doi:10.1016/j.nbd.2005.04.012
Copyright © 2005 Elsevier Inc. All rights reserved.
Apolipoprotein D is a component of compact but not diffuse amyloid-beta plaques in Alzheimer's disease temporal cortex
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Purnima P. Desaia, 1, Milos D. Ikonomovicb, c, 1, Eric E. Abrahamsonb, Ronald L. Hamiltond, Barbara A. Isanskib, Caroline E. Hopeb, William E. Klunkc, Steven T. DeKoskya, b, c and M. Ilyas Kamboha, c, 
, 
aDepartment of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA
bDepartment of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
cDepartment of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
dDepartment of Neuropathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
Received 15 December 2004;
revised 31 March 2005;
accepted 9 April 2005.
Available online 23 May 2005.
Abstract
Apolipoprotein D (apoD) is elevated in Alzheimer's disease (AD) cortex, localizing to cells, blood vessels, and neuropil deposits (plaques). The role of apoD in AD pathology and the extent of its co-distribution with diffuse (amorphous) and compact (dense fibrillar) amyloid-beta (Aβ) plaques are currently unclear. To address this issue, we combined apoD and Aβ immunohistochemistry with ThioS/X-34 staining of the β-pleated sheet protein conformation in temporal cortex from 36 AD patients and 12 non-demented controls. ApoD-immunoreactive, Aβ-immunoreactive, and ThioS/X-34-stained plaques were detected exclusively in AD tissue. Dual-immunolabeling showed that 63% of Aβ plaques co-localized apoD. All apoD plaques contained Aβ protein and ThioS/X-34 fluorescence. Compared to controls, AD cases showed elevated vascular and intracellular apoD immunostaining which localized primarily to cells clustered within plaques and around large blood vessels. ApoD-immunoreactive cells within plaques morphologically matched MHC-II- and CD-68-immunoreactive microglia, and did not contain the astrocytic marker GFAP, which labeled a subset of apoD-immunoreactive cells surrounding plaques. These data suggest that neuropil deposits of apoD localize only to a subset of Aβ plaques, which contain compact aggregates of fibrillar Aβ. Elevated apoD in AD brain may influence Aβ aggregation, or facilitate phagocytosis and transport of Aβ fibrils from plaques to cerebral vasculature.
Keywords: Alzheimer's disease; Amyloidosis; Apolipoprotein; Astrocyte; Microglia; Neuritic plaque; Neurodegeneration; Temporal cortex
Fig. 1. Immunohistochemical and Western blot characterization of the apoD antibody used in this investigation. (A) AD temporal cortex with a typical pattern of apoD immunoreactivity in glia (arrowheads), isolated neurons (double arrowhead), and perivascular or neuropil plaque deposits (arrows). (B) An adjacent tissue section incubated with apoD antibody preabsorbed with apoD protein shows no immunostaining (an unstained blood vessel is indicated by arrows). (C) Western blot analysis demonstrates that apoD and Aβ antibodies, used in the co-localization experiments in this study, specifically recognize appropriate antigens and do not cross react. (D) Western blot analysis of temporal cortex gray matter from two representative non-demented controls (C1–2) and two AD cases shows considerable variability in apoD protein levels in controls and an increase in AD, further confirming the specificity of the apoD antibody. Scale bar = 50 μm.
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Fig. 2. ApoD immunoreactivity in the temporal cortex from non-demented control (A) and AD subjects (B–G). (A) In control subject, apoD immunoreactivity is localized to glial cells and blood vessels. (B) AD temporal cortex shows more intense apoD labeling of glial and vascular elements, as well as numerous apoD-immunoreactive plaques and perivascular aggregates. (C,D) Higher-power photomicrographs of apoD plaque immunostaining reveal round-shaped apoD-immunoreactive cells inside apoD plaques. The density of these cells in plaques is proportional to the degree of the overall plaque and vascular apoD immunostaining. An AD case with minimal vascular immunostaining shows apoD plaques with lightly increased neuropil immunostaining and a few plaque-associated (arrows) or free-standing (arrowheads) glia (C). An AD case with more prominent vascular immunostaining displays darker apoD plaques with numerous, intensely apoD-immunoreactive glia clustered within plaques or around large blood vessel lumen (arrows), while many apoD-immunoreactive glia are seen free-standing (arrowheads) in the neuropil (D). (E–G) Three adjacent sections from temporal cortex of an AD case, immunostained with apoD (E represents the area boxed in D) or microglia markers, illustrate the similarity between apoD- and MHC-II- or CD-68-immunoreactive cells clustering inside plaques (arrowheads in G). Scale bar = 100 μm (A,B), 50 μm (C,D), and 25 μm (E–G).
Fig. 3. Photomicrographic composites on adjacent sections of AD temporal cortex immunostained for the neuronal antigen NeuN (A), which delineates cortical layers I–VI, apoD (B), and 6E10 (C) antibodies. There is a similar overall distribution pattern of apoD and Aβ plaques across cortical layers. In layers III–IV, apoD plaques show lighter immunoreactivity compared to Aβ plaques. WM = white matter.
Fig. 4. Paired light/fluorescent microscopic images of dual apoD immunohistochemistry (IHC)/fluorescent histochemistry (HC) in the temporal cortex of an AD subject. (A–B) All apoD deposits (arrows) are labeled with X-34, while numerous apoD-immunoreactive capillaries are X-34 negative. (C,D) Higher-magnification images of a large plaque associated with apoD-immunoreactive glia that also contains ThioS fluorescence. Scale bar = 200 μm (A,B), 100 μm (C,D).
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Fig. 5. Co-distribution of apoD with Aβ (A–C) or GFAP (D–F) in the temporal cortex of AD patients. (A,B) Paired fluorescent microscopic images of dual immunofluorescence for apoD (green) and the carboxy terminus of Aβ (red) show that the two peptides are localized to the same plaque. ApoD-immunoreactive cell elements are clustered in the plaque center (arrow) or scattered in the neuropil (arrowheads), while Aβ40/42 staining is distributed throughout the plaque. (C) Dual chromogen immunohistochemistry for apoD (blue) and the amino terminus of Aβ (brown) shows co-distribution of the peptides in large compact plaques, with apoD concentrated in the center (arrows). Smaller diffuse Aβ plaque is free of apoD (arrowhead). (D,E) Fluorescence and bright field microscopy of double immunohistochemistry for apoD and GFAP (D: apoD-green/GFAP-red; E: apoD-blue/GFAP-brown) illustrate that GFAP-immunoreactive astrocytes are restricted to the periphery of apoD plaques, while GFAP-negative apoD-immunoreactive cells are mainly inside plaques (double arrowhead in D). GFAP-immunoreactive astrocytes contain granular apoD material (arrows in D), while some apoD+/GFAP− cells are observed in the neuropil (arrowheads in D). (F) Bright field of dual-label immunohistochemistry for apoD (blue) and GFAP (brown) shows apoD immunostaining in the wall of a large-caliber blood vessel, surrounded by GFAP-immunoreactive astrocytes. Scale bar = 75 μm (A–E), 100 μm (F).
Table 1.
Summary of histofluorescent markers and antisera used for histological, immunohistochemical, and Western blot analyses
b = Biotinylated.
a Clone.
Table 2.
Demographic data, results of plaque analyses, and apoD protein measurements in temporal cortex from AD subjects and non-demented controls
n = Number of cases; PMI = postmortem interval; WB = Western blot; R.O.D. = relative optical density.
a Plaque pathology scored as absent (0), mild (1), moderate (2), or severe (3).
b P2-tailed < 0.01 (R.O.D. normalized to control levels).
Support: NIA AG13672 and NIA AG05133.
Corresponding author. Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA. Fax: +1 412 383 7844.
1 Both authors contributed equally to this work.
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