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. 2001 Jul 17;98(15):8850-5.
doi: 10.1073/pnas.151261398. Epub 2001 Jul 3.

Peripheral anti-A Beta Antibody Alters CNS and Plasma A Beta Clearance and Decreases Brain A Beta Burden in a Mouse Model of Alzheimer's Disease

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Peripheral anti-A Beta Antibody Alters CNS and Plasma A Beta Clearance and Decreases Brain A Beta Burden in a Mouse Model of Alzheimer's Disease

R B DeMattos et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Active immunization with the amyloid beta (A beta) peptide has been shown to decrease brain A beta deposition in transgenic mouse models of Alzheimer's disease and certain peripherally administered anti-A beta antibodies were shown to mimic this effect. In exploring factors that alter A beta metabolism and clearance, we found that a monoclonal antibody (m266) directed against the central domain of A beta was able to bind and completely sequester plasma A beta. Peripheral administration of m266 to PDAPP transgenic mice, in which A beta is generated specifically within the central nervous system (CNS), results in a rapid 1,000-fold increase in plasma A beta, due, in part, to a change in A beta equilibrium between the CNS and plasma. Although peripheral administration of m266 to PDAPP mice markedly reduces A beta deposition, m266 did not bind to A beta deposits in the brain. Thus, m266 appears to reduce brain A beta burden by altering CNS and plasma A beta clearance.

Figures

Figure 1
Figure 1
Anti-Aβ antibody m266 acts as an Aβ sink. (A) An in vitro assay was developed to identify the relative efficiency of Aβ binding proteins on sequestering soluble CNS Aβ. One milliliter of human CSF was placed in the top chamber of a tube separated by a 25-kDa dialysis membrane from a bottom chamber that contained 75 μl of PBS. Human CSF used in these studies contained, on average, 10 ng/ml of AβTotal (2.5 pmol/ml). (B) The % Aβ removed from the top chamber was determined by Aβ ELISA analysis of both the top and bottom chamber (n = 4 per condition) after a 3-h incubation at 37°C. The bottom chamber contained PBS ± 20 μg of the listed proteins (IgG and m266, 133 pmol; BSA, 332 pmol; apoE4, 585 pmol). Affinity-purified, astrocyte-secreted apoE4 lipoproteins, a known Aβ binding protein, sequestered significantly more Aβ (3.86%, P < 0.05) to the bottom chamber than nonspecific mouse IgG (2.18%) or BSA (2.64%). The Aβ antibody m266 was dramatically more efficient at sequestering CSF Aβ (48.91%) to the bottom chamber as compared with all other conditions tested (P < 0.001). Data were analyzed with ANOVA followed by post hoc Tukey's test.
Figure 2
Figure 2
m266 sequesters Aβ in vivo. (A) Three-month-old PDAPP+/+ mice were treated with PBS (n = 7) or 500 μg of m266 (n = 9) i.v. Twenty-four hours after m266 administration, plasma was analyzed for AβTotal by RIA. Before analysis, all plasma samples were first treated with protein G to remove Aβ complexed to m266. (B) In the first three lanes, 20 μl of plasma from PDAPP mice (24 h after administering 500 μg of m266 or PBS i.v.) was run on acid-urea gels followed by Western blotting for Aβ with m6E10 (Senetek, Napa, CA). Lanes 4 and 5 demonstrate that the plasma Aβ, from 20 μl of m266-injected mice, can be completely cleared with protein G treatment for animals treated with m266. In lanes 6 and 7, the protein-G beads from the 20 μl of plasma from lanes 4 and 5 were washed, denatured in formic acid, and analyzed. Lane 8 represents the protein G beads from 20 μl of plasma from a PBS-injected mouse. (C) Tissues collected from adult PDAPP+/+ mice were homogenized in a buffer containing 1% SDS. One hundred micrograms of total protein from each lysate was subjected to reducing 8% SDS/PAGE and analyzed by Western blotting for human APP with m6E10. (D) CSF Aβ40 and Aβ42 was determined by RIA from 3-month-old PBS- (n = 23) and m266-treated PDAPP mice 3 (n = 9), 24 (n = 5), and 72 h (n = 9) after i.v. injection of m266 as above. There was significantly greater Aβ40 in the CSF of the m266-treated mice at 3, 24, and 72 h and Aβ42 at 24 and 72 h (*, P < 0.05; **, P < 0.0001, ANOVA followed by post hoc Tukey's test.). Two microliters of CSF from each mouse per treatment group was collected, pooled (15 μl total), and subjected to acid urea gels followed by Western blotting with Aβ-specific antibodies.
Figure 3
Figure 3
i.v. m266 detects rapid efflux of exogenous and endogenous Aβ from CNS into plasma. (A) One microgram of Aβ40 was dissolved into 5 μl of rat CSF to keep it soluble and was then injected into the subarachnoid space of the cisterna magna of wild-type Swiss–Webster mice that had previously received either PBS (n = 3) or 200 μg of biotinylated m266 (n = 3, i.v.). At different time points after treatment, plasma AβTotal was determined by Aβ RIA. (B and C) Either 200 μg (n = 3) or 600 μg (n = 3) of m266 was injected i.v. into 3-month-old PDAPP+/+ mice. Before and at different time points after i.v. injection, the plasma concentration of Aβ bound to m266 was determined by RIA and each value is presented as mean ± SEM. (B) The amount of Aβ bound to m266 is illustrated up to 4 days after treatment. (C) The time course over the first several hours for all animals is shown.
Figure 4
Figure 4
Chronic administration of m266 decreases Aβ burden in PDAPP mice. Four-month-old PDAPP+/+ mice were treated every 2 weeks for 5 months with saline (n = 14), m266 (n = 14, 500 μg), or control mouse mAb (n = 13, control IgG, 100 μg, PharMingen), all administered i.p. The % area covered by Aβ immunoreactivity as identified with a rabbit pan-Aβ antibody (BioSource International, Camarillo, CA) was quantified in the cerebral cortex immediately overlying the dorsal hippocampus as described (35). (A) At 9 months of age, about half of each group had still not developed Aβ deposits. In PBS- and IgG-treated animals, 6/14 and 5/13 mice, respectively, had greater than 50% of the cortex covered by Aβ staining. In contrast, only 1/14 m266-treated mice had this level of Aβ staining. The proportion of mice with this level of Aβ staining was lowered by treatment with m266 (P = 0.02, χ2 test). Levels of PBS-soluble and insoluble Aβ in cortex of PBS and m266 treated groups were as follows (mean Aβ in ng/mg protein ± SEM): soluble AβTotal, PBS, 0.115 ± 0.019, m266, 0.06 ± 0.007, P = 0.01; insoluble AβTotal, PBS, 4.64 ± 1.62, m266, 1.4 ± 0.34, P = 0.06; soluble Aβ42, PBS, 0.026 ± 0.002, m266, 0.020 ± 0.002, P = 0.04; insoluble Aβ42, PBS, 2.66 ± 1.18, m266, 0.62 ± 0.166, P = 0.09. (B) The variability in Aβ deposition within the groups was related in some way to parental origin. Even though all mice used were PDAPP+/+ transgenic mice, plaque burdens of 50% or greater were only seen in mice derived from four of eight breeding pairs (high pathology litters). Most mice from the other breeding pairs were free of plaques (low pathology litters). Using parental origin as a covariate, there was a strong effect of m266 in reducing Aβ burden. When comparing m266 to controls in all groups (high and low pathology litters), P = 0.0082. When comparing m266 to controls in only the high pathology litters, P = 0.00025.

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