American Journal of Pathology, Vol. 141, No. 1, July 1992
Copyright (© American Association of Pathologists
Beta Amyloid Is Focally Deposited Within the Outer Basement Membrane in the Amyloid Angiopathy of Alzheimer's Disease An Immunoelectron Microscopic Study
H. Yamaguchi,*t T. Yamazaki," C. A. Lemere,*t M. P. Frosch,t and D. J. Selkoe*t From the Department ofNeurology, * Harvard Medical School, Boston, the Center for Neurologic Diseases,t and the Departments of Medicine (Neurology) and Pathology, Brigham and Women's Hospital, Boston, Massachusetts; and the Department of Neurology,." Gunma Unitersity School of Medicine, Maebashi, Japan
The fine structure of cerebral amyloid angiopathy, especially in small and presumably early deposits, was examined by immunolabeling of the P/A4 protein in semithin and ultrathin sections from brains with Alzheimer's disease. The following findings emerged: 1) in large leptomeningeal arteries, smanl focal amyloid deposits appear to consist of clusters of delicate (-8 nm diameter) amyloid fibrils, not previously described, in the outermost part of the basement membrane (BM) at the media-adventitia junction; 2) in small leptomeningeal arteries and perforating cortical arterioles, small foci of delicate amyloid fibrils were observed within the BM. They appeared mostly in the outer portion of the BM, around intact smooth muscle cells, rather than in the subendothelial region. In larger and presumably more advanced deposits, coarse amyloidfibrils (-10 nm) occupied the abluminal BM, and adjacent smooth muscle cells showed degeneration; and 3) in capillaries, small amounts of delicate (-8 nm) amyloid fibrils, not previously described, were seen within the BM in the smallest discernible deposits. The BM at these sites was abnormally folded and layered In larger deposits, amyloidfibrils appeared to extravasatefrom the outerBM of the capillary into the neuropil and were surrounded by astrocytic foot processes and/or microglia Our results suggest that vascular amyloid fibrils may first be formed within
the abluminal vascular BM, that i outside of cells. The BM may trap degradative intermediates of the amyloid precursor protein that contain the /A4 region, and local proteases may then cleave them further to yield amyloidogenic fragments. (Am JPathol 1992, 141:249-259)
Amyloid angiopathy (AA) and senile plaques (SP) are prominent forms of ,B/A4 amyloid deposition in the human brain.14 AA is an age-related change and is not closely associated with the clinical expression of dementia, although it is almost invariably present to some degree in Alzheimer's disease (AD).9 AA occurs in both leptomeningeal arteries and intracortical arterioles and capillaries, and less frequently in veins.-7710 After the discovery of the ,/3A4 protein as the major constituent of cerebrovascular amyloid in AD,1 many investigators have attempted to characterize the mechanism of amyloid deposition. Recent research suggests that alternative degradative pathways for the /A4 amyloid precursor protein (3APP)11 exist in cultured cells1'214,32 and tissues1 5,16 and may lead to the production of the amyloid fragment, /A4 protein, or larger potentially amyloidogenic intermediates. To gain information about the fine structure of small and presumably early /A4 amyloid deposits, we examined small foci of amyloid fibrils within the walls of selected meningeal and cortical microvessels in sections of AD cerebral cortex by electron microscopy. The structure Supported in part by NIH grants AG 07911 (LEAD Award) and AG 06173 and by a gift from the Broad Family Foundation. Accepted for publication March 12, 1992. Address reprint requests to Dr. Dennis J. Selkoe, Center for Neurologic Diseases, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115.
249
250
Yamaguchi et al
AJP July 1992, Vol. 141, No. 1
of AA is simpler than that of the ,B/A4-containing senile plaques in the cerebral cortex, and meningovascular AA in particular presents a simple system devoid of surrounding neurons, astrocytes, and other neural elements. Moreover, the detailed study of AA should provide insights about the potential contribution of vascular and circulating factors to progressive amyloidogenesis, and these might be more amenable to therapeutic manipulation than factors arising within brain tissue. To assess the fine structure of small amyloid deposits in the cerebral microvasculature, we used two methods: anti-,B/A4 immunogold labeling on ultrathin sections, and anti-P/A4 labeling of semithin sections and EM examination of adjacent ultrathin sections.
Immunogold Labeling of Epon Sections Ultrathin sections from epon-embedded tissues on nickel grids were treated with 10% H202 for 10 minutes, 1% periodic acid for 10 minutes and 10% normal goat serum (NGS) for 30 minutes. One of two anti-,/A4 antisera was applied for 2 hours: R1280 against synthetic P/A41X4 at a dilution of 1:1000,15 or p28K against synthetic P/A41_28 at 1:1 00.3 This was followed by washing and application of a 10-nm colloidal gold-tagged secondary antibody (Probetec, Buckingham, PA) for 1 hour. After fixation with 1% glutaraldehyde for 10 minutes, sections were stained with lead citrate and uranyl acetate. Control sections were treated with primary antisera that had been preabsorbed with their corresponding synthetic peptides.
Immunogold Labeling of Lowicryl Sections
Methods Cerebral cortex and cerebellum from six autopsied patients, five with clinical and neuropathologic AD and one with Down's syndrome (trisomy 21) and neuropathologic AD (Table 1), were examined ultrastructurally by the methods listed. All AD patients died at a clinically advanced stage of the disease, and the Down's syndrome patient showed typical advanced AD histopathology. All six brains had large numbers of neocortical senile plaques.
Routine Electron Microscopy Postmortem tissue samples were fixed with 4% paraformaldehyde and 1% glutaraldehyde solution, then with OS04, and embedded in epon (PolyBed). Semithin sections (1 ,um) were labeled with anti-P/A4 and an ABC Elite kit (Vector, Burlingame, CA) after formic acid pretreatment, as previously reported.17 The fine structure of regions of blood vessels that showed P/A4-positivity was examined in immediately adjacent ultrathin sections.
Some tissue samples were fixed with 4% paraformaldehyde solution and embedded in hydrophilic methacrylate (Lowicryl), which was polymerized at -30°C. Ultrathin sections were placed on formvar-coated nickel grids. Postembedding immunogold labeling of the ultrathin sections began after the blocking step in 10% NGS, as outlined earlier.
Results We examined 38 blood vessels showing AA by ,B/A4 immunolabeling of semithin or ultrathin sections: 14 meningeal arteries or arterioles; 8 cortical arterioles; and 16 capillaries (Table 1). Among these 38 vessels, 10 showed circumferential P/A4 labeling, whereas 28 showed focal labeling. In this study, the fine structure of these small, focally positive areas is described.
Larger Leptomeningeal Arteries Larger leptomeningeal arteries have internal elastic laminae and many layers of smooth muscle cells. At the light
Table 1. Summary of Cases with Cerebral Amyloid Angiopathy
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6
Age
Sex
Diagnosis
59 65 81 86 88 90
F
Down AD AD AD AD AD
M M F F F
Site
Occip
Cerebell Hippoc
Occip Occip Temp
EM
Location of AA
Methods Used Epon Lowicryl G R,G
in the 38 microvessels examined
R R
R,G R,G R
G G
Men(4), Cor(1) Men(2) Cap(5) Men(5), Cap(2) Men(3), Cor(7), Cap(7) Men(2)
AD: Alzheimer's disease; R: regular EM with matched immunostaining of adjacent semithin section; G: immunogold on grid; Men: meningeal arteries; Cor: cortical arterioles; Cap: capillaries.
EM of Amyloid Angiopathy
251
AJP July 1992, Vol. 141, No. I
ill,
LJs S {X""'-
4
?S 11-
Figure 1. /A4 immunoperoxidase labeling ofsemithin sections ofAD brain, showing (a) small focal F/A4 deposits (arrow) and large, more extensive deposits (arrowhead) at the media-adventitia junction in a large leptomeningeal artery; (b) focal (arrows) and circumferential deposits (arrowheads) in small leptomeningeal arteries; and (c) smallfocal deposits (arrow) and extravasated amyloid deposits (arrouhead) in cortical capillaries (c). The deposits indicated by arrows in (a) and (b) are shoun at high power in immediately adjacent sections in Figures 2 and 3, respectively. (a) x220; (b,c) x290.
microscopic level, small foci of /A4 immunoreactivity were usually localized to the media-adventitia junction (Figure 1 a). The fine structure of small, presumably early amyloid foci consisted of small clusters of delicate amyloid fibrils (-8 nm) situated in the outermost (abluminal) part of the basement membrane (BM), around intact smooth muscle cells at the media-adventitia junction (Figure 2). In more widespread ,B/A4-positive areas, i.e., presumably more advanced stages, coarser amyloid fibrils (-10 nm) spread toward the internal elastic laminae on the luminal side of the BM. In such deposits, amyloid
+. ~ W+ ~ . ~ !a
>
¢ :C^
. >.
-" *
.,,.t''
''.
_.iz-u&|]2SFE,w.:efs>vl
Copyright (© American Association of Pathologists
Beta Amyloid Is Focally Deposited Within the Outer Basement Membrane in the Amyloid Angiopathy of Alzheimer's Disease An Immunoelectron Microscopic Study
H. Yamaguchi,*t T. Yamazaki," C. A. Lemere,*t M. P. Frosch,t and D. J. Selkoe*t From the Department ofNeurology, * Harvard Medical School, Boston, the Center for Neurologic Diseases,t and the Departments of Medicine (Neurology) and Pathology, Brigham and Women's Hospital, Boston, Massachusetts; and the Department of Neurology,." Gunma Unitersity School of Medicine, Maebashi, Japan
The fine structure of cerebral amyloid angiopathy, especially in small and presumably early deposits, was examined by immunolabeling of the P/A4 protein in semithin and ultrathin sections from brains with Alzheimer's disease. The following findings emerged: 1) in large leptomeningeal arteries, smanl focal amyloid deposits appear to consist of clusters of delicate (-8 nm diameter) amyloid fibrils, not previously described, in the outermost part of the basement membrane (BM) at the media-adventitia junction; 2) in small leptomeningeal arteries and perforating cortical arterioles, small foci of delicate amyloid fibrils were observed within the BM. They appeared mostly in the outer portion of the BM, around intact smooth muscle cells, rather than in the subendothelial region. In larger and presumably more advanced deposits, coarse amyloidfibrils (-10 nm) occupied the abluminal BM, and adjacent smooth muscle cells showed degeneration; and 3) in capillaries, small amounts of delicate (-8 nm) amyloid fibrils, not previously described, were seen within the BM in the smallest discernible deposits. The BM at these sites was abnormally folded and layered In larger deposits, amyloidfibrils appeared to extravasatefrom the outerBM of the capillary into the neuropil and were surrounded by astrocytic foot processes and/or microglia Our results suggest that vascular amyloid fibrils may first be formed within
the abluminal vascular BM, that i outside of cells. The BM may trap degradative intermediates of the amyloid precursor protein that contain the /A4 region, and local proteases may then cleave them further to yield amyloidogenic fragments. (Am JPathol 1992, 141:249-259)
Amyloid angiopathy (AA) and senile plaques (SP) are prominent forms of ,B/A4 amyloid deposition in the human brain.14 AA is an age-related change and is not closely associated with the clinical expression of dementia, although it is almost invariably present to some degree in Alzheimer's disease (AD).9 AA occurs in both leptomeningeal arteries and intracortical arterioles and capillaries, and less frequently in veins.-7710 After the discovery of the ,/3A4 protein as the major constituent of cerebrovascular amyloid in AD,1 many investigators have attempted to characterize the mechanism of amyloid deposition. Recent research suggests that alternative degradative pathways for the /A4 amyloid precursor protein (3APP)11 exist in cultured cells1'214,32 and tissues1 5,16 and may lead to the production of the amyloid fragment, /A4 protein, or larger potentially amyloidogenic intermediates. To gain information about the fine structure of small and presumably early /A4 amyloid deposits, we examined small foci of amyloid fibrils within the walls of selected meningeal and cortical microvessels in sections of AD cerebral cortex by electron microscopy. The structure Supported in part by NIH grants AG 07911 (LEAD Award) and AG 06173 and by a gift from the Broad Family Foundation. Accepted for publication March 12, 1992. Address reprint requests to Dr. Dennis J. Selkoe, Center for Neurologic Diseases, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115.
249
250
Yamaguchi et al
AJP July 1992, Vol. 141, No. 1
of AA is simpler than that of the ,B/A4-containing senile plaques in the cerebral cortex, and meningovascular AA in particular presents a simple system devoid of surrounding neurons, astrocytes, and other neural elements. Moreover, the detailed study of AA should provide insights about the potential contribution of vascular and circulating factors to progressive amyloidogenesis, and these might be more amenable to therapeutic manipulation than factors arising within brain tissue. To assess the fine structure of small amyloid deposits in the cerebral microvasculature, we used two methods: anti-,B/A4 immunogold labeling on ultrathin sections, and anti-P/A4 labeling of semithin sections and EM examination of adjacent ultrathin sections.
Immunogold Labeling of Epon Sections Ultrathin sections from epon-embedded tissues on nickel grids were treated with 10% H202 for 10 minutes, 1% periodic acid for 10 minutes and 10% normal goat serum (NGS) for 30 minutes. One of two anti-,/A4 antisera was applied for 2 hours: R1280 against synthetic P/A41X4 at a dilution of 1:1000,15 or p28K against synthetic P/A41_28 at 1:1 00.3 This was followed by washing and application of a 10-nm colloidal gold-tagged secondary antibody (Probetec, Buckingham, PA) for 1 hour. After fixation with 1% glutaraldehyde for 10 minutes, sections were stained with lead citrate and uranyl acetate. Control sections were treated with primary antisera that had been preabsorbed with their corresponding synthetic peptides.
Immunogold Labeling of Lowicryl Sections
Methods Cerebral cortex and cerebellum from six autopsied patients, five with clinical and neuropathologic AD and one with Down's syndrome (trisomy 21) and neuropathologic AD (Table 1), were examined ultrastructurally by the methods listed. All AD patients died at a clinically advanced stage of the disease, and the Down's syndrome patient showed typical advanced AD histopathology. All six brains had large numbers of neocortical senile plaques.
Routine Electron Microscopy Postmortem tissue samples were fixed with 4% paraformaldehyde and 1% glutaraldehyde solution, then with OS04, and embedded in epon (PolyBed). Semithin sections (1 ,um) were labeled with anti-P/A4 and an ABC Elite kit (Vector, Burlingame, CA) after formic acid pretreatment, as previously reported.17 The fine structure of regions of blood vessels that showed P/A4-positivity was examined in immediately adjacent ultrathin sections.
Some tissue samples were fixed with 4% paraformaldehyde solution and embedded in hydrophilic methacrylate (Lowicryl), which was polymerized at -30°C. Ultrathin sections were placed on formvar-coated nickel grids. Postembedding immunogold labeling of the ultrathin sections began after the blocking step in 10% NGS, as outlined earlier.
Results We examined 38 blood vessels showing AA by ,B/A4 immunolabeling of semithin or ultrathin sections: 14 meningeal arteries or arterioles; 8 cortical arterioles; and 16 capillaries (Table 1). Among these 38 vessels, 10 showed circumferential P/A4 labeling, whereas 28 showed focal labeling. In this study, the fine structure of these small, focally positive areas is described.
Larger Leptomeningeal Arteries Larger leptomeningeal arteries have internal elastic laminae and many layers of smooth muscle cells. At the light
Table 1. Summary of Cases with Cerebral Amyloid Angiopathy
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6
Age
Sex
Diagnosis
59 65 81 86 88 90
F
Down AD AD AD AD AD
M M F F F
Site
Occip
Cerebell Hippoc
Occip Occip Temp
EM
Location of AA
Methods Used Epon Lowicryl G R,G
in the 38 microvessels examined
R R
R,G R,G R
G G
Men(4), Cor(1) Men(2) Cap(5) Men(5), Cap(2) Men(3), Cor(7), Cap(7) Men(2)
AD: Alzheimer's disease; R: regular EM with matched immunostaining of adjacent semithin section; G: immunogold on grid; Men: meningeal arteries; Cor: cortical arterioles; Cap: capillaries.
EM of Amyloid Angiopathy
251
AJP July 1992, Vol. 141, No. I
ill,
LJs S {X""'-
4
?S 11-
Figure 1. /A4 immunoperoxidase labeling ofsemithin sections ofAD brain, showing (a) small focal F/A4 deposits (arrow) and large, more extensive deposits (arrowhead) at the media-adventitia junction in a large leptomeningeal artery; (b) focal (arrows) and circumferential deposits (arrowheads) in small leptomeningeal arteries; and (c) smallfocal deposits (arrow) and extravasated amyloid deposits (arrouhead) in cortical capillaries (c). The deposits indicated by arrows in (a) and (b) are shoun at high power in immediately adjacent sections in Figures 2 and 3, respectively. (a) x220; (b,c) x290.
microscopic level, small foci of /A4 immunoreactivity were usually localized to the media-adventitia junction (Figure 1 a). The fine structure of small, presumably early amyloid foci consisted of small clusters of delicate amyloid fibrils (-8 nm) situated in the outermost (abluminal) part of the basement membrane (BM), around intact smooth muscle cells at the media-adventitia junction (Figure 2). In more widespread ,B/A4-positive areas, i.e., presumably more advanced stages, coarser amyloid fibrils (-10 nm) spread toward the internal elastic laminae on the luminal side of the BM. In such deposits, amyloid
+. ~ W+ ~ . ~ !a
>
¢ :C^
. >.
-" *
.,,.t''
''.
_.iz-u&|]2SFE,w.:efs>vl
