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The Journal of Neuroscience, September 15, 2002, 22(18):7873-7878
Non-Fc-Mediated Mechanisms Are Involved in Clearance of
Amyloid- In Vivo by Immunotherapy
Brian J.
Bacskai1,
Stephen T.
Kajdasz1,
Megan
E.
McLellan1,
Dora
Games2,
Peter
Seubert2,
Dale
Schenk2, and
Bradley T.
Hyman1
1 Alzheimer's Disease Research Laboratory,
Massachusetts General Hospital, Charlestown, Massachusetts 02129, and
2 Elan Pharmaceuticals, South San Francisco, California
94080
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ABSTRACT |
Transgenic (Tg) mouse models overexpressing amyloid
precursor protein (APP) develop senile plaques similar to those
found in Alzheimer's disease in an age-dependent manner. Recent
reports demonstrated that immunotherapy is effective at preventing or removing amyloid- deposits in the mouse models. To characterize the
mechanisms involved in clearance, we used antibodies of either IgG1
(10d5) or IgG2b (3d6) applied directly to the brains of 18-month-old Tg2576 or 20-month-old PDAPP mice. Both 10d5 and 3d6 led to
clearance of 50% of diffuse amyloid deposits in both animal models
within 3 d. Fc receptor-mediated clearance has been shown to be
important in an ex vivo assay showing antibody-mediated
clearance of plaques by microglia. We now show, using in
vivo multiphoton microscopy, that FITC-labeled
F(ab')2 fragments of 3d6 (which lack the Fc region of the
antibody) also led to clearance of 45% of the deposits within 3 d, similar to the results obtained with full-length 3d6 antibody. This
result suggests that direct disruption of plaques, in addition to
Fc-dependent phagocytosis, is involved in the antibody-mediated clearance of amyloid- deposits in vivo. Dense-core
deposits that were not cleared were reduced in size by ~30% with
full-length antibodies and F(ab')2 fragments 3 d after
a topical treatment. Together, these results indicate that clearance of
amyloid deposits in vivo may involve, in addition to
Fc-dependent clearance, a non-Fc-mediated disruption of plaque structure.
Key words:
amyloid; transgenic; Alzheimer; multiphoton; imaging; senile plaque; microglia; immunotherapy; antibody
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INTRODUCTION |
Alzheimer's disease (AD) is a
debilitating neurodegenerative disease characterized by the presence of
senile plaques in the brain (Hyman and Trojanowski, 1997 ; Markesbery,
1997; Powers, 1997). Amyloid-, a 40-42 amino acid peptide, is the
primary component of these plaques, and genetic causes of AD lead to
dramatically increased amyloid- plaque deposition (Selkoe, 1996;
Rubinsztein, 1997). Although the exact role of amyloid- deposits in
AD is unknown, senile plaques remain a primary target for drug
development aimed at prevention or reversal of the disease. Transgenic
(Tg) mouse models overexpressing amyloid precursor protein (APP)
develop senile plaques in an age-dependent manner, similar to those
found in the human disease (Games et al., 1995; Hsiao et al., 1996). The transgenic mice are valuable as a model system for evaluating therapeutic approaches toward removing or preventing formation of
amyloid- deposits.
Recently, an approach involving immunization of PDAPP mice with
amyloid- was shown to be effective at prevention of plaque deposition (Schenk et al., 1999). In these experiments, a peripheral immune response resulted in an alteration of amyloid- deposition in
the brain. A subsequent report demonstrated that anti-amyloid- antibodies, given peripherally, were also effective at prevention of
amyloid- deposition in these mice (Bard et al., 2000). This result,
using a "passive" immunotherapeutic approach, suggests that an
active T-cell-mediated immune response is not necessary for
alterations of amyloid- deposits in the brain. Moreover, we showed
recently that a single application of anti-amyloid- antibodies to
the surface of the brain in these mice led to clearance of existing
senile plaques in the remarkably short time frame of 3-8 d (Bacskai et
al., 2001). Together, these results demonstrated that immunotherapy
prevents formation of new plaques in PDAPP mice and can lead to their
clearance. The mechanism of this antibody-mediated clearance of plaques
appears to be mediated, at least in part, by Fc receptor-mediated
phagocytosis; however, additional mechanisms might also be involved.
Specifically, ex vivo experiments demonstrated that Fc
receptor-mediated phagocytosis is at least one mechanism involved in removing the amyloid- in the brain (Bard et al., 2000).
F(ab')2 fragments were unable to stimulate
microglial cells, and the process was blocked by anti-Fc receptor
antibodies, demonstrating that clearance was mediated by Fc. The close
association of microglial cells with dense-core plaques, as well as
results showing activation of these cells after topical antibody
treatment (Bacskai et al., 2001), supports this idea. Similarly, a
recent report in double transgenics expressing APP and TGF-
demonstrated clearance of amyloid- deposits via upregulation of
microglia (Wyss-Coray et al., 2001). Together, clearance of amyloid-
deposits by an Fc-mediated mechanism involving active cellular removal
seems likely. On the other hand, in vitro experiments have
shown that antibodies binding to amyloid- deposits may disrupt
-pleated sheet conformation and lead to plaque disaggregation
directly (Solomon et al., 1997). Finally, an additional possibility for
plaque clearance involves activation of complement systems that could
enhance degradation or clearance. Our current experiments used an
in vivo approach to examine these possible mechanisms.
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MATERIALS AND METHODS |
Animal preparation. Eighteen-month-old Tg2576
mice (Hsiao et al., 1996) or 20-month-old homozygous PDAPP mice (Games
et al., 1995) were used for in vivo imaging, as described
previously (Bacskai et al., 2001). Briefly, the animals were
anesthetized with avertin (1.3% tribromoethanol and 0.8%
tert-pentylalcohol in distilled water; 250 mg/kg, i.p.). and
immobilized in custom-built stage-mounted ear bars and a nosepiece,
similar to a stereotaxic apparatus. A 2-3 cm incision was made between
the ears, and the scalp was reflected to expose the skull. Four
circular craniotomies (~1-1.2 mm in diameter; on either side of
sagittal suture and just posterior to coronal suture) were made using a
high-speed drill (Fine Science Tools, Foster City, CA) and a dissecting
microscope (Leica, Wetzlar, Germany) for gross visualization. Heat and
vibration artifacts were minimized during drilling by frequent
application of artificial CSF (ACSF) (in mM: 125 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 1 CaCl2, and 25 glucose). The dura was carefully removed from each site with fine
forceps. An ACSF reservoir was created within the opened scalp over the
open skull preparations to accommodate the long working distance, water
immersion dipping objectives (Olympus Optical, Tokyo, Japan) of an
Olympus Optical BX-50 microscope. Texas Red dextran [0.05 ml; 35 mg/ml; 70,000 molecular weight (MW); Molecular Probes, Eugene,
OR] was injected into a lateral tail vein to create a
fluorescent angiogram used to facilitate image alignment from session
to session. Thioflavin S (thioS) (0.005% in ACSF; Sigma, St. Louis,
MO) and 8 µl of 1 mg/ml fluorescein-labeled antibody was then applied
to the surface of the brain for 20 min. Sites were washed with ACSF and
imaged. After imaging, the animals were sutured and allowed to recover.
Three days later, the animals were reanesthetized and prepared for
imaging. Thioflavin S and Texas Red dextran were reapplied. A
different, noncompeting labeled antibody was used in the second imaging
session to ensure that the original epitopes of amyloid- were not
simply masked at the second session.
In vivo imaging. Two-photon fluorescence was generated with
750 nm excitation from a mode-locked Ti:Sapphire laser Tsunami (Spectra-Physics, Mountain View, CA), mounted on a commercially available multiphoton imaging system (Bio-Rad 1024ES; Bio-Rad, Hercules, CA). Custom-built external detectors containing three photomultiplier tubes (Hamamatsu Photonics, Bridgewater, NJ) collected emitted light in the range of 380-480, 500-540, and 560-650 nm. Thioflavin S, FITC-labeled antibody, and Texas Red angiograms were
separated spectrally into the three imaging channels. At the end of the
experiment, the animal was killed, and the brain fixed in 4% paraformaldehyde.
Image analysis. Three-dimensional imaging volumes were
analyzed using custom macros written for Scion Image (Scion, Frederick, MD) or rendered in three-dimension using Voxblast (VayTek, Fairfield, IA). Stacks of two-dimensional images were projected using maximal intensity and aligned from one imaging session to another using blood
vessels as independent fiduciary markers. Thioflavin S-positive plaques
were measured as described previously (Christie et al., 2001). Local
image quality was assessed using the angiogram fluorescence as a
positive control. A threshold was applied to the images, and diameters
were obtained from the maximal cross-section of each plaque. Plaques
were identified by thioflavine S staining and morphological appearance.
Ambiguous deposits <5 µm in diameter were not counted. This
technique allowed us to determine whether the thioflavine S deposit
present in the original imaging session was cleared in the second
imaging session. If the plaque was not cleared, the size of the
identified plaque before and after treatment was compared. Diffuse
deposits were analyzed by measuring antibody-positive, thioflavine
S-negative amyloid- in projections of the three-dimensional imaging
volumes from each animal. Clearance was expressed as a percentage of
amyloid burden within each site from each animal after 3 d.
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RESULTS |
Our previous report demonstrated antibody-mediated clearance of
existing amyloid- deposits (Bacskai et al., 2001) in PDAPP transgenic mice, which overexpress a minigene containing the human APPV717F mutation (Games et al., 1995). In vivo imaging of
the brain in an intact animal is achieved with multiphoton microscopy, which offers significant advantages over other modes of fluorescence or
confocal fluorescence, particularly in thick biological specimens (Denk
et al., 1990; Christie et al., 2001). Multiphoton fluorescence microscopy achieves spatial resolution that is comparable with visible
light confocal microscopy, i.e., on the order of 1 µm. This
resolution is at least two orders of magnitude superior to other
imaging modalities, such as magnetic resonance imaging or positron
emission tomography scanning (Yang et al., 1998) and allows
discrimination of single cells, as well as subcellular structures. This
technology permits highly sensitive visualization of microscopic
structures deep within biological tissue. Chronic imaging of the live
mice with multiphoton microscopy showed that antibody application to
the surface of the brain led to clearance of plaques within a few days.
To generalize the efficacy of such a treatment in different transgenic
mouse models, a similar approach was used in the Tg2576 mouse. This
mouse overexpresses the Swedish mutation of the human APP695 gene
driven by the hamster prion protein promoter (Hsiao et al., 1996). Mice
were surgically prepared as described in Materials and Methods. A
craniotomy was performed, and the dura was removed in up to four sites
per animal. FITC-labeled antibody (10d5; Elan Pharmaceuticals, South
San Francisco, CA) as well as thioS were applied to the exposed brain
in ACSF for 20 min and then washed off. Texas red dextran (70,000 MW;
Molecular Probes) was injected into a tail vein of each animal to
permit simultaneous recording of a fluorescent angiogram.
Three-dimensional image volumes of 615 × 615 µm and up to 200 µm deep to the surface of the brain were obtained in each of the
cranial sites using a 20× water immersion objective (Olympus Optical;
numerical aperture of 0.5). Dense-core plaques were defined as those
plaques labeled with thioS (fluorescence in channel 1), whereas the
labeled antibody (channel 2) binds to both dense-core and diffuse
plaques. Both thioS and antibody label amyloid angiopathy. The
angiogram (channel 3) is recorded to facilitate alignment of volumes
from sequential imaging sessions within each cranial site, independent
of plaque location. Figure 1 illustrates
an example of antibody-mediated clearance of existing amyloid deposits
in a Tg2576 mouse. The initial imaging session reveals dense-core and
diffuse plaques, as well as amyloid angiopathy labeled with the
fluorescent antibody. The subsequent imaging session of the same volume
of brain obtained 3 d later shows a marked diminution of diffuse
deposits. Figure 2 compares the clearance
of amyloid deposits with a single 10d5 application at 3 d in
Tg2576 and PDAPP mice. This antibody is remarkably effective at
mediating removal of plaques in both transgenic mouse models within
3 d. The Tg2576 and PDAPP mouse models differ in their expression
of diffuse versus dense-core plaques. The Tg2576 mouse exhibits more
frequent and larger dense-core plaques, with comparatively sparse
diffuse amyloid deposits. The PDAPP mouse model develops ~10-fold
more diffuse deposits than the Tg2576 mouse at comparable ages. Our
imaging approach allows us to measure identified amyloid deposits
within the same animal before and after treatment, permitting direct
measurement of clearance of existing amyloid deposits regardless of
their prevalence. These results demonstrate that clearance is
independent of the genetic strain of animal, the specific mutation in
the APP gene, or the promoter-driving expression of the transgene.
These results are consistent with recent reports using different
transgenic mouse models in which immunization with amyloid- peptides
prevented behavioral deficits (Janus et al., 2000; Morgan et al.,
2000).
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Figure 1.
Topical antibody application leads to clearance of
diffuse amyloid- in Tg2576 mice. These images are projections of
three-dimensional volumes from the cortex of a representative Tg2576
mouse treated with FITC-labeled 10d5 antibody. The images were acquired
in the anesthetized mouse using multiphoton microscopy. The
left shows labeled amyloid deposits at the initial
application of antibody. Numerous diffuse deposits as well as amyloid
angiopathy can be seen. The right is the same volume
3 d later, labeled with 3d6 antibody, which recognizes amyloid-
independently of 10d5. A majority of the amyloid- deposits have been
cleared in this 3 d period.
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Figure 2.
10d5 antibody is equally effective at clearing
diffuse amyloid- deposits in both PDAPP and Tg2576 transgenic mouse
models. These plots represent the percentage of amyloid clearance in
3 d from paired volumes of cortex within the same animals after
3 d. A single application of antibody to the cortex was given at
day 0. Approximately one-half of the diffuse deposits are cleared under
these conditions. These results are the means ± SE for
n = 9-15 sites from at least three animals in each
group. Percentage of clearance ranged from 91.2 to  7.9% in PDAPP
mice and 82.9 to 4.5% in Tg2576 mice. Not statistically different;
Student's t test.
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The determination of clearance of diffuse amyloid deposits in
vivo depends on the addition of a second labeled antibody after treatment with the initial labeled antibody. When using 10d5 as the
treatment antibody, 3d6 was used to detect remaining amyloid deposits
that were not cleared. These antibodies have neighboring epitopes on
amyloid- but do not compete with each other at the concentrations
used here, as described by Bacskai et al. (2001) and shown in Figure
3. In this experiment,
paraformaldehyde-fixed tissue sections were first treated with
FITC-labeled 10d5 at 1 mg/ml for 20 min, washed, and then subsequently
incubated with rhodamine-labeled 3d6 at 1 mg/ml for 20 min. Confocal
microscopy shows that both labeled antibodies are bound to amyloid
deposits in these sections, and they colocalize identically. Similar
results are obtained in cryostat sections and in vivo.
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Figure 3.
10d5 and 3d6 antibodies colocalize at
noncompetitive binding sites on amyloid- plaques.
Paraformaldehyde-fixed tissue sections from an 18-month-old Tg2576
mouse brain were treated sequentially with FITC-labeled 10d5
antibodies, followed by rhodamine-labeled 3d6 antibodies. Images were
obtained with a Bio-Rad confocal microscope using 488 nm excitation for
FITC (A) and 568 nm excitation for rhodamine
(B). A color-merged image
(C) shows that 10d5 (green)
and 3d6 (red) are both able to bind to the plaques and
colocalize everywhere, producing a yellow color. Scale
bar, 50 µm.
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The following experiments were performed to determine whether the
mechanism of amyloid- clearance in vivo was specific to the 10d5 antibody. 3d6 monoclonal antibody, which recognizes amino acids 1-5 of amyloid-, was compared with 10d5 antibody, which recognizes a close but distinct epitope of amyloid-, residues 3-6.
As can be seen in Figure 4, each of these
antibodies was similarly effective after 3 d when applied
topically to the PDAPP mouse. Diffuse amyloid burdens were measured in
the identical volumes within each site before and after a single
treatment. The amyloid burdens were decreased with 10d5 and 3d6 by 50 and 48%, respectively, with each antibody treatment. These results suggest that the mechanism of clearance is not dependent on a single
antibody but can be generalized to other antibodies directed toward the
N terminus of amyloid-. Likewise, 10d5 is of the IgG1 isotype,
whereas 3d6 is an IgG2b. Therefore, antibody-mediated clearance is not
dependent on any one specific IgG subtype. Because IgG1 antibodies do
not activate complement in the mouse, this result demonstrates that
activation of the complement cascade is not necessary for clearance of
amyloid- in vivo.
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Figure 4.
10d5 and 3d6 antibodies are equally effective at
clearing diffuse amyloid- deposits in the PDAPP mouse model after
3 d. 10d5 and 3d6 recognize the N-terminus residues 3-6 and 1-5,
respectively, but their epitopes do not physically overlap. 10d5 (IgG1)
and 3d6 (IgG2b) are different isotypes as well, suggesting that
clearance in vivo is not specific to any one class of
antibody. These data represent n = 9 sites from
four animals. Not statistically different; Student's t
test.
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Previous reports have shown that amyloid- clearance in an ex
vivo assay proceeds through Fc-mediated phagocytosis by microglial cells, because F(ab')2 fragments that lack the Fc
portion of the antibody were ineffective (Bard et al., 2000). To
evaluate this mechanism in vivo, we treated animals with
FITC-labeled F(ab')2 fragments of 3d6 and
measured the clearance of amyloid- deposits. The quantitative
results are presented in Figure 5.
F(ab')2 fragments of 3d6 were as effective as
full-length antibodies at clearing diffuse amyloid- deposits in the
PDAPP mice. The FITC-labeled F(ab')2 fragments
labeled amyloid-deposits in vivo, as shown in Figure
6, and, within 3 d, over one-half of
the labeled deposits were cleared, similar to the results obtained with
full-length 3d6 or 10d5. The F(ab')2 preparation
was examined by Western blot, and no Fc could be detected. Moreover,
sections of animals treated with F(ab')2 were
immunostained for the presence of Fc on plaques, and none could be
detected. Histological examination of the tissue after the experiments
showed some activation of microglia in all cases, probably resulting
from the surgical preparation, and no difference between F(ab')2 and
16b5-treated animals. These results demonstrate that, in addition to
mechanisms involving Fc-mediated phagocytosis of antibody-labeled
amyloid deposits, an alternative and efficacious mechanism exists for
clearance of amyloid deposits that does not depend on Fc. Direct
biophysical interaction of antibodies with amyloid- deposits may be
responsible for clearance by direct disaggregation of the deposits.
Prevention of fibril formation, disaggregation, and inhibition of
toxicity with antibodies has been demonstrated in vitro
(Solomon et al., 1996, 1997; Frenkel et al., 2000), and our current
results indicate that antibody-mediated disaggregation may occur
in vivo. The F(ab')2 results
demonstrate that a mechanism not requiring receptor-mediated cellular
activation is involved in clearance by immunotherapy with topical
application in vivo.
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Figure 5.
F(ab')2 fragments of 3d6 antibody are
equally effective at leading to clearance of diffuse amyloid-
deposits after 3 d in the PDAPP mouse model. F(ab')2
fragments were purified and analyzed for Fc contamination by Western
blot analysis and immunohistochemistry. No evidence for trace Fc was
detected with either assay. Approximately one-half of the diffuse
amyloid- deposits were cleared 3 d after a single topical
application to the cortex of the transgenic mice. Error bars
represent means ± SE from n = 15 or 11 sites
from four animals from each group. Not statistically different;
Student's t test.
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Figure 6.
F(ab')2 fragments of 3d6 antibody are
equally effective at leading to clearance of diffuse amyloid-
deposits after 3 d in the PDAPP mouse model. These images are
projections of three-dimensional volumes from the cortex of a
representative PDAPP mouse treated with FITC-labeled 3d6 or
3d6-F(ab')2 antibodies. The images were acquired in the
anesthetized mouse using multiphoton microscopy. The left
images shows labeled amyloid deposits at the initial
application of antibody. Numerous diffuse deposits as well as amyloid
angiopathy can be seen, pseudocolored green. A
fluorescent angiogram (red) fills the blood vessels in
each imaging session to facilitate image alignment. The right
images are the same volumes 3 d later, immediately after
application of labeled 10d5 antibody, which recognizes amyloid-
independently of 3d6. A majority of the amyloid- deposits have been
cleared in this 3 d period with both full-length (top
row) and F(ab')2 fragments (bottom
row) of antibody. Scale bar, 100 µm.
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The results above focus on clearance of diffuse amyloid- deposits.
We also tested the ability of antibodies to lead specifically to the
clearance of dense-core deposits in PDAPP mice. All plaques labeled
with thioS were considered "dense-core" plaques. Animals were
imaged with thioS, treated topically with antibody, and then imaged
again with thioS 3 d later. thioS-positive plaques present in the
initial imaging session were identified in the subsequent imaging
session. If a plaque could not be found, then it was scored as "not
found." The fluorescent angiogram was used as a positive control for
the quality of the imaging in the immediate volume of the plaque. The
angiogram also permitted identifying the relative three-dimensional
location of each plaque within the brain. The number of plaques not
found divided by the total number of plaques identified in the initial
imaging session was used to establish the percentage of cleared
plaques, as shown in Figure 7. The
ability of antibodies to lead to rapid clearance of thioS plaques is
more variable. 10d5 was able to clear nearly 35% of identified
dense-core plaques compared with control antibodies and 3d6 and
3d6-F(ab')2, which showed ~20% of plaques that
could not be found at the second imaging session.
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Figure 7.
Dense-core plaques are cleared with 10d5 after
3 d in PDAPP mice. thioS-positive plaques were identified and
counted in animals at the initial imaging-treatment session. In the
subsequent session, after 3 d, the presence or absence of the
individual plaques was scored in the identical locations within each
animal, using the fluorescent angiogram as both a three-dimensional
fiduciary marker, as well as a positive control for imaging quality.
Two independent observers scored the presence or absence of
identifiable plaques in the PDAPP mice. 16b5 is a fluorescently labeled
control antibody that recognizes human tau. 3d6 and F(ab')2
fragments of 3d6 did not lead to appreciable clearance of thioS plaques
after 3 d, whereas 10d5 led to the removal of 35% of identified
plaques. Bars represent the percentage of identified
plaques that were not found after 3 d. At least 45 plaques from
8-12 sites in four animals were scored for each group.
*p < 0.05 indicates statistically significant by
t test.
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We wondered whether the 20% of plaques scored as "not
found" in the control treatment
reflected technical difficulties in reimaging plaques from session to
session or a true change in plaques attributable to nonspecific
stimuli associated with preparing a craniotomy and imaging. We reasoned
that remaining plaques would shrink between the first and second
imaging if affected by a process that ultimately would lead to their
removal, whereas their size would remain constant if a failure to
reimage the same plaques was attributable to technical issues. We
therefore measured the size of the identified plaques that were not
cleared. The results are shown in Figure
8 and demonstrate that the plaques that
were not cleared were reduced in size by ~30% for 10d5, 3d6, and
F(ab')2 fragments of 3d6, but the measured plaque
size was unchanged with the control antibody, 16b5. These results
demonstrate that different antibodies and F(ab')2
fragments of 3d6 are effective at clearing or reducing the size of
dense-core plaques, as well as clearing diffuse amyloid- deposits
within 3 d in vivo.
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Figure 8.
Remaining dense-core plaques in PDAPP mice are
reduced in size 3 d after treatment with anti-amyloid-
antibodies. The identified plaques that were imaged in the first
imaging session and remained after 3 d after a single antibody
treatment were measured as described previously (Christie et al.,
2001). Plaques that were cleared completely would have been measured as
100% change in size but are not included in this analysis. 10d5, 3d6,
and F(ab')2 fragments of 3d6 were equally effective at
reducing the size of remaining dense-core plaques compared with control
antibody 16b5. Error bars are mean ± SE from at least 12 plaques in four animals from each group. *p < 0.05 indicates statistically significant by Student's t
test.
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DISCUSSION |
Transgenic mouse models that overexpress APP develop senile
plaques over time that resemble those found in human Alzheimer's disease. These animals are a valuable tool toward understanding the
physiology and pathology of senile plaques in living tissue and are
ideal for evaluating therapeutics aimed at clearance of amyloid-
deposits in the brain. With the recent success using immunotherapy for
prevention of amyloid- deposits in these animals (Schenk et al.,
1999; Bard et al., 2000), as well as clearance of existing plaques
(Bacskai et al., 2001), this treatment seems very promising. Recent
reports have also indicated that immunotherapy may have positive
effects on behavioral deficits exhibited in transgenic mouse models
(Janus et al., 2000; Morgan et al., 2000). These findings are important
for predicting whether anti-amyloid therapies will prove beneficial not
just in arresting deposition of amyloid- but also in prevention of
the associated dementia. Microglial cells were implicated in the
alterations of amyloid- deposition by immunotherapy. Clearance of
amyloid- deposits in tissue sections by cultured microglia in an
ex vivo system was shown to be Fc receptor dependent (Bard
et al., 2000). However, additional or alternative mechanisms for
clearance of amyloid- peptide are possible in vivo.
Activation of the complement cascade may play a role in clearance.
Direct interaction of antibodies with amyloid- may lead to
disruption of aggregates, as has been shown in vitro
(Solomon et al., 1997), and this process should not depend on Fc
receptor activation. A combination of Fc-dependent and Fc-independent
mechanisms may be involved.
We used in vivo imaging using multiphoton microscopy to
study the mechanisms of immunotherapeutic approaches toward removal of
amyloid- deposits in the brains of transgenic mice overexpressing APP. Our current results extend our observations showing
antibody-mediated clearance of plaques in PDAPP mice (Bacskai et al.,
2001) in several ways. First, the direct demonstration of clearance of
amyloid- deposits in vivo with anti-amyloid-
antibodies in another mouse model, Tg2576, was shown. This result
suggests that the previous work in PDAPP mice can be extended to other
animal models and is not dependent on the strain of animal, specific
mutation in APP, or the specific patterns of expression of the
transgene; behavioral studies in other mouse models in which
immunotherapy proved beneficial also support this conclusion (Janus et
al., 2000; Morgan et al., 2000). Second, an additional mouse monoclonal antibody of a different IgG isotype also led to effective clearance of
previously deposited amyloid- plaques in vivo. This
result complements the observation that both of these antibodies can prevent deposition of amyloid- after passive administration over a 6 month period in PDAPP mice (Bard et al., 2000), as well as both of the
efficacy of the antibodies for the targeted digestion of plaques
in an ex vivo preparation (Bard et al., 2000). The direct
demonstration of clearance of existing, identified plaques in
vivo with different antibodies is important in determining the
specificity of the response. IgG1s do not activate complement in the
mouse and may exhibit other specific differences that provide insight
into the mechanism of clearance, particularly in the short time frame
of 3 d. The difference in isotype may contribute to the
effectiveness of the two antibodies at clearing dense-core plaques in
the mice. Although both 3d6 and 10d5 were equally effective at clearing
diffuse deposits in the animals and both antibodies led to similar
decreases in size of remaining, identified dense-core plaques, 10d5 was
more effective at completely clearing dense-core plaques in 3 d.
It is possible that this subtle difference in efficacy is dependent on
either the dose or time course of the treatment. Although both
antibodies were added at the same dose for the same duration, they may
have unequal affinities or effective actions at this concentration.
Nonetheless, it is possible that complement activation or other
differences between IgG1 and IgG2b contribute to the apparently
enhanced rate of 10d5 clearance of dense-core plaques. This result
suggests that even very similar antibodies may have subtly different efficacy.
Surprisingly, the F(ab')2 fragments of 3d6 were
as effective at clearance of diffuse amyloid- deposits as
full-length 3d6. F(ab')2s lack the Fc portion of
the antibody and therefore prohibit Fc-mediated phagocytosis of
amyloid- by microglia. Previous work with primary cultures of
microglial cells and frozen tissue sections has demonstrated that this
Fc-mediated mechanism is dominant in removal of amyloid- deposits
(Bard et al., 2000). The current results suggest that an alternative
mechanism besides Fc-mediated phagocytosis can be involved in the
clearance of amyloid- deposits after topical administration in
vivo. Other cellular mechanisms include activation of scavenger
receptors (Huang et al., 1999; Bornemann et al., 2001) or receptors for
advanced glycation end products (Munch et al., 1997; Tabaton et al.,
1997; Bornemann et al., 2001). However, neither of these
receptor-dependent mechanisms would be activated with
F(ab')2 fragments. Direct biophysical interaction
of antibodies with amyloid- may have led to disaggregation and
removal of deposits in vivo, similar to results shown
in vitro (Solomon et al., 1997). Likewise, a recent report
demonstrated that peripheral antibodies may act as a sink for
amyloid-, removing it from the CNS and preventing plaque deposition
in the brain (DeMattos et al., 2001).
Clearance may depend on multiple mechanisms, one involving direct
interaction of antibodies [or F(ab')2
fragments] with the deposits resulting in disaggregation and the
second involving cell-mediated clearance, possibly involving Fc
receptor activation. The two mechanisms may occur independently, or may
operate in tandem, with cellular removal of amyloid- after
disaggregation. Passive redistribution of soluble amyloid- in CSF or
plasma after disaggregation may occur (DeMattos et al., 2001).
Characterization of these mechanisms will lead to an optimized
therapeutic plan for efficient clearance of amyloid- deposits.
| |
FOOTNOTES |
Received April 12, 2002; revised June 11, 2002; accepted June 14, 2002.
This work was supported by National Institute on Aging Grants AG08487,
P01 AG15453, and T32GM07753, as well as the Fidelity Foundation and the
Walters Family Foundation.
Correspondence should be addressed to Dr. Bradley T. Hyman,
Alzheimer's Disease Research Unit, Charlestown Navy Yard 2450, Massachusetts General Hospital, 114 16th Street, Charlestown, MA 02129. E-mail: bhyman{at}partners.org.
| |
REFERENCES |
-
Bacskai BJ,
Kajdasz ST,
Christie RH,
Carter C,
Games D,
Seubert P,
Schenk D,
Hyman BT
(2001)
Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy.
Nat Med
7:369-372[ISI][Medline].
-
Bard F,
Cannon C,
Barbour R,
Burke RL,
Games D,
Grajeda H,
Guido T,
Hu K,
Huang J,
Johnson-Wood K,
Khan K,
Kholodenko D,
Lee M,
Lieberburg I,
Motter R,
Nguyen M,
Soriano F,
Vasquez N,
Weiss K,
Welch B,
Seubert P,
Schenk D,
Yednock T
(2000)
Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.
Nat Med
6:916-919[ISI][Medline].
-
Bornemann KD,
Wiederhold KH,
Pauli C,
Ermini F,
Stalder M,
Schnell L,
Sommer B,
Jucker M,
Staufenbiel M
(2001)
Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice.
Am J Pathol
158:63-73[Abstract/Free Full Text].
-
Christie RH,
Bacskai BJ,
Zipfel WR,
Williams RM,
Kajdasz ST,
Webb WW,
Hyman BT
(2001)
Growth arrest of individual senile plaques in a model of Alzheimer's disease observed by in vivo multiphoton microscopy.
J Neurosci
21:858-864[Abstract/Free Full Text].
-
DeMattos RB,
Bales KR,
Cummins DJ,
Dodart JC,
Paul SM,
Holtzman DM
(2001)
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.
Proc Natl Acad Sci USA
98:8850-8855[Abstract/Free Full Text].
-
Denk W,
Strickler JH,
Webb WW
(1990)
Two-photon laser scanning fluorescence microscopy.
Science
248:73-76[Abstract/Free Full Text].
-
Frenkel D,
Solomon B,
Benhar I
(2000)
Modulation of Alzheimer's beta-amyloid neurotoxicity by site-directed single-chain antibody.
J Neuroimmunol
106:23-31[ISI][Medline].
-
Games D,
Adams D,
Alessandrini R,
Barbour R,
Berthelette P,
Blackwell C,
Carr T,
Clemens J,
Donaldson T,
Gillespie F,
Guido T,
Hagoplan S,
Johnson-Wood K,
Khan K,
Lee M,
Leibowitz P,
Lieberburg I,
Little S,
Masliah E,
McConlogue L
(1995)
Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein.
Nature
373:523-527[Medline].
-
Hsiao K,
Chapman P,
Nilsen S,
Eckman C,
Harigaya Y,
Younkin S,
Yang F,
Cole G
(1996)
Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice.
Science
274:99-102[Abstract/Free Full Text].
-
Huang F,
Buttini M,
Wyss-Coray T,
McConlogue L,
Kodama T,
Pitas RE,
Mucke L
(1999)
Elimination of the class A scavenger receptor does not affect amyloid plaque formation or neurodegeneration in transgenic mice expressing human amyloid protein precursors.
Am J Pathol
155:1741-1747[Abstract/Free Full Text].
-
Hyman BT,
Trojanowski JQ
(1997)
Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease.
J Neuropathol Exp Neurol
56:1095-1097[ISI][Medline].
-
Janus C,
Pearson J,
McLaurin J,
Mathews PM,
Jiang Y,
Schmidt SD,
Chishti MA,
Horne P,
Heslin D,
French J,
Mount HT,
Nixon RA,
Mercken M,
Bergeron C,
Fraser PE,
St George-Hyslop P,
Westaway D
(2000)
A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease.
Nature
408:979-982[Medline].
-
Markesbery WR
(1997)
Neuropathological criteria for the diagnosis of Alzheimer's disease.
Neurobiol Aging
18 [Suppl 4]:S13-S19[ISI][Medline].
-
Morgan D,
Diamond DM,
Gottschall PE,
Ugen KE,
Dickey C,
Hardy J,
Duff K,
Jantzen P,
DiCarlo G,
Wilcock D,
Connor K,
Hatcher J,
Hope C,
Gordon M,
Arendash GW
(2000)
A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease.
Nature
408:982-985[Medline].
-
Munch G,
Thome J,
Foley P,
Schinzel R,
Riederer P
(1997)
Advanced glycation endproducts in ageing and Alzheimer's disease.
Brain Res Brain Res Rev
23:134-143[Medline].
-
Powers JM
(1997)
Diagnostic criteria for the neuropathologic assessment of Alzheimer's disease.
Neurobiol Aging
18 [Suppl 4]:S53-S54[Medline].
-
Rubinsztein DC
(1997)
The genetics of Alzheimer's disease.
Prog Neurobiol
52:447-454[ISI][Medline].
-
Schenk D,
Barbour R,
Dunn W,
Gordon G,
Grajeda H,
Guido T,
Hu K,
Huang J,
Johnson-Wood K,
Khan K,
Kholodenko D,
Lee M,
Liao Z,
Lieberburg I,
Motter R,
Mutter L,
Soriano F,
Shopp G,
Vasquez N,
Vandevert C,
Walker S,
Wogulis M,
Yednock T,
Games D,
Seubert P
(1999)
Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.
Nature
400:173-177[Medline].
-
Selkoe DJ
(1996)
Cell biology of the beta-amyloid precursor protein and the genetics of Alzheimer's disease.
Cold Spring Harb Symp Quant Biol
61:587-596[Abstract/Free Full Text].
-
Solomon B,
Koppel R,
Hanan E,
Katzav T
(1996)
Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.
Proc Natl Acad Sci USA
93:452-455[Abstract/Free Full Text].
-
Solomon B,
Koppel R,
Frankel D,
Hanan-Aharon E
(1997)
Disaggregation of Alzheimer beta-amyloid by site-directed mAb.
Proc Natl Acad Sci USA
94:4109-4112[Abstract/Free Full Text].
-
Tabaton M,
Perry G,
Smith M,
Vitek M,
Angelini G,
Dapino D,
Garibaldi S,
Zaccheo D,
Odetti P
(1997)
Is amyloid beta-protein glycated in Alzheimer's disease?
NeuroReport
8:907-909[Medline].
-
Wyss-Coray T,
Lin C,
Yan F,
Yu G-Q,
Rohde M,
McConlogue L,
Masliah E,
Mucke L
(2001)
TGF-B1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice.
Nat Med
7:612-618[ISI][Medline].
-
Yang X,
Renken R,
Hyder F,
Siddeek M,
Greer CA,
Shepherd GM,
Shulman RG
(1998)
Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI.
Proc Natl Acad Sci USA
95:7715-7720[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22187873-06$05.00/0
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|
|
|
|
|
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INTRODUCTION
