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The Journal of Neuroscience, November 15, 2002, 22(22):9785-9793
Evidence That Synaptically Released  -Amyloid Accumulates as
Extracellular Deposits in the Hippocampus of Transgenic Mice
Orly
Lazarov1,
Michael
Lee2,
Daniel A.
Peterson3, and
Sangram S.
Sisodia1
1 Department of Neurobiology, Pharmacology, and
Physiology, The University of Chicago, Chicago, Illinois 60637, 2 Department of Pathology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205, and
3 Department of Neuroscience, The Chicago Medical School,
The Finch University, North Chicago, Illinois 60064
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ABSTRACT |
A neuropathological hallmark of Alzheimer's disease is the
deposition of amyloid- (A) peptides in senile plaques in the hippocampus and cerebral cortex. A is derived from larger integral membrane proteins termed amyloid precursor proteins (APP). We demonstrated previously that APP, synthesized by neurons in the entorhinal cortex, is transported via the perforant pathway to presynaptic terminals in the dentate gyrus. We reported that, although
full-length APP and membrane-tethered, C-terminal APP derivatives
(APP-CTFs) accumulate at terminal fields, the production of A
peptides at these sites was indeterminate. To test the hypothesis that
APP-CTFs, generated from axonally transported APP, are further metabolized to A peptides that are subsequently released and deposited proximal to nerve terminals, we created unilateral knife lesions of the perforant pathway of transgenic mice that exhibit hippocampal amyloid deposits. We observed pronounced reductions in
amyloid burden in the ipsilateral dentate gyrus, findings that lead us
to conclude that axonally transported APP gives rise to A peptides
that are released from presynaptic sites in the dentate gyrus and
deposited in extracellular plaques. Moreover, our findings are
consistent with the view that A deposits are dynamic structures and
that the perforant path lesion alters the equilibrium between A
production-deposition toward clearance as a consequence of blocked
axonal transport of APP from the entorhinal cortex to terminal fields
in the hippocampus.
Key words:
Alzheimer's disease; amyloid precursor protein; amyloid
deposition; perforant pathway; hippocampus; synapse
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INTRODUCTION |
Alzheimer's disease (AD) is
characterized by the deposition of the amyloid- (A) peptides in
the brain parenchyma and cerebrovasculature of affected individuals.
A are 39-43 amino acid peptides derived from amyloid precursor
proteins (APP). Familial, early-onset forms of Alzheimer's disease
(FAD) are caused by the expression of genes encoding mutant variants of
APP and presenilins (PS1 and PS2) (Price and Sisodia, 1998 ; Selkoe,
2001); FAD-linked APP or presenilin variants cause disease by enhancing
the levels, length, or fibrillogenic properties of A peptides
(Borchelt et al., 1996; Duff et al., 1996; Scheuner et al., 1996;
Citron et al., 1997) and their subsequent deposition.
We and others have documented that neuronally expressed APP is
subject to fast anterograde transport to nerve terminals (Koo et al.,
1990; Ferreira et al., 1993; Sisodia et al., 1993; Amaratunga and Fine,
1995; Buxbaum et al., 1998; Kaether et al., 2000; Kamal et al., 2000,
2001). We reported that APP, synthesized in the entorhinal cortex (EC),
is transported via the perforant pathway to the hippocampus and dentate
gyrus (Buxbaum et al., 1998). In our studies, a set of APP derivatives,
including soluble APP and membrane-tethered APP C-terminal
fragments (APP-CTFs) that harbor the entire A sequence, accumulate
at these presynaptic sites (Buxbaum et al., 1998). However, it has not
been established whether the APP-CTFs, generated from axonally
transported APP, are further metabolized to A peptides and whether
these peptides are subsequently released and deposited proximal to
nerve terminals. To address this important issue, we exploited
transgenic mice coexpressing FAD-linked APP and presenilin 1 variants
that exhibit amyloid deposition throughout the neocortex and
hippocampal formation (Borchelt et al., 1997).
To examine whether APP transported via the perforant pathway is a major
contributor to accumulation of A deposits in the hippocampus, we
performed unilateral lesions of the perforant pathway of transgenic
mice that express both the FAD-linked human PS1- E9 variant (Lee et
al., 1996) and a chimeric mouse-human APP Swedish (APPswe) (Borchelt
et al., 1997) and assessed amyloid burden in the hippocampal formation
after the lesion (Fishman et al., 2001). Our assumption, based on
compelling evidence that amyloid deposition is in equilibrium with
clearance (Holtzman et al., 1999; Schenk et al., 1999), was that we
should be able to shift the equilibrium toward clearance simply by
abrogating APP transport (and subsequent A production-deposition)
at terminal fields. Fulfilling this prediction, we now show that
amyloid deposits in the ipsilateral (lesioned) hippocampus of
FAD-linked transgenic mice are cleared within 1 month after the
perforant pathway lesion. Importantly, clearance was most pronounced in
a region of the ipsilateral hippocampus corresponding to the dentate
gyrus. Finally, we assessed amyloid deposition in mice in which
perforant pathway lesions were performed before the onset of amyloid
deposition. Four months after the lesion, amyloid burdens in the
lesioned and unlesioned hippocampus are essentially identical. Hence,
it is likely that amyloid deposition in the ipsilateral hippocampus is
the consequence of release of A from reactive presynaptic terminals
that have reinnervated the ipsilateral hippocampus.
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MATERIALS AND METHODS |
Transgenic mice. Mice coexpressing FAD mutant human
PS1-E9 and a chimeric mouse-human APP695 harboring a human A
domain and mutations (K595N, M596L) linked to Swedish FAD pedigrees
(APPswe) have been described previously (Borchelt et al., 1996, 1997;
Lee et al., 1997). The background strains for APPswe are
{C3H/HeJ × C57BL/6J F3} × C57BL/6J n1, and PS1-E9
are C3H/HeJ × C57BL/6J F3.
Perforant pathway lesion. Animals were deeply anesthetized
using a mixture of ketamine (75 mg/kg) and xylazine (4 mg/kg; Henry Schein, Melville, NY) and placed on a Kopf stereotaxic apparatus adjusted to mice (David Kopf Instruments, Tujunga, CA). A wire knife
assembly was used for precision perforant pathway lesion. Coordinates
for knife cut lesion relative to bregma are as follows (in mm):
anteroposterior,  4.6; mediolateral, ±3.3; dorsoventral, 4.5 to 3.5 (from dura), with the nose bar set at 3.5. An
opening in the scalp at the above coordinates was performed using
electric drill, and the dura was exposed. The assembly is set to the
ventral extent of the lesion (4.5 mm). After penetration, the knife
was extended to form a hook that extends medially for 2 mm, the
assembly was raised to the dorsal extent of the lesion (3.5 mm), and
the knife was retracted, reextended, and again lowered to the
ventral extent. The knife hook was raised and lowered five times to
verify complete transection of the perforant pathway; each retraction and reextension was performed to ensure that flexible axons that were
stretched by the knife hook were cut. To verify the accuracy of the
lesion, a crystal of the fluorescent dye Fluoro-Ruby (Molecular Probes,
Eugene, OR) were dissolved in saline and introduced into the injury
site concomitantly with knife penetration. The injury site was
subsequently visualized by Olympus Optical (Melville, NY)
Fluoview confocal laser scanning microscope.
Histology. Animals were perfused transcardially under deep
anesthesia with a saline solution, followed by fixative solution composed of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer. The brains were removed and
kept in fixative overnight, followed by flotation in 30% sucrose
solution. Brains were sectioned horizontally (50 µm) using a
frozen-stage equipped microtome and then placed at 20°C in
cryoprotectant solution (glycerol and ethylene glycol-based
PBS). Every sixth tissue section was subject to
immunocytochemistry, as described below, and analyzed by confocal
microscopy. Sections containing Fluoro-Ruby-labeled injury sites were
analyzed using a MetaMorph imaging program (Universal Imaging
Corporation, West Chester, PA). Equivalent sections of nonlesioned
mice, were also analyzed in this manner.
Immunohistochemistry. Free floating horizontal brain
sections (50 µM) were rinsed three times for 10 min each in Tris-buffered saline (TBS), blocked using
blocking-permeabilizing solution (5% donkey serum and 0.25% Triton
X-100) for 1-3 hr, and incubated with mouse anti-S100 (1:10,000;
Swant, Bellinzona, Switzerland), goat anti-GFAP (1:5000; Dako,
Cambridgeshire, UK), rabbit anti-neurofilament (NF-200 kDa) (1:500;
Chemicon, Temecula, CA), mouse anti-Bassoon (1:1000; a gift from
Dr. Craig C. Garner, University of Alabama at Birmingham, Birmingham,
AL), mouse anti-human A 6E10 (1:1000), or rabbit anti-A42
antibodies (FCA3542; 1:1000; a gift from Dr. Frederic Checler, Centre
National de la Recherche Scientifique, Valbonne, France) for 72 hr at
4°C. Tissues were then rinsed in blocking solution for 2 hr. Sections
were incubated with secondary antibodies (1:250; Jackson
ImmunoResearch, West Grove, PA) at room temperature. Sections were
then washed three times for 10 minutes each in TBS and mounted on
gelatin-coated slides using polyvinyl
alcohol-1,4-diazabicyclo-[2.2.2]octane (PVA-DABCO; Sigma, St.
Louis, MO) mounting solution.
Imaging and quantification of amyloid burden.
Immunofluorescence in sections were visualized and imaged using an
Olympus Optical Fluoview confocal laser scanning microscope. Images at
each wavelength were collected separately, using a separate and
specific excitation filter. Images were taken and recorded using a
Fluoview 2.1 program. Fluorescent images were either used for
quantification of amyloid burden or processed for collage by Dell
(Round Rock, TX) OptiPlex GX1 running Photoshop 5.0 (Adobe Systems,
Mountain View, CA). Z series of 10 µm depth were imaged from each
section with 1 µm intervals between images. Volume of amyloid burden
was quantified using Multiscan 500PS (Sony, Tokyo, Japan) equipped with
MetaMorph 4.1 (Universal Imaging Corporation). Z series images were
converted in all planes into binary images, after calibration of pixel
size. Single components were considered noise and removed uniformly from all planes. Images were converted to gray scale by thresholding, and the burden volume was estimated.
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RESULTS |
Quantitative analysis of amyloid burden in the hippocampus of
transgenic mice
To examine amyloid deposition and clearance in the hippocampus, we
used transgenic mice that coexpress FAD-linked mutant human PS1-E9
(Lee et al., 1997) and APPswe (Borchelt et al., 1996, 1997). The
expression of mutant PS1-E9 in the singly transgenic line S9 (Lee et
al., 1997) is ~1.5-fold higher than endogenous PS1, whereas
expression of APPswe in the singly transgenic line C3-3 (Borchelt et
al., 1996, 1997) is ~2.5-fold over endogenous APP. In contrast to the
singly transgenic C3-3 line, which exhibits sparse amyloid deposits at
between 18 and 20 months of age (Borchelt et al., 1997), the doubly
transgenic mice that coexpress APPswe and PS1-E9 exhibit amyloid
deposits, most prominently in the hippocampal formation, at the age of
5-6 months. Figure 1 shows representative images of brain sections from mice stained with the
human A-specific antibody 6E10 and visualized by indirect immunofluorescence using laser confocal microscopy. The left
and right panels depict A antibody-immunoreactive
deposits in the dentate gyrus and hippocampus, respectively, of
transgenic mice at the age of 6 months (A), 8 months
(B), and 13 months (C).
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Figure 1.
Representative images of amyloid immunoreactivity
in the dentate gyrus (left panels; DG)
and hippocampus (right panels; HIPP) of
unlesioned transgenic mice (A, 6-month-old mouse;
B, 8-month-old mouse; C, 13-month-old
mouse). Amyloid immunoreactivity in the left side (DG-I,
HIPP-I) was compared with that detected in
the contralateral (right) side (DG-C,
HIPP-C). Amyloid was detected by immunolabeling with
6E10 antibodies. No significant difference in amyloid immunoreactivity
could be detected. Scale bars, 250 µm.
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Whereas the extent of amyloid deposition in the hippocampus increases
with age, as expected (Borchelt et al., 1997), there is no obvious
differences in the A burden between the left and right hemispheres
in either the dentate gyrus or the hippocampus of nonlesioned
mice. To quantify amyloid burden in the hippocampus and dentate gyrus
of unlesioned transgenic mice, we performed a quantitative analysis of
the volume of A-immunoreactive structures. Z series of hippocampal
images were obtained using a confocal microscope, and the volume of
amyloid burden was analyzed using MetaMorph software. The hippocampal
area that was analyzed includes the dentate gyrus, regio superior, and
regio inferior, whereas the dentate gyrus subfield that was subject to
analysis included the granule cell layer (stratum granulare) and the
outer molecular layer (stratum moleculare). Morphometric quantification
of A immunoreactivity confirmed that there are no significant
differences in amyloid deposition between the left and right
hemispheres in animals between the ages of 5 and 13 months (Fig.
2).
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Figure 2.
Quantitative analysis of amyloid burden in the
hippocampus of unlesioned mice. For each individual animal analyzed,
amyloid burden in the left side was compared with the burden in the
right side in both the hippocampus (left) and the
dentate gyrus (right). The ratio between the hemispheres
is indicated. No difference in amyloid burden could be observed.
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Perforant pathway transection leads to reduced amyloid burden in
the hippocampus
Having established that amyloid deposition occurs to the same
extent in both hemispheres of unlesioned animals, we then asked whether
unilateral transection of the perforant pathway would impact on amyloid
burden in the hippocampus. The rationale of this experiment is that,
after transection, delivery of axonally transported APP from the
entorhinal cortex to the terminal fields of the hippocampus would not
occur, thus shifting the balance between A production-deposition
toward clearance. The perforant pathway provides the major neuronal
input to the hippocampus from the EC and involves afferent projections
from layers II and III of the EC that terminate in the outer molecular
layer of granule cell dendrites in the dentate gyrus (Hjorth-Simonsen
and Jeune, 1972; Tamamaki and Nojyo, 1993). Thus, transection of the
perforant pathway through the presubiculum from its medial surface
through the splenium of the corpus callosum completely disconnects the hippocampus from the entorhinal cortex.
We assessed amyloid burden by confocal microscopy of brain sections of
lesioned mice 1 month after the transection. Figure 3 shows representative images of amyloid
immunoreactivity in both the ipsilateral (lesioned) and contralateral
(unlesioned) dentate gyrus (left panels) or hippocampus
(right panels) of 7 month (A), and two 10 month (B, C) animals. We observed a qualitative
reduction of amyloid immunoreactivity in the ipsilateral hippocampus
and dentate gyrus compared with the contralateral side.
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Figure 3.
Representative images of amyloid immunoreactivity
in the dentate gyrus (left panels; DG)
and hippocampus (right panel; HIPP) of
perforant pathway-lesioned mice (A, 7-month-old mouse;
B, C, 10-month-old mice). Amyloid
immunoreactivity in the ipsilateral (lesioned) side
(DG-I, HIPP-I) was compared with
that in the contralateral side (DG-C,
HIPP-C) 1 month after perforant pathway lesion. Amyloid
was detected by immunolabeling with 6E10 antibodies.
Immunoreactivity of amyloid deposits is reduced in the ipsilateral
hippocampus and dentate gyrus compared with the contralateral side.
Scale bars, 250 µm.
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To quantify the magnitude of change in amyloid burden 1 month after
transection, we used morphometric approaches, as described in Materials
and Methods. Figure 4 is a quantitative
analysis of amyloid burden in the ipsilateral versus contralateral side of individual animals between the ages of 7 and 10 months (at the time
of lesion). In all brain sections of lesioned mice, the amyloid burden
in the entire ipsilateral hippocampus was approximately twofold to
threefold lower than the burden in the entire contralateral hippocampus
(Fig. 4). However, comparison of the amyloid burden of the dentate
gyrus alone revealed that the reduction in amyloid burden on the
ipsilateral side was between 5.5- and 10-fold lower than the
contralateral side (Fig. 4). Parallel analysis of other brain areas
that are not directly affected by the lesion, including the cortex,
failed to show differences in amyloid burden between left and right
hemispheres after the lesion (data not shown).
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Figure 4.
Quantitative analysis of amyloid burden in the
hippocampus of perforant pathway-lesioned mice 1 month after lesion.
Analysis revealed a significant reduction in amyloid burden in the
ipsilateral hippocampus (left) and dentate gyrus
(right) compared with the contralateral side (the ratio
between the hemispheres is indicated). The reduction in amyloid burden
in the dentate gyrus was most pronounced.
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Although clearance of amyloid deposits after perforant pathway lesion
occurs in a region-specific manner, it was conceivable that
lesion-induced astroglial activation and subsequent inflammatory reactions may play a critical role in the process of amyloid clearance. To examine this issue, we introduced a stab wound lesion into the
perforant pathway of transgenic mice, one that does not cause substantial transection of the axons that perforate the dentate gyrus.
One month later, we quantified amyloid burden in brain sections of
these mice. These studies revealed that there were no significant
differences in amyloid burden between the ipsilateral and the
contralateral hippocampi or dentate gyrus of the stab-wounded mice
(Fig. 5). Thus, perforant pathway
lesion-mediated clearance of amyloid deposition occurs in a highly
selective, region-specific manner, consistent with the notion that
axonal delivery of APP from the entorhinal cortex is responsible for
A production and deposition in the dentate gyrus.
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Figure 5.
Amyloid burden in the ipsilateral and
contralateral hippocampus and dentate gyrus of stab wound-treated mice
1 month after treatment. Top panels show 6E10-labeled
hippocampus of a stab wound-treated mouse. Bottom panels
shows quantitative analysis of amyloid burden in the ipsilateral
(HIPP-I) and contralateral
(HIPP-C) hippocampus and dentate gyrus of stab
wound-treated mice 1 month after treatment. No significant difference
in amyloid burden was found between the ipsilateral and the
contralateral hippocampus or dentate gyrus in these animals. Scale bar,
250 µm.
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Amyloid clearance attenuates astrogliosis and
neuritic dystrophy
As has been reported previously, we observed the presence
of both activated astrocytes and microglia in brain regions with amyloid deposition. In unlesioned animals, immunocytochemical staining
with antibodies against S100, the major low-affinity Ca+2 binding protein in astrocytes and
microglial cells (Garbuglia et al., 1999; Adami et al., 2001),
disclosed the presence of extensively ramified glial cells (Fig.
6A,B).
Higher-magnification confocal images revealed some overlap in A
immunoreactivity and S100-positive glial processes (Fig.
6C-E), suggesting that these cells are actively reacting to
amyloid deposition. In contrast, in hippocampal sections from mice with
perforant pathway lesions, S100 immunoreactivity was markedly
reduced in the ipsilateral hippocampus, with few, albeit
morphologically quiescent, glial cells that remained (Fig. 6F). In contrast, reactive glia were prominent around
amyloid deposits in the contralateral side (Fig. 6G). Hence,
it is reasonable to assume that the presence of insoluble amyloid
aggregates induces glial activation and that the activated phenotype of
these cells returns to a quiescent state once the deposited A
peptides are no longer present. However, it should be noted that
microglia and astrocytes have been shown to become activated in the
terminal zones of lesioned entorhinal afferents to remove degenerating axons and terminals, and, hence, it is conceivable that clearance may,
in part, be mediated by these lesion-activated cells.
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Figure 6.
A, B, Reactive
gliosis in the hippocampus and dentate gyrus of unlesioned mice as
could be detected by immunostaining using S100 antibodies in dentate
gyrus in the left hemisphere (A) and in the right
one (B). S100 immunoreactivity (in
red) was most pronounced around amyloid deposits
(immunostained using 6E10 antibodies; in green). Some
overlap between S100-labeled glia (D) and
amyloid deposits (6E10 antibodies; C) could be detected
(E, see arrows). F,
G, Reactive morphology of glia is reduced in the
ipsilateral hippocampus (F) but not in the
contralateral one (G) 1 month after perforant
pathway lesion. Scale bar, 150 µm.
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In addition to changes in glial morphology and immunoreactivity, we
also observed marked alterations in the general organization of
neurofilament-positive neuronal processes. The presence of dystrophic
neurites around amyloid deposits has been observed in specimens from
human AD subjects, as well as in transgenic mouse models (Vickers et
al., 1996; Irizarry et al., 1997a,b; Holtzman et al., 2000; Pigino et
al., 2001), but the mechanism regulating this process is unclear. It
has been suggested that fibrillar plaques induce local neuritic
alterations and the subsequent modifications of cytoskeletal proteins
within associated neuronal processes (Dickson and Vickers, 2001). To
examine possible changes in neurofilament morphology during the process
of amyloid clearance, brain sections of lesioned and unlesioned mice
were immunolabeled with antibodies specific for neurofilament H (NF-H).
In the unlesioned (contralateral) hippocampus of the transgenic mice,
we observed marked NF-H-positive neuritic dystrophy (Fig.
7A,B).
This phenotype was reversed to a normal pattern 1 month after perforant
pathway lesion (Fig. 7C). Hence, lesion-induced clearance of
amyloid deposition in the hippocampus results in the reversal of
neuritic dystrophy and attenuation of astrocyte-glial activity.
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Figure 7.
Neurofilament morphology in the dentate
gyrus-hippocampus as detected by immunolabeling 1 month after
perforant pathway lesion. A, B,
Contralateral hippocampus (B, high power).
C, Ipsilateral hippocampus. Brain sections are double
immunolabeled for neurofilament (in green) and
-amyloid (6E10 antibodies; in red). Neuritic atrophy
could be detected in the contralateral side. In contrast, neurofilament
morphology in the ipsilateral side seems to be recovered, at least in
part. Scale bars, 250 µm.
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Perforant pathway lesion-induced reinnervation and subsequent
deposition of A in the dentate gyrus
Previous studies have established that the terminal arbors of
entorhinal fibers in the dentate gyrus undergo a cycle of deinnervation and subsequent reinnervation after perforant pathway lesions. Deinnervation is completed 7-10 d after perforant pathway lesion, a
process that creates a zone of deinnervated postsynaptic elements that
become receptive to new axonal growth. This process, termed reactive
synaptogenesis, occurs progressively over the next 60-90 d (Frotscher
et al., 1997; Turner et al., 1998). Sprouting and reactive
synaptogenesis of intrinsic hippocampal fibers takes place in the of
the outer two-thirds of the molecular layer of the dentate gyrus. In
addition, the deinnervated field in the dentate gyrus also receives
inputs from the contralateral entorhinal cortex and predominantly from
cells in layer II that normally project to the ipsilateral dentate
gyrus (Steward, 1976). The reinnervating cells are located in the
dorsal half of the entorhinal cortex and concentrated in the medial
most portion of layer II (Steward and Vinsant, 1978). Finally, it is
highly likely that, in addition to intrinsic and contralateral
afferents, cholinergic neurons of the septohippocampal pathway will
also reinnervate the denervated zone, as has been described previously
(Nadler et al., 1974, 1977). In any event, we hypothesized that, if
A deposition at terminal fields is the result of A released from synaptic sites, then lesions of the perforant pathway in mice before
the onset of deposition should lead to initial deinnervation and
subsequent recruitment of reinnervating fibers to the outer molecular
layers of the dentate gyrus. In turn, newly formed synaptic termini
should provide a new "reservoir" from which A is subsequently released. To examine the hypothesis that reinnervation of terminal zones could lead to presynaptic release and deposition of amyloid, we
performed unilateral perforant pathway lesions in transgenic mice at 4 months of age, a time at which amyloid deposition is still
undetectable. Four months later, these animals were killed, and
brain sections were examined for the presence of A deposits by
indirect immunofluorescence using the human A-specific antibody 6E10. As we predicted, quantitative analysis of amyloid burden in the
dentate gyrus and hippocampus of these perforant pathway-lesioned mice
revealed little, if any, difference between the ipsilateral (lesioned)
and contralateral hemispheres (Fig. 8).
In addition, we did not detect any difference in reactive gliosis
between the ipsilateral side and the contralateral hemispheres (data
not shown).
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Figure 8.
Quantitative analysis of amyloid burden in the
hippocampus of mice after reinnervation of the outer molecular layer of
the dentate gyrus. Mice were subject to perforant pathway lesion at 4 months of age. Four months later, brain sections were analyzed for
amyloid burden. The ratio between the hemispheres is indicated. No
difference in amyloid burden could be observed between the lesioned
hippocampus and the unlesioned one.
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To confirm that reactive synaptogenesis had occurred in the dendritic
arbors of dentate neurons of these mice, brain sections were examined
for the presence of Bassoon (tom Dieck et al., 1998), a protein that is
specifically localized to presynaptic active zones (Zhai et al., 2001).
Brain sections of animals killed 1 week, 1 month, or 4 months after
lesion were analyzed. Bassoon immunoreactivity was readily detectable
in the ipsilateral hippocampus of 4 month postlesioned mice but was
markedly lower in brain sections of 1 month and barely detected in
sections of 1 week postlesioned mice (Fig.
9). These findings are consistent with
the reported rate of reinnervation and synaptogenesis in the dentate
gyrus after perforant pathway lesions (Turner et al., 1998). To examine the relationship between presynaptic structures and amyloid deposits in
the lesioned hippocampus, we performed double labeling with antibodies
specific for Bassoon and the highly fibrillogenic form of A, termed
A42 (FCA3542) (Barelli et al., 1997). These studies revealed
the presence of presynaptic elements in immediate proximity to amyloid
deposits, findings we interpret to suggest that deposited A is
released from presynaptic sites and deposited in the extracellular milieu (Fig. 9E-G). Notably, we also observed
colocalization of numerous Bassoon-immunoreactive structures with
A42 (Fig. 9G, arrows). These coimmunoreactive
structures likely represent dystrophic terminals in proximity to
plaques, findings that offer the notion that A42 deposited in
extracellular sites are generated at presynaptic terminals. Additional
ultrastructural and immuno-EM studies will be required to confirm these
observations. Essentially identical distributions of Bassoon and A42
were observed in the contralateral hippocampus of lesioned mice or
nonlesioned mice with amyloid deposits (data not shown).
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Figure 9.
Reactive synaptogenesis in the outer molecular
layer of mice 4 months after perforant pathway lesion. Brain sections
were examined for the presence of the presynaptic marker Bassoon 1 week
(A), 1 month (B), or 4 months (C, D) after lesion.
Immunoreactivity for Bassoon was easily detected in the ipsilateral
hippocampus of 4 month postlesion mice, less pronounced in brain
sections of 1 month mice, and barely detected in sections of 1 week
postlesioned mice. D, Bassoon and synapsin
double-labeled synapses in the dentate gyrus of 4 month postlesion
mice. E-G, Synapses could be detected, surrounding
amyloid deposits. This close proximity may suggest that the source of
deposited material is synapse released. E, Bassoon
immunoreactivity in the dentate gyrus of 4 month postlesion mice.
F, Amyloid immunoreactivity at the same area as
E, as detected by FCA3542 antibodies. G,
Merged image of Bassoon and amyloid immunoreactivities.
Arrows in G indicate overlapping staining
of immunoreactivity for A42 and Bassoon. Scale bars, 250 µm.
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DISCUSSION |
The hippocampal formation is a critical neuroanatomical structure
responsible for certain aspects of learning and declarative memory.
These neuropsychological parameters are primarily diminished in
AD and likely a reflection of extensive deposition of amyloid in senile
plaques in the hippocampus (Braak et al., 1994; Braak and Braak, 1996).
The hippocampus, and specifically, the granule cell dendrites in the
dentate gyrus, receives major afferent projections from the EC, a
region that receives significant cortical input and serves as a relay
station to the hippocampal formation. Neuropathological studies have
revealed that neurons in the EC and the hippocampus are particularly
vulnerable in AD, with abundant amyloid deposition and neurofibrillary
pathology in both regions (Van Hoesen et al., 1991; Braak and Braak,
1996; Gomez-Isla et al., 1996).
In previous efforts, we used a metabolic radiolabeling paradigm to
document that APP synthesized by EC neurons are axonally transported
via the perforant pathway to the hippocampal formation (Buxbaum et al.,
1998). We also demonstrated that these APP species, and a set of
membrane-tethered APP derivatives, termed APP-CTFs, accumulate in the
dentate gyrus. However, we were unsuccessful in demonstrating that APP
or APP-CTFs were further processed to A peptides. Hence, it remained
uncertain whether A peptides that accumulate in amyloid deposits in
the hippocampal formation were derived from APP that is synthesized
locally or by catabolism of APP (or APP-CTFs) that are synthesized at
distal sites and axonally transported to synaptic terminals. In an
attempt to establish that APP and/or APP-CTFs could be processed to
A peptides in vivo, we used transgenic mice that express
a chimeric mouse-human APP polypeptide harboring the familial Swedish
double mutation (Borchelt et al., 1996). In brain of line C3-3, this
polypeptide, termed APPswe, is expressed at approximately twofold
higher levels than endogenous APP, and sandwich ELISA studies indicate
that the steady-state levels of A in brain of these animals is
~20-25 pmol/gm (Borchelt et al., 1996) (D. Borchelt, personal
communication). Unfortunately, we did not succeed in detecting
A in the hippocampus 2 hr after the injection of up to 1 mCi of
[35S]methionine into the entorhinal
cortex or in cortical tissue after a bolus injection of 1 mCi of
[35S]methionine into the frontal cortex
(data not shown) of these animals. Our calculations reveal that, if all
newly synthesized APP is labeled in the 2 hr period and if 10% of APP
molecules are metabolized to A peptides, it would take nearly 1 year
to detect the presence of radiolabeled A by autoradiography.
Moreover, the rapid turnover of newly generated A (estimated
t1/2 of ~1-2 hr)(Savage et al.,
1998), likely served to further compromise our ability to detect
radiolabeled A species.
To examine the contribution of EC-derived, axonally transported APP in
A production and deposition in the hippocampus, we chose to exploit
transgenic mice coexpressing FAD-linked APP (APPswe) and PS1 that
exhibit amyloid deposits throughout the hippocampus and cortex by the
age of ~5-6 months. In these animals, the steady-state levels of
A42 peptides are slightly elevated, but these species contribute a
small fraction (~15-20%) to total A levels in the brains of
these animals. Our strategy involved unilateral transection of the
perforant pathway and assessment of hippocampal amyloid deposition
after disconnection of the EC and the terminal fields. Our rationale
was that disconnection of the hippocampus from the entorhinal cortex
might lead to clearance of preexisting deposits because the incoming
entorhinal afferents would not be able to transport APP to terminal
fields. In the present report, we provide several novel insights that
are consistent with the notion that axonally transported APP is a
principal contributor to amyloid deposition in the hippocampal
formation. First, we document that, after unilateral lesions of the
perforant pathway, amyloid burden in the ipsilateral hippocampus is
diminished, and this reduction in amyloid burden is associated with a
highly selective clearance in the dentate gyrus. Second, and in support
of these latter findings, reactive gliosis and neuritic dystrophy are
markedly reduced in the ipsilateral hippocampus compared with extensive
neuropathology observed in the contralateral side. These findings offer
the conclusion that occluding the transport of APP, APP-CTFs, or A
transport to terminal fields reduces amyloid burden simply by shifting
the equilibrium from A production and synaptic deposition toward clearance.
The mechanism of amyloid clearance is not presently clear but may
involve active phagocytosis of deposited A peptides by microglia or
by the binding of A peptides by the protease inhibitor  2
macroglobulin and clearance via the low-density lipoprotein receptor-related protein LRP (Narita et al., 1997; Shibata et al.,
2000). With regard to the former model, we provided evidence for the
codistribution of A-immunoreactive structures and glial processes,
similar to the description of the close apposition of microglia and
congophilic amyloid plaques in the brains of APP23 transgenic mice that
overexpress an FAD-linked human APP (Bornemann et al., 2001). Our
studies support several previous conclusions that amyloid deposits are
dynamic structures (Schenk et al., 1999; DeMattos et al., 2001, 2002)
and may suggest that turnover of amyloid deposits is an ongoing process
in the brain.
Finally, and to assess the contribution of synaptically released A
to extracellular amyloid deposition, in vivo, we performed unilateral lesions in transgenic mice at a time before the onset of
deposition. In contrast to the massive reduction in amyloid burden in
the ipsilateral hippocampus of mice with preexisting amyloid deposits 1 month after lesion, we failed to detect differences in amyloid burden
between the ipsilateral and the contralateral dentate gyrus and
hippocampus 4 months after lesions in mice in which amyloid deposition
is not apparent before the lesion. These findings strongly suggest that
reinnervation of the outer two-thirds of the molecular layer of the
dentate gyrus by intrinsic hippocampal fibers, afferent fibers from the
contralateral entorhinal cortex, and/or septal cholinergic neuron
afferents leads to release of A from synaptic termini and subsequent
deposition in the extracellular space.
Although our data support our view that synaptically released A are
the species deposited in the extracellular space, several important
questions remain. For example, it is not clear whether the deposited
A peptides are derived from full-length APP, APP-CTFs that are in
transit along axons, or APP-related species that are subject to
proteolytic processing at nerve terminals. Recent studies have provided
evidence that A peptides can be generated in vitro using
membranes prepared from mouse sciatic nerve, and these peptides appear
to be present within axonal membranes that undergo rapid anterograde
transport (Kamal et al., 2001). Unfortunately, we have not been
successful in detecting radiolabeled A derived from neuronally
synthesized and axonally transported APP in the perforant pathway of
rats (Buxbaum et al., 1998) or the transgenic mice used in this study
(see above; data not shown).
Second, we have not formally proven that A deposited in the terminal
fields of entorhinal afferents are derived from APP transported from
entorhinal neurons. In this regard, we cannot rule out the possibility
that A production-secretion and subsequent deposition at terminal
fields of entorhinal afferents might be a function of synaptic
activity-dependent release of A from dendritic spines of dentate
granule cells. As a corollary, it could be argued that the perforant
path lesion limits entorhinal-derived synaptic input and, in so doing,
leads to a reduction in amyloid burden via normal clearance processes.
However, our previous demonstration that radiolabeled APP-CTFs, the
penultimate precursors of A and A-related peptides, are detected
in the hippocampus after injections of
[35S]methionine in the entorhinal cortex
strongly argue that A is generated from axonally transported APP.
Finally, it should be noted that, although we see marked reductions in
amyloid burden in the dentate gyrus, we also observed reductions,
albeit limited, in amyloid burden in the hippocampal CA1 and CA3
subfields after perforant pathway lesions. Again, it could be argued
that the perforant pathway lesion diminishes synaptic drive through the entire trisynaptic circuit, i.e., from granule cell afferents to CA3
mossy fibers and subsequently projections from CA3 projections via
Shaeffer collaterals to dendritic terminals of CA1 pyramidal neurons.
On the other hand, it is now well established that the dendritic arbors
of CA1 and CA3 pyramidal neurons receive considerable innervation from
ipsilateral afferents of layer III neurons in the entorhinal cortex
(Steward and Scoville, 1976), in addition to projections from the
septum and brainstem. Thus, this reduction in amyloid burden in the
CA1-CA3 sector is not incompatible with our view that disconnection of
afferents from layer II and layer III neurons from the hippocampus
leads to clearance of amyloid deposits, specifically in the dendritic
fields of granule cell neurons and CA1-CA3 pyramidal neurons,
respectively. Recognizing the limitations of our conclusion that
synaptic release of A is a principal contributor to amyloid
deposition, we add that our findings are consistent with the compelling
in vivo demonstrations that, in diffuse plaques of AD
patients and aged nonhuman primates, A is present along neuronal
dendrites and around the soma of neurons included in the plaques
(Martin et al., 1991; Probst et al., 1991).
| |
FOOTNOTES |
Received May 22, 2002; revised Aug. 16, 2002; accepted Aug. 16, 2002.
This work was supported by National Institutes of Health Grants
AG-021494 (S.S.S.) and AG-20047-02 (D.A.P.) and the Ellison Medical
Foundation and the Ruth Broad Medical Research Foundation (S.S.S.).
Correspondence should be addressed to Dr. Sangram S. Sisodia,
Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, IL 60637. E-mail:
ssisodia{at}drugs.bsd.uchicago.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
