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, 188 (3), 431-8

Complement-dependent Proinflammatory Properties of the Alzheimer's Disease Beta-Peptide

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Complement-dependent Proinflammatory Properties of the Alzheimer's Disease Beta-Peptide

B M Bradt et al. J Exp Med.

Abstract

Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the beta-amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement system mediates the cellular changes that surround fibrillar Abeta deposits in NP. We report that Abeta peptides directly and independently activate the alternative complement pathway as well as the classical complement pathway; trigger the formation of covalent, ester-linked complexes of Abeta with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells. These findings provide inflammation-based mechanisms to account for the presence of complement components in NP in association with damaged neurons and increased numbers of activated glial cells, and they have potential implications for the therapy of AD.

Figures

Figure 1
Figure 1
ELISA demonstration of complement-mediated formation of complexes of Aβ with C3 activation fragments (a– d  ). Complexes were captured, detected, and quantitated as described in Materials and Methods. NHS or purified ACP proteins only do not contain Aβ. (a) Preaggregated Aβ 1–42 (100 μM and 50 μM) was incubated in NHS, captured with mAb to C3b, and detected with rabbit Ab to Aβ. (b) Two preaggregated Aβ 1–42 preparations (Aβ 1 and Aβ 2, at 20 μM) were incubated in NHS, captured with mAb to iC3b, and detected with rabbit Ab to Aβ. (c) Preaggregated Aβ 1–42 (58 μM) was incubated in NHS, or in NHS containing 10 mM EDTA, captured with mAb to Aβ, and detected with rabbit Ab to C3. (d  ) Preaggregated Aβ 1–42 was incubated in NHS at the indicated final concentrations, captured with mAb to Aβ, and detected with rabbit Ab to C3. (e) Preaggregated Aβ 1–42 (58 μM) was incubated in NHS, factor B–depleted NHS (B-dpl  ), or C1q-depleted NHS (C1q-dpl  ), captured with mAb to Aβ, and detected with rabbit Ab to C3. (f   ) Preaggregated Aβ 1–42 (58 μM) was incubated with the six purified ACP proteins (PAP) or NHS, captured with mAb to Aβ, and detected with rabbit Ab to C3. Background levels obtained in EDTA controls containing Aβ and purified ACP proteins or NHS, in the various experiments described above, were subtracted. (  g) Specificity of complement activation. Preaggregated Aβ preparations (20 μM) and the same concentrations of monomeric Aβ (mono), insulin B chain (Ins.B), neuropeptide Y-porcine (NPY), urotensin I (Uro.), exendin 3 (Exen), amyloid precursor peptide 657–676 (APP-pep.), and adenovirus penton base 50-residue peptide (PB50) were incubated with NHS. Complement activation was assessed by the CH50 method. Correlation coefficients for the CH50 determinations ranged from 0.995 to 1.000.
Figure 1
Figure 1
ELISA demonstration of complement-mediated formation of complexes of Aβ with C3 activation fragments (a– d  ). Complexes were captured, detected, and quantitated as described in Materials and Methods. NHS or purified ACP proteins only do not contain Aβ. (a) Preaggregated Aβ 1–42 (100 μM and 50 μM) was incubated in NHS, captured with mAb to C3b, and detected with rabbit Ab to Aβ. (b) Two preaggregated Aβ 1–42 preparations (Aβ 1 and Aβ 2, at 20 μM) were incubated in NHS, captured with mAb to iC3b, and detected with rabbit Ab to Aβ. (c) Preaggregated Aβ 1–42 (58 μM) was incubated in NHS, or in NHS containing 10 mM EDTA, captured with mAb to Aβ, and detected with rabbit Ab to C3. (d  ) Preaggregated Aβ 1–42 was incubated in NHS at the indicated final concentrations, captured with mAb to Aβ, and detected with rabbit Ab to C3. (e) Preaggregated Aβ 1–42 (58 μM) was incubated in NHS, factor B–depleted NHS (B-dpl  ), or C1q-depleted NHS (C1q-dpl  ), captured with mAb to Aβ, and detected with rabbit Ab to C3. (f   ) Preaggregated Aβ 1–42 (58 μM) was incubated with the six purified ACP proteins (PAP) or NHS, captured with mAb to Aβ, and detected with rabbit Ab to C3. Background levels obtained in EDTA controls containing Aβ and purified ACP proteins or NHS, in the various experiments described above, were subtracted. (  g) Specificity of complement activation. Preaggregated Aβ preparations (20 μM) and the same concentrations of monomeric Aβ (mono), insulin B chain (Ins.B), neuropeptide Y-porcine (NPY), urotensin I (Uro.), exendin 3 (Exen), amyloid precursor peptide 657–676 (APP-pep.), and adenovirus penton base 50-residue peptide (PB50) were incubated with NHS. Complement activation was assessed by the CH50 method. Correlation coefficients for the CH50 determinations ranged from 0.995 to 1.000.
Figure 2
Figure 2
Assessment of bonds mediating binding of Aβ to C3 activation fragments. (a) Preaggregated Aβ 1–42 (25 μM) was incubated in NHS, and complexes were captured on 10D5-coated wells; NHS only does not contain Aβ. Replicate samples were treated with pH 9.5 buffer, or with the same buffer containing 1 M hydroxylamine (NH2OH ), and remaining bound C3 was then detected and quantitated as described in Materials and Methods. The control containing Aβ, NHS, and EDTA has been subtracted. (b) Replicate wells subjected to treatment with the pH 9.5 buffer or hydroxylamine were evaluated for residual bound Aβ as described in Materials and Methods. (c) Preaggregated Aβ 1–40 was incubated in NHS in the presence or absence of EDTA; lane 5 contains NHS but no Aβ. After centrifugation and washing, fibrillar Aβ pellets were incubated with pH 7.4 buffer (lanes 1 and 2), pH 9.5 buffer (lane 3), or 1 M hydroxylamine in pH 9.5 buffer (lane 4). After further washing, samples were subjected to SDS-PAGE under nonreducing conditions followed by blotting for the presence of C3 and, after stripping, for Aβ. Arrow, The C3 band at ∼180 kD. (d) Preaggregated Aβ 1–42 (50 μM) was incubated in NHS alone, and in the presence of deferoxamine (Defer.; 1 mM), glutathione (Glut.; 1 mM), dimethylthiourea (DMTU; 30 mM), catalase (CAT; 2 × 104 U/ml), SOD (10 μM), or catalase plus SOD, and the Aβ complexes with C3 activation fragments were then detected as described in Materials and Methods.
Figure 2
Figure 2
Assessment of bonds mediating binding of Aβ to C3 activation fragments. (a) Preaggregated Aβ 1–42 (25 μM) was incubated in NHS, and complexes were captured on 10D5-coated wells; NHS only does not contain Aβ. Replicate samples were treated with pH 9.5 buffer, or with the same buffer containing 1 M hydroxylamine (NH2OH ), and remaining bound C3 was then detected and quantitated as described in Materials and Methods. The control containing Aβ, NHS, and EDTA has been subtracted. (b) Replicate wells subjected to treatment with the pH 9.5 buffer or hydroxylamine were evaluated for residual bound Aβ as described in Materials and Methods. (c) Preaggregated Aβ 1–40 was incubated in NHS in the presence or absence of EDTA; lane 5 contains NHS but no Aβ. After centrifugation and washing, fibrillar Aβ pellets were incubated with pH 7.4 buffer (lanes 1 and 2), pH 9.5 buffer (lane 3), or 1 M hydroxylamine in pH 9.5 buffer (lane 4). After further washing, samples were subjected to SDS-PAGE under nonreducing conditions followed by blotting for the presence of C3 and, after stripping, for Aβ. Arrow, The C3 band at ∼180 kD. (d) Preaggregated Aβ 1–42 (50 μM) was incubated in NHS alone, and in the presence of deferoxamine (Defer.; 1 mM), glutathione (Glut.; 1 mM), dimethylthiourea (DMTU; 30 mM), catalase (CAT; 2 × 104 U/ml), SOD (10 μM), or catalase plus SOD, and the Aβ complexes with C3 activation fragments were then detected as described in Materials and Methods.
Figure 2
Figure 2
Assessment of bonds mediating binding of Aβ to C3 activation fragments. (a) Preaggregated Aβ 1–42 (25 μM) was incubated in NHS, and complexes were captured on 10D5-coated wells; NHS only does not contain Aβ. Replicate samples were treated with pH 9.5 buffer, or with the same buffer containing 1 M hydroxylamine (NH2OH ), and remaining bound C3 was then detected and quantitated as described in Materials and Methods. The control containing Aβ, NHS, and EDTA has been subtracted. (b) Replicate wells subjected to treatment with the pH 9.5 buffer or hydroxylamine were evaluated for residual bound Aβ as described in Materials and Methods. (c) Preaggregated Aβ 1–40 was incubated in NHS in the presence or absence of EDTA; lane 5 contains NHS but no Aβ. After centrifugation and washing, fibrillar Aβ pellets were incubated with pH 7.4 buffer (lanes 1 and 2), pH 9.5 buffer (lane 3), or 1 M hydroxylamine in pH 9.5 buffer (lane 4). After further washing, samples were subjected to SDS-PAGE under nonreducing conditions followed by blotting for the presence of C3 and, after stripping, for Aβ. Arrow, The C3 band at ∼180 kD. (d) Preaggregated Aβ 1–42 (50 μM) was incubated in NHS alone, and in the presence of deferoxamine (Defer.; 1 mM), glutathione (Glut.; 1 mM), dimethylthiourea (DMTU; 30 mM), catalase (CAT; 2 × 104 U/ml), SOD (10 μM), or catalase plus SOD, and the Aβ complexes with C3 activation fragments were then detected as described in Materials and Methods.
Figure 3
Figure 3
Aβ-mediated complement activation generates C5a and the MAC. (a) Preaggregated Aβ 1–42 was incubated in NHS, and C5a generation was then quantitated as described in Materials and Methods. (b) C5b-9 was quantitated after NHS was incubated with varying concentrations of preaggregated Aβ 1–42 or 1–40. SC5b-9 formation was quantitated as described in Materials and Methods.
Figure 4
Figure 4
C5b-9 generated by Aβ 1–42-mediated complement activation is functionally active. (a) NT2 cells were incubated with preaggregated Aβ 1–42 at the designated concentrations in NHS, or in NHS containing 10 mM EDTA; serum only lacks Aβ. Flow cytometric analyses with a rabbit Ab to C5b-9 neoantigens are shown. Numbers (right), Percentage of C5b-9+ cells, as determined by their relationship to the marker (arrow, dashed line). (b) Density plot analyses of propidium iodide and C5b-9 (FITC C5b-9) reactivities are shown. For clarity, only live cells are depicted.
Figure 4
Figure 4
C5b-9 generated by Aβ 1–42-mediated complement activation is functionally active. (a) NT2 cells were incubated with preaggregated Aβ 1–42 at the designated concentrations in NHS, or in NHS containing 10 mM EDTA; serum only lacks Aβ. Flow cytometric analyses with a rabbit Ab to C5b-9 neoantigens are shown. Numbers (right), Percentage of C5b-9+ cells, as determined by their relationship to the marker (arrow, dashed line). (b) Density plot analyses of propidium iodide and C5b-9 (FITC C5b-9) reactivities are shown. For clarity, only live cells are depicted.

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