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The Journal of Neuroscience, May 1, 2003, 23(9):3745
Intracranially Administered Anti-A Antibodies Reduce
-Amyloid Deposition by Mechanisms Both Independent of and Associated
with Microglial Activation
Donna M.
Wilcock1,
Giovanni
DiCarlo1,
Debbi
Henderson1,
Jennifer
Jackson1,
Keisha
Clarke1,
Kenneth E.
Ugen2,
Marcia N.
Gordon1, and
Dave
Morgan1
Departments of 1 Pharmacology and 2 Medical
Microbiology and Immunology, Alzheimer's Research Laboratory,
University of South Florida, Tampa, Florida 33612
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ABSTRACT |
Active immunization against the  -amyloid peptide (A) with
vaccines or passive immunization with systemic monoclonal anti-A antibodies reduces amyloid deposition and improves cognition in APP
transgenic mice. In this report, intracranial administration of
anti-A antibodies into frontal cortex and hippocampus of Tg2576 transgenic APP mice is described. The antibody injection resulted initially in a broad distribution of staining for the antibody, which
diminished over 7 d. Although no loss of immunostaining for
deposited A was apparent at 4 hr, a dramatic reduction in the A
load was discernible at 24 hr and was maintained at 3 and 7 d. A
reduction in the thioflavine-S-positive compact plaque load was delayed
until 3 d, at which time microglial activation also became
apparent. At 1 week after the injection, microglial activation returned
to control levels, whereas A and thioflavine-S staining remained
reduced. The results from this study suggest a two-phase mechanism of
anti-A antibody action. The first phase occurs between 4 and 24 hr, clears primarily diffuse A deposits, and is not associated
with observable microglial activation. The second phase occurs between
1 and 3 d, is responsible for clearance of compact amyloid
deposits, and is associated with microglial activation. The results are
discussed in the context of other studies identifying coincident
microglial activation and amyloid removal in APP transgenic animals.
Key words:
Alzheimer's disease; A; antibody; immunization; intracranial; phagocytosis
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Introduction |
Alzheimer's disease (AD) is a
neurodegenerative disorder characterized by progressive cognitive
deficits. There are several pathological characteristics to the disease
process, including congophilic amyloid plaques that contain the
-amyloid peptide (A) and intracellular inclusions of
neurofibrillary tangles that consist of hyperphosphorylated tau
protein. Another characteristic of AD is the initiation and
proliferation of a brain-specific inflammatory response that consists
of activated microglia and astrocytes. Amyloid deposition is thought to
be the key step in the pathogenesis of AD (Selkoe, 1991 ; Hardy and
Selkoe, 2002); this is why development of potential therapies focuses
on clearance of amyloid.
Vaccination with A1-42 was first described
by Schenk et al. (1999). Their report demonstrated that active
immunization with A1-42 in the PDAPP
transgenic mouse reduced levels of A deposits dramatically. This
immunization protected APP+PS1 transgenic mice (Morgan et al., 2000)
and TgCRND8 transgenic mice (Janus et al., 2000) from memory deficits.
More recent studies showed that treatment with a passive immunization
regimen that consisted of anti-A antibodies resulted in a dramatic
reduction in A (Bard et al., 2000; DeMattos et al., 2001) and
reversal of memory deficits (Dodart et al., 2002; Kotilinek et al.,
2002) in the PDAPP mouse.
In this experiment, we show that intracranially administered
anti-A antibodies have both an early microglia-independent and a
later, possibly microglia-dependent mechanism of action. A levels
were dramatically reduced 24 hr after administration in the absence of
microglial activation. However, 72 hr after antibody administration,
thioflavine-S-positive compact plaques were reduced concomitant with a
striking activation of microglia.
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Materials and Methods |
Transgenic Tg2576 APP mice (Hsiao et al., 1996) were obtained
after breeding of Tg2576 APP mice with line 5.1 PS1 mice (Duff et al.,
1996), which yielded four different genotypes: nontransgenic, transgenic APP, transgenic PS1, and doubly transgenic APP+PS1 mice.
Animals were provided food and water ad libitum and were kept on a 12 hr light/dark cycle; they were housed in groups if possible until before the surgery, when they were all singly housed until they were killed. We used two cohorts of mice in this study, the
first cohort of 19-month-old APP mice (n = 16) and the
second cohort of 16-month-old APP mice (n = 22).
Mice from the first cohort all received anti-A antibodies
(Biosource, Camarillo, CA; mouse anti-A
IgG1, recognizing amino acids 1-16). Mice from
the second group were assigned to groups that received anti-A
antibodies, control antibody (anti-HIV, ID6; K. Ugen, Department of
Medical Microbiology and Immunology, University of South Florida,
Tampa, FL) (n = 5), or vehicle (0.02% thimerosal in
PBS; Sigma-Aldrich, St. Louis, MO) (n = 5). All mice were injected in both the frontal cortex and hippocampus of the right hemisphere, whereas the left hemisphere remained untreated
as an internal control. Those mice that received anti-A antibodies
were assigned survival times of 4 (n = 5), 24 (n = 7), 72 (n = 8), or 168 (n = 6) hr. Mice receiving either control antibody or
vehicle were examined after a 72 hr survival time. A third group of
untreated 17-month-old APP mice (n = 5) were killed
without having been injected or manipulated to assess differences between the right and left sides of the brain.
On the day of surgery, the mice were weighed, anesthetized with
isoflurane, and placed in a stereotaxic apparatus (51603 dual manipulator laboratory standard; Stoelting, Wood Dale,
IL). A midsagittal incision was made to expose the cranium, and two
burr holes were drilled with a dental drill over the right frontal cortex and hippocampus to the following coordinates: cortex,
anteroposterior, +1.5 mm; lateral, -2.0 mm; hippocampus,
anteroposterior, -2.7 mm; lateral, -2.5 mm, all taken from
bregma. A 26 gauge needle attached to a 10 µl syringe
(Hamilton, Reno, NV) was lowered 3 mm ventral to bregma,
and a 2 µl injection was made over a 2 min period. The incision was
cleaned with saline and closed with surgical staples.
On the day they were killed, the mice were overdosed with pentobarbital
100 mg/kg (Nembutal sodium solution; Abbott Laboratories, North Chicago, IL) and perfused intracardially with 25 ml of 0.9% sodium chloride and 50 ml of freshly prepared 4% paraformaldehyde, pH
7.4. The brains were collected and postfixed for 24 hr in 4% paraformaldehyde. The brains were then incubated for 24 hr in 10, 20, and 30% sucrose sequentially to cryoprotect them. Horizontal sections
of 25 µm thickness were then collected with a sliding microtome and
stored at 4°C in Dulbecco's PBS buffer with sodium azide to prevent
microbial growth. Six to eight sections ~100 µm apart were selected
that spanned the injection site and were stained by free-floating
immunohistochemistry methods for total A (rabbit antiserum
primarily reacting with the N terminus of the A peptide;
1:10,000), CD45 (Serotec, Raleigh, NC; 1:3000), and major
histocompatibility complex class II (MHC-II; BD
PharMingen, Palo Alto, CA; 1:3000) as described previously
(Gordon et al., 2002). For immunostaining, some sections were omitted
from the primary antibody to assess nonspecific immunohistochemical
reactions. Immunohistochemical methods were used to stain for the
injected antibody with anti-mouse IgG conjugated to horseradish
peroxidase (Sigma-Aldrich; 1:1000). Adjacent sections were
mounted on slides and stained with 4% thioflavine-S
(Sigma-Aldrich) for 10 min. Selected sections stained for
CD45 were counterstained for Congo red (Sigma-Aldrich) to
detect amyloid deposits on these sections.
The immunohistochemical reaction product on all stained sections was
measured with a videometric V150 image analysis system (Oncor, San Diego, CA) in the injected area of cortex and
hippocampus and corresponding regions on the contralateral side of the
brain. Data are presented as the average ratio of injected side to
noninjected side for A, thioflavine-S, and CD45, whereas data for
MHC-II are expressed as area occupied by positive stain, because many values on the contralateral side were close to zero.
To assess possible treatment-related differences, the measurement for
either cortex or hippocampus of each subject were analyzed by ANOVA
with StatView software version 5.0.1 (SAS Institute Inc., Raleigh, NC) followed by Fisher's least significant difference means comparisons.
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Results |
Immunohistochemistry against mouse IgG was performed to trace the
diffusion of anti-A antibodies after injection into the hilus of the
dentate gyrus. The injected anti-A antibody showed diffuse
distribution throughout the entire hippocampus at 4 hr, with a focal
concentration in the outer molecular areas of the dentate and Ammons'
horn near the hippocampal fissure (Fig.
1A). By 24 hr, the
diffuse pattern remained broad, but the focal concentration began
shifting toward the granule cell layers of the dentate gyrus (Fig.
1B). At 72 hr, staining for the injected antibody was
lighter and became concentrated at the granule cell layer of the
dentate gyrus (Fig. 1C). Interestingly, by the 1 week time
point, the injected antibody staining had cleared for the most part,
with some residual staining in the outer molecular layer of the ventral (ventricular) blade of the dentate gyrus and the glial limitans. A
similar time course of staining was seen in the frontal cortex (data
not shown).
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Figure 1.
Time course of injected anti-A antibody
distribution in the hippocampus from 4 hr to 7 d.
Immunohistochemical staining is shown for the injected antibody in the
hippocampus at 4 (A), 24 (B), 72 (C), and 168 (D) hr. Orientation and locations of hippocampal
subregions are as in Figure 2D. Magnification,
40×. Scale bar, 120 µm. A high-resolution color version of this
micrograph can be obtained by e-mail from D. Morgan
(dmorgan{at}hsc.usf.edu).
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A immunohistochemistry in APP transgenic mice resembled that
reported previously by others and ourselves (Hsiao et al., 1996; Gordon
et al., 2002). In both cortex (Fig.
2A) and hippocampus (Fig. 2C), there were a few intensely stained deposits and a
number of smaller, less intensely stained deposits. In previous work, we found the intensely stained A deposits were usually also stained with thioflavine-S or Congo red (Holcomb et al., 1998; Gordon et al.,
2001), which indicates they were analogous to compact deposits
containing fibrillar amyloid, whereas the less intense deposits were
analogous to diffuse, nonfibrillar deposits observed commonly in AD
tissue. Whereas the deposits were distributed fairly uniformly within
the cortex, in the hippocampus they were concentrated in the outer
molecular layers of the dentate gyrus and Ammon's horn (Fig.
2C). The subiculum also appeared more rich in deposits than
other areas.
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Figure 2.
Reduction in A immunohistochemistry 1 d
after anti-A antibody injections. Immunohistochemical staining is
shown for A in the frontal cortex (A,
B) and hippocampus (C, D).
A and C are from an animal injected with
control antibody, whereas animals for which stains are shown in
B and D received the anti-A
antibody. Magnification, 40×. Scale bar, 120 µm. B,
FCX, frontal cortex; STR, striatum. D, CA1, cornu
ammonis 1; CA3, cornu ammonis 3; DG, dentate gyrus. A high-resolution
color version of this micrograph can be obtained by e-mail from D. Morgan (dmorgan{at}hsc.usf.edu).
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The injection of anti-A antibody into brain did not result in a
rapid loss of signal in postmortem immunohistochemical reactions, because we did not observe a change in A staining 4 hr after injection in either cortex (Fig.
3A) or hippocampus (Fig.
3B). However, A staining was reduced at the injection
sites in frontal cortex and hippocampus 24 hr after administration of
anti-A antibody (Fig. 2B,D, respectively) and
remained reduced to roughly the same extent through the 1 week time
point (Fig. 3). The reduction in the frontal cortex was 60% compared
with both the 4 hr time points and the two control groups of vehicle
and anti-HIV antibody (Fig. 3A; p < 0.001).
The reduction in the hippocampus was 50% compared with the 4 hr time
points and the control groups (Fig. 3B; p < 0.005).
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Figure 3.
Quantification of reduced A load after
anti-A antibody injections. Data are expressed as the ratio of
A staining in the injected hemisphere to the control hemisphere. The
three bars on the left indicate the A load in the untreated group
(None) and the vehicle (VEH) and anti-HIV antibody (Cont-Ab) groups at
72 hr. The line shows the ratio of A immunohistochemical staining
at 4, 24, 72, and 168 hr survival times. Reduced A load was
observed in the frontal cortex (A) and
hippocampus (B) at 24, 72, and 168 hr compared
with 4 hr and both control groups (**p < 0.005).
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An interesting phenomenon was that the ratio of A staining on the
right to left sides in untreated mice was >1, which indicates more
A deposition on the right side than the left (Fig. 3). It appears
that this pattern of A deposition is a consistent property of the
APP mice. The A distribution seen in the mice administered control
injections at 3 d and anti-A antibody at the 4 hr time point
is the typical distribution found in APP transgenic mice of this age.
As expected, considerably fewer deposits were stained with
thioflavine-S than by A immunohistochemistry. Nonetheless, the regional distribution of these deposits approximately paralleled that
of A-positive deposits in the cortex and hippocampus (Fig. 4A,C). In contrast to
the A, thioflavine-S-positive staining at the injection site was
not reduced until 72 hr after administration of anti-A antibody
(Fig. 4B,D) and remained reduced at the 1 week time
point (Fig. 5). The reduction in frontal
cortex was 80% compared with the 4 and 24 hr time points and the
control groups (Fig. 5A; p < 0.001). The
reduction in hippocampus was 60% compared with both the 4 and 24 hr
time points and the control groups (Fig. 5B;
p < 0.005).
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Figure 4.
Reduction in thioflavine-S staining 3 d after
anti-A antibody injections. Thioflavine-S staining is shown in
frontal cortex (A, B) and hippocampus
(C, D). Mice in A and
C received control antibody, whereas those in
B and D received anti-A antibody.
Magnification, 40×. Scale bar, 120 µm. Orientation and locations of
major subregions are as in Figure 2, B and
D. A high-resolution color version of this micrograph
can be obtained by e-mail from D. Morgan (dmorgan{at}hsc.usf.edu).
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Figure 5.
Anti-A antibody injections result in a
reduction of thioflavine-S-positive plaques. Data are expressed as the
ratio of thioflavine-S staining in the injected hemisphere to the
control hemisphere. The three bars show thioflavine-S-positive staining
in the untreated group (None) and the vehicle (VEH) and anti-HIV
antibody (Cont-Ab) groups at 72 hr. The line shows the ratio of
thioflavine-S staining at 4, 24, 72, and 168 hr survival times. Reduced
thioflavine-S staining was observed in the frontal cortex
(A) and hippocampus (B) at
72 and 168 hr compared with 4 and 24 hr and both control groups
(**p < 0.005).
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In untreated mice, activated microglia stained with CD45 or MHC-II
antibodies were found only in the immediate periphery of compacted
plaques. In the injected control groups, some microglial activation was
detected at the 72 hr survival time by CD45 antibodies, and this was
restricted primarily to the injection site (Fig. 6A,C, arrows;
quantified in Fig. 7). Very little staining for CD45 was detected on
the uninjected side of the brain, which
led to inflated right-to-left ratios with relatively small
increases in staining. MHC-II had a lower overall level of expression
than that of CD45 and was mostly unaffected in mice administered
control injections (Fig.
8A,C; quantified in
Fig. 9).
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Figure 6.
CD45 immunohistochemistry is increased 3 d
after anti-A antibody injections. CD45 immunohistochemistry is
shown in frontal cortex (A, B) and
hippocampus (C, D). Mice in
A and C received control antibody,
whereas those in B and D received
anti-A antibody. Magnification, 40×. Scale bar, 120 µm. Arrows
indicate the site of injection identified from the needle tract. A
high-resolution color version of this micrograph can be obtained by
e-mail from D. Morgan (dmorgan{at}hsc.usf.edu).
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Figure 7.
Anti-A antibody injections result in
increased CD45 immunohistochemistry 3 d after injection. Data are
expressed as the ratio of CD45 staining in the injected hemisphere to
the control hemisphere. The three bars indicate CD45 expression in the
untreated group (None) and the vehicle (VEH) and anti-HIV antibody
(Cont-Ab) groups at 72 hr. The line shows the ratio of CD45 staining at
4, 24, 72, and 168 hr survival times. Increased CD45 staining was
observed in the frontal cortex (A) and
hippocampus (B) at 72 hr compared with 4, 24, and
168 hr and both control groups (**p < 0.005).
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Figure 8.
MHC-II immunohistochemistry is increased 3 d
after anti-A antibody injections. MHC-II immunohistochemistry is
shown in frontal cortex (A, B) and
hippocampus (C, D). Mice in
A and C received control antibody,
whereas those in B and D received
anti-A antibody. Magnification, 40×. Scale bar, 120 µm. A
high-resolution color version of this micrograph can be obtained by
e-mail from D. Morgan (dmorgan{at}hsc.usf.edu).
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In contrast, 72 hr after the injection of
anti-A antibodies, the microglial activation detected with CD45
antibodies was more widespread, detected not only at the injection site
but also away from the injection site in the frontal cortex (Fig.
6B) and throughout the dentate gyrus, with a
concentration within the granule cell layer at the 72 hr time point
(Fig. 6D). MHC-II staining revealed a similar
pattern, although not as extensive as that found with CD45 staining
(Fig. 8B,C).
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Figure 9.
Anti-A antibody injections result in an
increase in MHC-II immunohistochemistry 3 d after injection. Data
are expressed as percentage area occupied by MHC-II-positive staining
in the injected hemisphere. The three bars indicate MHC-II expression
in the untreated (None) group and the vehicle (VEH) and anti-HIV
antibody (Cont-Ab) groups at 72 hr. The line shows the amount of MHC-II
staining at 4, 24, 72, and 168 hr survival times. Increased MHC-II
staining was observed in the frontal cortex (A)
and hippocampus (B) at 72 hr compared with 4, 24, and 168 hr and both control groups (**p < 0.01).
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Quantification of these results indicated that the injection of
anti-A antibodies increased expression of the microglial marker
CD45 significantly only at the 72 hr time point compared with all other
time points and control groups in both cortex (Fig. 7A;
p < 0.005) and hippocampus (Fig. 7B;
p < 0.005). Also, the injection of anti-A
antibodies increased the expression of the microglial marker MHC-II at
the 72 hr time point compared with all other time points and control
groups in both cortex (Fig. 9A; p < 0.01)
and hippocampus (Fig. 9B; p < 0.005). The expression of CD45 and MHC-II in the frontal cortex
at the anti-A injection site increased more than eightfold over
that of all other time points, including 1 week, and both of the
control groups. The expression of CD45 in the hippocampus at the
anti-A injection site increased more than twofold, whereas the
increase in expression of MHC-II was more than eightfold. As in our
previous work, there was considerable variability among samples with
both microglial markers; however, all anti-A-injected animals had
values that were greater than the means for the control groups.
There were few remaining amyloid deposits near the injection sites in
the anti-A antibody-injected mice at 72 hr (Figs. 4, 5). These
residual deposits were relatively faint when stained with Congo red and
were contacted by rounded, CD45-positive microglial cells (Fig.
10B). In contrast,
the more abundant amyloid deposits on the contralateral side and in the
control animals were contacted by microglia with long processes that
were stained for CD45, whereas the cell body stained only faintly for
this marker of microglial activation (Fig. 10A).
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Figure 10.
Anti-A antibody injections result in rounded
microglia in association with remaining congophilic amyloid deposits
3 d after injection. CD45 immunostaining counterstained with Congo
red is shown in the hippocampus at the 72 hr time point.
A, Typical intensely stained congophilic deposit
surrounded by CD45 immunostained microglial processes, with faintly
stained somata (arrow). B, Faintly stained congophilic
deposit in the anti-A antibody-injected hippocampus. Note the two
rounded intensely CD45-positive cells in contact with the faintly
stained deposit (arrow). Magnification, 600×. Scale
bar, 8.33 µm.
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Discussion |
Intracranial anti-A antibody injections reduced A load
substantially in the vicinity of the injection in both anterior cortex and hippocampus over a 7 d time frame. By 4 hr after the
injection, there was a broad distribution of injected antibody that
filled a volume of ~0.5 mm3, as
estimated from anti-IgG immunohistochemistry. In addition to the broad
pattern of diffusion, the antibody was concentrated in the outer
molecular layers of Ammon's horn and the dentate gyrus, a zone that
mostly overlaps with the distribution of A staining in transgenic
mice of this age (Fig. 2C). Thus, it appears the injected
antibody was binding to in situ A at this early time
point but was also spread throughout the hippocampus. By 24 hr, there
was a reduction in the A immunostaining in the vicinity of the
antibody injection in both cortex and hippocampus. This reduction in
A load is unlikely to be an artifact caused by the injected
antibody masking the epitope of the primary antibody used for
immunohistochemistry, because the reduced load was not detected 4 hr
after administration, and by 24 hr, the injected IgG appeared to be
concentrated closer to the granule cell region than the outer molecular
layer in the hippocampus. Furthermore, the stoichiometry of injected
antibody (13 pmol) to A in deposits (estimated at 250 pmol in 0.5 mg; Chapman et al., 1999) is likely too low to interfere substantially
with the histochemical reaction. This early reduction in A load
occurred in the absence of the expression of microglial activation
markers CD45 and MHC-II. Although this does not preclude some rapid
response of the microglia, it does suggest that the role of microglia
is qualitatively different at this early postsurvival time from when
markers of activation are being expressed.
Between 24 and 72 hr after injection of anti-A antibodies, there
were parallel reductions in fibrillar amyloid deposits detected by
thioflavine-S and increases in microglial activation evaluated by CD45
and MHC-II staining. Although control injections of anti-HIV antibody
and vehicle caused some elevation of the CD45 marker, activation was
restricted to the immediate vicinity of the injection site and was
likely caused by mechanical injury associated with needle insertion and
fluid compression of the tissue. Occasionally, in the anti-A
antibody-injected mice, some remaining wisps of amyloid could be found
in the vicinity of the antibody injection at 72 hr, and these were in
contact with rounded CD45-immunopositive cells suggestive of phagocytic
microglia or macrophages. Also at the 72 hr time point, there was a
concentration of staining for both the injected antibody and the
microglia near the granule cell layer of the dentate gyrus. The
temporal association of fibrillar amyloid loss with microglial
activation suggests some causal role for microglial activation in this
process. One possibility is that between 1 and 3 d, activated
microglia near the deposits in the outer molecular layer phagocytosed
opsonized amyloid via Fc receptor- or complement-mediated mechanisms
and migrated toward the granule cell layer. CD45-positive microglia
could be detected readily in the outer molecular layer near the fissure
at 3 d, although they were concentrated most heavily near the
granule cell layer at that time point (Fig. 6D). A
second option is that after dissolution of the A deposits, the
antibodies diffused to the granule cell region independently of the
microglia. Possibly, the fibrillar deposits simply required more time
to dissolve than the more diffuse material. More detailed time course
studies of the period between 1 and 3 d coupled with
immunoelectron microscopy will likely be required to decide between
these options. Remarkably, the microglial activation was terminated
rapidly and returned to normal levels by the 1 week time point in
conjunction with a significant reduction in staining for the injected
IgG and A.
An accumulating body of evidence finds an association between
microglial activation and amyloid reductions in transgenic mouse models
of amyloid deposition. Schenk et al. (1999) noted in the first study
evaluating A vaccines that the clearance of amyloid was associated
with enhanced microglial activity around the remaining deposits.
Wilcock et al. (2001) mostly confirmed this observation in a different
transgenic model. Nakagawa et al. (2000) unexpectedly found that fluid
percussion injury activates microglia and results in reduced amyloid
deposition as mice grow older. Lim et al. (2001) noted that transgenic
mice treated with curcumin had a reduced amyloid load but an increase
in the activation state of microglia surrounding plaques. Similarly,
Jantzen et al. (2002) found a reduced amyloid load in transgenic mice
treated with a nitric oxide-releasing nonsteroidal anti-inflammatory
drug, NCX-2216, which was also associated with increased
microglial activation. Wyss-Coray et al. (2001) found that the crossing
of APP transgenic mice with mice that overexpressed TGF- led to
increased microglial activation and reduced amyloid loads. Conversely,
Wyss-Coray et al. (2002) found that blocking of complement activation
with soluble complement receptor-related protein Y overexpression
diminished the microglial reaction in APP transgenic mice and led to
elevated amyloid loads. DiCarlo et al. (2001) attempted to activate
microglia directly by injecting lipopolysaccharide and found that this
was associated with clearance of A in the vicinity of the injection. However, Qiao et al., (2001) injected lipopolysaccharide chronically into young transgenic mice before normal amyloid deposition and found
that it could stimulate A deposition. It is also the case that
careful serial section electron microscopy failed to detect internalized amyloid in microglia associated with amyloid deposits in
untreated APP23 transgenic mice (Stalder et al., 2001), although mice
treated to provoke microglial activation have yet to be examined. Nonetheless, there is a growing body of literature that associates the
activation of microglia with a reduction in A deposition in the
transgenic mouse models.
A number of studies have demonstrated that cultured microglial cells
are capable of internalizing A1-42 aggregates (Paresce et al.,
1996; Webster et al., 2001). A can also be cleared from unfixed
brain sections by anti-A antibodies in a microglia-dependent manner
(Bard et al., 2000). Direct imaging of amyloid deposits in
vivo by multiphoton microscopy has shown clearance of plaque after
application of an anti-A antibody in association with an upregulation of activated microglia (Bacskai et al., 2001). Suggested alternative mechanisms to microglial phagocytosis include a physical interaction between antibody and A that results in disaggregation of deposits, which was demonstrated in vitro with monoclonal
anti-A antibodies (Solomon, 2001). Consistent with this idea,
Bacskai et al. (2002) demonstrated recently that
F(ab')2 fragments prepared from an anti-A
antibody reduced amyloid deposits as effectively as the intact antibody
when applied topically to the cortex of transgenic mice through a
craniotomy. Although there was no measurement of microglial activation
in this study, it is plausible that this occurred in the absence of
microglial involvement. This antibody-mediated dissolution hypothesis
is consistent with the early phase of A reduction described here and
may still be found to be responsible for the second phase of fibrillar
deposit reduction.
A major unresolved question is how this antibody-mediated clearance of
A might apply to the human condition. Increasingly, AD has been
argued to involve inflammation as a component of its pathogenesis
(McGeer and McGeer, 2001). The early stages of the A vaccine trials
resulted in a small fraction of patients developing adverse reactions
consistent with inflammation of the CNS, presumably including
microglial activation (Hock et al., 2002; Schenk and Yednock, 2002).
Although adverse reactions to immunotherapy have been rare in the
transgenic models (Pfeifer et al., 2002), it remains the best
experimental system in which to understand the different components of
the immune reactions to vaccines and to identify those components that
cause adverse outcomes. Certainly, identification of immunotherapies
that avoid the problem of deleterious CNS inflammation will be
necessary if this treatment approach is to find use in the clinical
setting. A better understanding of the mechanisms of antibody-mediated
clearance of A in the transgenic models of amyloid deposition should
benefit this effort.
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FOOTNOTES |
Received Sept. 17, 2002; revised Jan. 21, 2003; accepted Jan. 29, 2003.
This work was supported by National Institutes of Aging, National
Institutes of Health Grants AG15490 (M.N.G.), AG18478 (D.M.), and
AG20227(K.E.U.). D.M.W. is a Benjamin Scholar in Alzheimer's Disease Research.
Correspondence should be addressed to Dave Morgan, 12901 Bruce B. Downs
Boulevard, MDC 9, Tampa, FL 33612. E-mail: dmorgan{at}hsc.usf.edu.
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TOP
ABSTRACT
Introduction
