglia perikarya, dystrophic neurites, and APP immunoreactivity was evaluated. Nonphosphorylated neurofilament-positive neurons were detected within 40% of DPs in both AD and HPND sections (Fig. 4, Table 1). The association between neuronal perikarya and NDP was low in both AD (6.6%) and HPND (3.3%) sections. Neuronal perikarya within DPs showed normal trigonal perikarya and apical dendrites (Fig. 5A). Confocal analysis also detected many nonphosphorylated neurofilament-positive dendrites within NDPs.

FIG. 1. Distribution of bA4 immunoreactivity in free-floating sections (30 µm thick) of prefrontal cortex from AD (A) and HPND (B) brain. Dark reaction product indicates greater bA4 load in AD section. Scale bars, 600 µm. 93

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FIG. 2. The amyloid load in HPND control and AD sections. bA4 deposits are subdivided into those associated with diffuse and nondiffuse plaques. AD sections contain a significantly greater bA4 load (P , 0.05) and NDP density (P , 0.05) than HPND sections. See Table 1 for individual values.

GFAP-positive astrocyte perikarya were detected in less than 2.5% of NDPs and DPs in both AD and HPND sections (Fig. 4, Table 1). Activated astrocytes were often present at the outer edge and extended multiple processes into NDPs (Fig. 5B). Many astrocytes oriented the majority of their processes into NDPs. Astrocytes with smaller cell bodies and thinner and shorter processes than those associated with NDPs were located next to and extended processes into DPs. The association between astrocytes and bA4 deposits did not appear different in AD and HPND sections. There was no obvious relationship between astrocytes and small amyloid deposits. Microglia perikarya, identified by ferritin antibodies, were detected within 62.1% of the NDPs and 10.3% of the DP in AD sections, and in 42.8% of the NDPs and 6.3% of the DPs in HPND sections (Fig. 4, Table 1). The association between microglia and nondiffuse plaques was significantly greater (P , 0.05) than that between microglia and DPs in both AD and HPND sections. Although NDPs in AD sections contained more microglia than those in HPND sections, the difference did not reach significance (P , 0.055). Similarly, AD sections contained more microglia-positive DPs than HPND sections, but the difference was not statistically significant (P , 0.052). Microglia associated with NDPs had hypertrophied perikarya which extended irregularly shaped, asymmetrical, thick processes. These morphological changes indicate that these cells are activated. The relationship between microglia and nondiffuse plaques was variable and suggested that microglia are actively involved in core plaque formation. NDPs containing small irregular bA4-negative cavities often had activated microglia

associated with their outer edge (Fig. 5C). These microglia cells extended processes into the plaques and many bA4-negative regions were occupied by their processes. NDPs with larger bA4-negative cavities contained one or more microglia perikarya within these cavities (Fig. 5D). Many of these cells extended processes within the bA4-rich regions. NDPs with amyloid cores contained multiple microglia in the bA4-negative wreath (Fig. 5E). Microglia perikarya and/or processes often surrounded the amyloid core. Microglia within the wreath of these plaques also extended processes throughout the outer bA4 regions and into the neuropil. To identify alterations in neuronal processes, sections were immunostained with ubiquitin antibodies, an accepted marker for neuropil threads and dystrophic neurites. Neuropil threads appeared as thin structures with intense and homogenous ubiquitin staining (Fig. 6). Neuropil threads were abundant and evenly distributed throughout the cortex in AD sections. Neuropil threads were less abundant in HPND sections. In addition to neuropil threads, larger ubiquitin-positive dystrophic neurites (Figs. 6, 7A, and 7B) were invariably associated with NDPs in both AD and HPND sections. Two types of dystrophic neurites were identified. One had a tortuous fusiform structure with intense homogenous ubiquitin staining (Fig. 7) which was similar in appearance to that found in neuropil threads. These structures were more abundant in AD than in HPND sections and were concentrated in lower layers of the cortex. Other dystrophic neurites (Fig. 7B) were globular in shape, contained granular ubiquitin immunoreactivity, and were present in both AD and HPND brains. Fusiform and globular dystrophic neurites were clustered within the cavities of NDPs. Single dystrophic neurities were also detected within DPs at the same density as those scattered in the neuropil. Only clusters of three or more dystrophic neurites were included in our quantitative analysis. Clustered dystrophic neurites were detected in 98.4% of the NDPs in AD sections and in 95.5% of the NDPs in HPND sections (Fig. 4, Table 1). Clustered dystrophic neurites were not associated with DPs in either AD or HPND sections (Table 1). Intense APP immunoreactivity was detected in 95.9% of the NDPs in AD sections and in 91.5% of the NDPs in HPND sections, but was not detected within DPs in either AD or HPND sections (Fig. 4, Table 1). The distribution of APP immunoreactivity in NDP was similar to that of ubiquitin-positive dystrophic neurites (Fig. 7C). Weaker, punctate APP immunoreactivity was also detected in neuronal perikarya and proximal dendrites in both AD and HPND sections. The frequency and distribution of APP in neuronal perikarya appeared similar in AD and HPND sections.

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FIG. 3. bA4 immunoreactivity in AD brains. Diffuse amyloid deposits (A–C) consist of large plaques (A) and smaller filamentous deposits (B, arrowheads) which often have a branched appearance (C). Confocal analysis of nondiffuse plaques (D–F) demonstrates intense bA4 immunoreactivity. bA4-negative regions in nondiffuse plaques can be small and randomly distributed (D), larger and more confluent (E), or surround amyloid cores (F). Scale bars: (A) 25; (B,C) 12.5; (D–F) 20 µm.

DISCUSSION

This study investigated amyloid burden and the association of neural elements with amyloid plaques in AD brains and brains from nondemented individuals who contained significant amyloid deposition. While we found significantly greater amyloid deposition and NDPs in AD brains, no significant differences were found in the association of neuronal elements (neuronal, astrocytic, microglia perikarya, dystrophic neurites, or APP) and amyloid plaques. Our studies confirmed a significant association between NDPs, activated microglia, and APP-positive dystrophic neurites and support the hypothesis that ‘‘activated’’ microg-

lia play a role in the formation of core plaques and dystrophic neurites. The total amount of bA4 deposits was significantly greater in AD than in HPND sections, although HPND subjects displayed sufficient amyloid deposits in their neocortex to meet both the age adjusted NIA/ADRDA (36) as well as the CERAD (50) criteria of AD. Possibly more relevant to the etiology of dementia, AD sections contained significantly more NDPs (13.6% of total bA4 deposits) than HPND sections (5.2% of total bA4 deposits). Since the amyloid load in AD section was twice that in HPND sections, the number of nondiffuse plaques is approximately five times greater in AD than in HPND. Our findings are in agreement with previous studies

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FIG. 4. The association of neural elements with 200 diffuse and 200 nondiffuse plaques in AD and HPND sections. Although no significant differences were found between AD and HPND sections, differences were detected between diffuse and nondiffuse plaques in both AD and HPND sections.

which concluded that DPs are age-related and not associated with dementia (10, 12–14, 49, 74) and that NDPs with associated dystrophic neurites, synaptic loss, and activated microglia correlate with clinical status (63). While the computer-based image analysis procedure used to measure the cortical area occupied by bA4 deposits was similar to the method used by Cummings et al. (11), the total cortical area occupied by bA4 immunoreactivity in AD sections in our study (8.5% to 43.2%) was greater than that reported (1.6– 14.8%) by these authors. These differences may be due to our use of more aggressive pretreatment of sections with Triton X-100, microwave, and formic acid and to more sensitive detection of small bA4 deposits by immunofluorescence techniques when compared to DAB. Role of Microglia in Core Plaque Formation In normal mammalian brain, microglia form a network of process-bearing cells which covers the entire neuropil (24). Each microglia cell occupies a neuropil domain and processes of neighboring cells rarely over-

lap. In AD and HPND sections, microglia often occurred in clusters of two to five cells and double-labeling studies with ferritin and bA4 detected most of these clusters in NDPs. We identified many NDPs with three to five microglia and one plaque contained nine microglia. Our results also support the hypothesis that microglia play a role in formation of core plaques. Increases in the bA4-negative regions of NDPs were accompanied by an increase in the number of microglia present within the plaque. These observations suggest that NDPs are formed by mechanisms which do not require multiple microglia. NDPs, however, appear to attract microglia (Figs. 3C, 3D, and 3E). Initially, these plaques may have few bA4-negative regions which are occupied by dendrites, axons, and astrocyte processes, but few neuronal or astrocytic perikarya. Microglia perikarya can be detected at the perimeter of NDPs which contain few bA4-free regions and these microglia can extend processes into the plaque (Fig. 3C). NDPs with larger bA4-negative cavities contain multiple microglia perikarya. In NDPs with amyloid cores, microglia are intimately associated with the amyloid core. A signifi-

FIG. 5. Confocal micrographs illustrating the distribution of bA4 (red), Neurons (A, green), astrocytes (B, green), and microglia (C–E, green). Neurons and dendrites (A) are detected in diffuse amyloid deposits but were rare in nondiffuse plaques. Reactive astrocytes extended processes to and into nondiffuse plaques (B). Microglia were associated with most nondiffuse plaques. Microglia were often located at the edge of plaques with few bA4-negative cavities (C). Nondiffuse plaques with larger, more confluent bA4-negative cavities (D) and plaques with amyloid cores (E) contained multiple microglia cells. Scale bars, 10 µm. FIG. 7. Dystrophic neurites and nondiffuse plaques. Sections double-labeled with bA4 (red) and ubiquitin (B, C, green) detected clustered dystrophic neurites in almost all nondiffuse plaques. Some dystrophic neurites had diffuse ubiquitin staining (A) while others had more punctate staining (B). Many dystrophic neurites were also stained by APP antibodies (C, green). Scale bars, 10 µm.

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FIG. 6. Distribution of ubiquitin immunoreactivity in section (30 µm thick) from AD brain. Thin lines of ubiquitin staining (neuropil threads) occurred throughout the cortex (small arrows). Larger ubiquitin-positive structure occur in clusters (arrowheads). Many of these structures are round and represent dystrophic neurites. Isolated dystrophic neurites are also present throughout the cortex (large arrow). Scale bar, 50 µm.

cant percentage of the bA4-negative regions of NDPs are occupied by dystrophic neurites, astrocytic processes, dendrites, and axons. Previous reports have provided evidence that activated microglia are involved in core plaque formation (25, 27–29, 59, 77, 82, 84). As CNS resident monocytes, microglia can be ‘‘activated’’ by a number of mechanisms including nerve degeneration (72), demyelination (8), and breakdown of the blood–brain barrier (21, 61). Two criteria can be used to identify activated microglia: changes in cell shape and expression of monocyte activation markers. Microglia associated with NDPs display both criteria. NDP-associated microglia have larger cell bodies and fewer, thicker, and less symmetrical processes than microglia in normal brain. NDPs, however, do not appear to transform microglia into debris-laden foamy macrophages as happens during brain trauma or demyelination (8). This indicates that activation mechanisms may be more specialized than those operating during more classical phagocytosis. In support of this hypothesis, recent in vitro studies reported that exposure of bA4 peptides to rat microglia activated three distinct tyrosine kinases, elevated levels of protein tyrosine phosphorylation, and the production of reactive oxygen species (G. Landreth, personal communication). Previous studies have detected several activation markers in microglia associated with NDPs including MHC class II, IL-1, and phosphotyro-

sine (34, 46, 48, 64). While our results are most consistent with the hypothesis that microglia react to NDPs, we cannot rule out the possibility that microglia secrete bA4 or play a role in concentrating diffuse bA4 into NDPs. Our confocal analysis supports a role for microglia in aggregation and redistribution of bA4 during core plaque formation. It is possible that microglia respond to certain sequences or conformation states of bA4 enriched in NDPs but not in DPs. In support of this possibility, electron microscopic studies of AD brains have shown an association between microglia and bA4 fibrils in core plaques (29, 47) and rodent microglia scavenge bA4 in vitro (1) and after injection into the CNS (18). Another cellular element consistently associated with NDPs was dystrophic neurites. While ubiquitin-positive neurites were detected throughout the neuropil of AD sections, NDPs consistently contained clusters of large dystrophic neurites. Clusters of ubiquitin-positive neurites were not detected outside of NDPs. Interestingly, previous studies (6) have demonstrated that bA4 deposition is a prerequisite event for the occurrence of dystrophic neurites which are found in association with senile plaques. In these investigations, dystrophic neurites expressing PHF or neuropeptides were never seen in the absence of bA4, suggesting a unique relationship between bA4 deposition and neuritic alterations. These findings support the notion that various

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types of dystrophic neurites, including perhaps those containing ubiquitin, are in fact a direct consequence of bA4 deposition and not merely the by-product of a more generalized process of neuronal degeneration. Although the precise role that ubiquitination of neurites plays in the development of NDPs remains a question of intense research activity, it is interesting to note that ubiquitin is a member of a family of low-molecularweight proteins whose expression increases in response to stress. As such, ubiquitin is considered to be a heat shock protein. Since stress is often associated with protein damage, it is reasonable to expect that synthesis of a protein such as ubiquitin, which is involved in energy-dependent degradation of abnormal or denatured proteins, would increase coincidentally in disease states related to alterations in the configuration of proteins such as amyloid. Furthermore, amyloid deposition may facilitate the formation of dystrophic neurites by acting as a trophic factor which initiates abnormal sprouting of presynaptic fibers (20). In fact, at the ultrastructural level, ubiquitin-immunoreactive neurites appear as swollen processes filled with membranous dense bodies possibly of lysosomal origin (15, 32). Thus, investigations of the interaction of ubiquitin and amyloid may provide clues as to the pathogenesis of NDPs in AD. Source of bA4 in AD One objective of this study was to determine if consistent associations between bA4 deposition and neural elements could provide clues to the source of the APP from which bA4 is produced. We found a significant correlation between neuronal perikarya and NDPs in both AD and HPND sections and many neurons contained weak punctate APP immunoreactivity in their perinuclear cytoplasm. Neurons are considered to be a major source of APP and bA4 in AD. The invariable presence of APP-positive dystrophic neurites with NDPs in our studies supports this hypothesis. However, it remains to be established if dystrophic neurites cause NDPs or whether NDPs cause dystrophic neurites. If the latter is the case, APP associated with dystrophic neurites could play a role in remodeling NDPs but not in their formation. It is possible that APP associated with dystrophic neurites is processed into bA4 peptides that differ from those produced from other CNS elements. While astrocyte cell bodies were not detected in bA4 deposits, GFAP-positive processes were present in most NDPs and DPs. Many astrocytes in AD brains are reactive and our confocal analysis indicates that they reorient and extend their processes around and into NDPs (29). This observation raises the possibility of neuronal dysfunction due to loss of astrocytic homeostasis of synaptic environments. Surprisingly little is known about APP in AD brains. Immunocytochemical

studies, including the present study, have been consistently disappointing in localizing APP. The reasons for this are unclear but may be due to rapid turnover or low, steady-state levels of APP. The development of more sensitive immunocytochemical techniques may help address this issue. Intense APP immunoreactivity has been detected in radial glia in fetal and early postnatal mammalian brain (78). Due to the common lineage of astrocytes and radial glial and to reports that reactive rodent astrocytes express APP immunoreactivity following stab wound (57) and excitotoxic damage to the hippocampus (67), the astrocyte remains a possible source of the APP which is processed to bA4 in AD brains. It should be stressed, however, that future studies must delineate APP expression from that of the APP-like proteins (69–71, 75, 81). Confirmation of APP expression will require verification by methods other than immunocytochemistry. Neuronal Dysfunction in AD Previous studies comparing the pathology of AD and HPND revealed that HPND cases contained: (i) predominantly diffuse plaques, (ii) less neurofibrillary pathology (e.g., PHF staining), and (iii) more earlystage pathology such as Alz-50-positive plaques and neurons (2–6). The present study revealed an increase in amyloid burden, especially NDPs and neuropil threads in AD. There was no clear association between diffuse amyloid deposits and neuropil threads. Our studies did not identify differences in the deposition and processing of bA4 in the CNS of AD and HPND sections. Senile plaques and amyloid deposits in AD and normal-aged brain have similar biochemical features with a predominance of bA4 (1– 42) and the association of bA4 (1– 40) with core plaques (19). The identification of several gene defects in familial AD (23, 39, 55, 80) indicates that amyloid deposition is a downstream event in a number of CNS insults associated with aging. The results presented here support the hypothesis that processing bA4 into NDPs may play an important role in the etiology of dementia in AD. Identification of the cell or cells responsible for APP and/or bA4 production may help elucidate different processing pathways that encourage formation of NDPs. ACKNOWLEDGMENTS This work was supported by NIH Grants NS 30451 (B.D.T.), AG 10668, AG 09466, and AG 10161 (E.J.M.). The authors thank Peter Hauer, David Cheng, and Judy Drazba for assistance in confocal analysis and Lois Becker for typing the manuscript.

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