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The Journal of Neuroscience, October 1, 1999, 19(19):8552-8559
Cerebral Amyloid Induces Aberrant Axonal Sprouting and Ectopic
Terminal Formation in Amyloid Precursor Protein Transgenic Mice
Amie L.
Phinney1,
Thomas
Deller2,
Martina
Stalder1,
Michael E.
Calhoun1,
Michael
Frotscher2,
Bernd
Sommer3,
Matthias
Staufenbiel3, and
Mathias
Jucker1
1 Department of Neuropathology, Institute of Pathology,
University of Basel, CH-4003 Basel, Switzerland,
2 Institute of Anatomy, University of Freiburg,
D-79001Freiburg, Germany, and 3 Central Nervous System
Research, Novartis Pharma, Inc., CH-4002 Basel, Switzerland
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ABSTRACT |
A characteristic feature of Alzheimer's disease (AD) is the
formation of amyloid plaques in the brain. Although this hallmark pathology has been well described, the biological effects of plaques are poorly understood. To study the effect of amyloid plaques on axons
and neuronal connectivity, we have examined the axonal projections from
the entorhinal cortex in aged amyloid precursor protein (APP)
transgenic mice that exhibit cerebral amyloid deposition in plaques and
vessels (APP23 mice). Here we report that entorhinal axons form
dystrophic boutons around amyloid plaques in the entorhinal termination
zone of the hippocampus. More importantly, entorhinal boutons were
found associated with amyloid in ectopic locations within the
hippocampus, the thalamus, white matter tracts, as well as surrounding
vascular amyloid. Many of these ectopic entorhinal boutons were
immunopositive for the growth-associated protein GAP-43 and showed
light and electron microscopic characteristics of axonal terminals. Our
findings suggest that (1) cerebral amyloid deposition has neurotropic
effects and is the main cause of aberrant sprouting in AD brain; (2)
the magnitude and significance of sprouting in AD have been
underestimated; and (3) cerebral amyloid leads to the disruption of
neuronal connectivity which, in turn, may significantly contribute to
AD dementia.
Key words:
Alzheimer's disease; hippocampus; PHAL; tracing; entorhinal cortex; axon; synapse; sprouting; CNS; neurodegeneration; vasculature; APP; mouse; aging
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INTRODUCTION |
The deposition of amyloid throughout
the brain is a characteristic pathology of Alzheimer's disease (AD),
although its pathophysiological significance remains unclear (Selkoe,
1991 ; Yankner, 1996). It has been proposed that neuronal circuits are
compromised in AD (Hyman et al., 1984; Kowall and Kosik, 1987; Geula,
1998), and one hypothesis is that amyloid plaques disrupt neuronal
connectivity in the brain, resulting in loss of function and dementia
(Knowles et al., 1998). This hypothesis is difficult to test because of the presence of other lesions, such as neurofibrillary tangles, along
with the deposition of amyloid in tissue of AD patients. In contrast,
mouse models of cerebral amyloidosis have opened new avenues into the
investigation of the specific role of amyloid deposition in the
pathogenesis of AD (Games et al., 1995; Hsiao et al., 1996;
Sturchler-Pierrat et al., 1997; Hsia et al., 1999).
To investigate the effect of amyloid deposition on axons and neuronal
connectivity, we studied the axonal projections from the entorhinal
cortex to the hippocampus in APP23 mice that overexpress mutated human
amyloid precursor protein (APP) and form amyloid plaques progressively
with age (Sturchler-Pierrat et al., 1997; Calhoun et al., 1998). The
entorhinal cortex and the hippocampus have a well known function in
learning and memory (Wallenstein et al., 1998) and exhibit early and
severe pathology in AD (Hyman et al., 1984; Braak and Braak, 1991). To
visualize the entorhinal axons, the sensitive anterograde tracer
Phaseolus vulgaris leucoagglutinin (PHAL), which allows for
the analysis of projections at the level of single axons (Gerfen and
Sawchenko, 1984), was injected into the entorhinal cortex of aged and
young APP23 mice as well as nontransgenic controls.
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MATERIALS AND METHODS |
Animals. The generation of APP23 transgenic mice is
described in detail elsewhere (Sturchler-Pierrat et al., 1997).
Briefly, a murine Thy-1 promoter element was used to drive
neuron-specific expression of human mutated APP751 (Swedish double
mutation 670/671 KM NL) in B6D2 mice. In this study we used six aged
(18-20 month) hemizygous APP23 mice and two age-matched nontransgenic
controls. In addition, two young (5 month) hemizygous APP23 mice were
used. The mice are from the F8-F10 generation of backcrossing to B6 mice and thus can be considered C57BL/6J-TgN(Thy1-APP670/671
KM NL) mice.
Anterograde tracing. For anterograde tracing of entorhinal
axons, mice received a unilateral injection of PHAL into the entorhinal cortex (Gerfen and Sawchenko, 1984; Deller et al., 1999). In brief, mice were deeply anesthetized using a combination of ketamine (10 mg/kg
body weight; Ketalar; Parke-Davis, Ann Arbor, MI) and xylazine (20 mg/kg; Rompun, Bayer, Germany) in saline. PHAL (2.5% in 10 mM PBS, pH 7.8; Vector Laboratories, Burlingame, CA) was delivered by iontophoretic injection via a stereotactically positioned glass micropipette (tip diameter, 15-30 µm). A 5 µA positive
current was applied for 15 min in a 5 sec on-5 sec off cycle. Two
adjacent injections were administered to the left entorhinal cortex at the following stereotaxic coordinates: (for mice up to 26 gm body weight) anteroposterior (bregma),  4.4; lateral, 2.9; dorsoventral (skull), 4.2; and anteroposterior, 4.6; lateral, 2.9; dorsoventral, 3.9; for mice with body weight >26 gm, coordinates were adjusted to
+0.1 anteroposterior, +0.1 lateral, and +0.1 dorsoventral.
Tissue preparation and immunocytochemistry. All mice were
killed 9 d after PHAL injections by an overdose of
pentobarbital (50 mg/ml Nembutal; Abbott Laboratories, Chicago, IL) and
subsequent transcardial perfusion with 0.01 M PBS
followed by 4% paraformaldehyde (PFA) in PBS. Some mice were perfused
with PFA plus 1% glutaraldehyde and 0.2% picric acid. Brains were
removed and post-fixed in PFA for 24 hr. Brains fixed with
glutaraldehyde were used for combined light microscopic and
ultrastructural analysis and were cut on a vibratome (50 µm section
thickness) and then processed for electron microscopy according to a
previously published protocol (Deller et al., 1996). Brains for light
microscopic analysis only were placed in 30% sucrose for 24 hr, frozen
in 2-methylbutane at 30°C, and sectioned on a freezing-sliding
microtome (25-100 µm section thickness).
Immunocytochemistry was performed on free-floating sections using the
avidin-biotin peroxidase method (ABC Elite kit; Vector Laboratories)
according to previously published protocols (Jucker et al., 1994;
Deller et al., 1996). In brief, sections were incubated in 1%
H2O2 followed by 0.3%
Triton X-100 and blocked in 5% goat or horse serum, all in
Tris-buffered saline (TBS). After an overnight incubation at 4°C with
the primary antibody in 2% serum and 0.3% Triton X-100, sections were
incubated in biotinylated secondary antibody (except for sections
incubated with biotinylated PHAL), followed by the avidin biotin
peroxidase complex solution (ABC Elite kit). The chromogen was
3',3-diaminobenzidine-dihydrochloride (DAB; 0.08%; Sigma, St. Louis,
MO). For double immunolabeling, sections were processed in a sequential
manner. Immediately after visualization of the first primary antibody
by DAB (brown reaction product), sections were incubated for 30 min in
1% H2O2, rinsed extensively in TBS, and immunoreacted as described above with the
second primary antibody. The peroxidase substrate for the second
peroxidase reaction was Vector-SG (blue-gray reaction product; Vector
Laboratories). Additional immunolabeling was performed on
paraffin-embedded sections that were prepared according to standard
protocols (Phinney et al., 1999).
For fluorescence immunocytochemistry, free-floating sections were
labeled using a protocol similar to that described above with the
following changes. TBS containing 0.5% bovine serum albumin was used
for all steps. The secondary antibodies were Cy2-conjugated goat
anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG (diluted 1:100
and 1:500, respectively; Dianova, Hamburg, Germany). For double
immunofluorescence labeling, sections were incubated in both primary
antibodies simultaneously while secondary antibodies were added
sequentially. For all immunocytochemistry, control sections were
processed in the absence of one or both primary antibodies to affirm specificity.
The following antibodies were used: polyclonal biotinylated goat
anti-PHAL (for peroxidase immunocytochemistry; diluted 1:800; Vector
Laboratories); polyclonal rabbit anti-PHAL (for immunofluorescence; 1:800; Vector Laboratories); polyclonal anti-A (NT-11; 1:2000) (Sturchler-Pierrat et al., 1997); polyclonal and monoclonal antibodies to growth associated protein-43 (GAP-43) (1:1000 and 1:10,
respectively; gift of P. Caroni) (Aigner et al., 1995); polyclonal
anti-calretinin (1:10,000; SWant, Bellinzona, Switzerland); polyclonal
anti-synaptophysin (1:2000; Dako, Glostrup, Denmark); monoclonal
anti-MAP2 (AP14; 1:500; gift of L. Binder) (Caceres et al., 1983); and
polyclonal antibody to spinophilin (1:2000; gift of P. Greengard)
(Allen et al., 1997).
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RESULTS |
PHAL tracing revealed a typical entorhinohippocampal projection in
young APP23 mice and nontransgenic controls, and single entorhinal
axons showed a normal morphology (Stanfield et al., 1979). No
immunoreactivity for amyloid- peptide (A) could be detected in
these animals (Fig. 1a,c).
Consistent with previous reports, aged APP23 mice showed a significant
amyloid load in brain (Fig. 1b,d) that is largely
congophilic in nature (Sturchler-Pierrat et al., 1997; Calhoun et al.,
1998). In areas free of amyloid plaques, as well as in areas of
dif fuse amyloid deposition, PHAL-labeled entorhinal fibers in
these mice were indistinguishable from controls. However, fibers in the
vicinity of compact amyloid deposits often appeared swollen and
tortuous and formed large balloon-like structures around the plaques
(Fig. 1d,e). Typically, axons maintained a normal morphology
until in the vicinity of a plaque. At this point, axons lined the
periphery of the plaque and characteristically formed one or more small
swellings followed by one or more larger, terminal swellings. Such
terminal swellings usually formed facing away from the plaque (Fig.
1e).
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Figure 1.
Entorhinal axonal projections in APP23 mice.
a, PHAL-labeled projection from the medial entorhinal
cortex to the middle molecular layer (MML;
arrow) of the dentate gyrus (DG) in an
18-month-old nontransgenic control mouse. Minor projections were
identified to CA3 and CA1 of the hippocampus and to the thalamus
(TH). The same pattern was observed in young
APP23 mice that lack amyloid plaque formation. b,
PHAL-labeled projection in an 18-month-old APP23 mouse. Main
termination pattern in the MML (arrow) is similar to
that seen in the control mouse with the most notable difference being
the hyperinnervation of the thalamus. Note the amyloid deposits
(blue-gray reaction product) throughout the
hippocampus, the thalamus, and within the alveus
(ALV). c, d, High
magnification of the inferior blade of DG from the control mouse shown
in a, and APP23 mouse shown in b. Note
the thickened PHAL-labeled axons (arrow) and ballooned,
spheroidal axon terminals (arrowheads) in the vicinity
of amyloid plaques. In the MML, amyloid plaques were completely
engulfed by entorhinal dystrophic terminals, whereas outside of the
main entorhinal termination zone only a subpopulation of dystrophic
boutons were PHAL-labeled. e, High magnification of
PHAL-labeled axons with the characteristic dystrophic terminals around
an amyloid plaque in the stratum-lacunosum moleculare of CA3. Many
PHAL-labeled axons appear normal until directly adjacent to the amyloid
(arrow), then typically curve around the amyloid
periphery, forming several small swellings followed by a large terminal
balloon-shaped swelling that turns away from the plaque
(arrowhead). GC, granule cell layer of
the dentate gyrus; IML, inner molecular layer;
OML, outer molecular layer. Scale bars:
a, 300 µm; c, e, 25 µm; panels a and b, and
c and d have the same
magnification.
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To further investigate the nature of the PHAL-labeled spheroid
structures surrounding the plaques, correlated light and electron microscopy was performed (Fig. 2). At the
ultrastructural level, PHAL-labeled structures could be
identified as swollen, or dystrophic, presynaptic boutons of varying
sizes that contain large amounts of unstained membranous material and
lysosomal dense bodies (Fig. 2b,d). Only a percentage of the
swollen boutons associated with the plaques were labeled, as would be
expected since the ipsilateral entorhinal neurons provide the major but
not exclusive innervation to this area (Stanfield et al., 1979). Many
of the labeled terminals were observed maintaining a synapse (Fig.
2e). These PHAL-labeled terminals strongly resemble the
plaque-associated dystrophic neurites described in AD (Wisniewski and
Terry, 1973; Masliah et al., 1991a; Peters et al., 1991) and
provide direct evidence that such dystrophic neurites are of axonal
origin. Further substantiating this conclusion, immunohistochemistry
for the dendritic marker microtubule-associated protein-2 (MAP-2)
(Caceres et al., 1983) and the dendritic spine protein spinophilin
(Allen et al., 1997) failed to label amyloid-associated dystrophic
neurites in APP23 mice (data not shown).
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Figure 2.
Ultrastructure of PHAL-labeled dystrophic
entorhinal boutons in the hippocampus. a, Light
micrograph of an amyloid plaque (asterisk) in the
molecular layer of the dentate gyrus surrounded by numerous large
PHAL-labeled entorhinal boutons (arrows).
b, Electron micrograph of the bottom half of the plaque
shown in a. The amyloid (asterisk) is
surrounded by several dystrophic PHAL-labeled entorhinal boutons
(arrows). c, Light micrograph of an
amyloid plaque (asterisk) in the molecular layer of the
dentate gyrus. The arrow points to a heavily
PHAL-labeled entorhinal bouton. d, Electron micrograph
of the PHAL-labeled entorhinal bouton illustrated in c.
The bouton is filled with numerous multilamellar bodies, characteristic
of dystrophic neurites. These structures are surrounded by electron
dense immunoprecipitate. e, PHAL-labeled entorhinal
bouton in the vicinity of a plaque. This dystrophic bouton contains
synaptic vesicles and forms a synapse with a spine. The
arrow points to the synaptic cleft. An unlabeled
dystrophic neurite (DN), as well as a normal axon
terminal (A) are also illustrated. Scale bars:
a, c, 10 µm; b, 2.5 µm; d, 1 µm; e, 0.5 µm.
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One of the most intriguing observations was that entorhinal fibers were
found outside of their normal termination zone in APP23 mice. Such
ectopic terminals were most notable in the strictly laminated dentate
gyrus (DG): entorhinal axons, normally restricted to the outer
molecular layer of the DG (even during regenerative sprouting)
(Frotscher et al., 1997), invade the inner molecular layer, which is
the zone of commissural fibers (Fig.
3a). Similarly, commissural
axons, normally restricted to the inner molecular layer of the DG,
entered the outer molecular layer which is the zone of entorhinal
fibers (Fig. 3b) (Stanfield et al., 1979). Thus, amyloid
deposition causes aberrations of both entorhinal and commissural fibers
and disrupt the normal laminar organization of the DG.
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Figure 3.
Plaque-associated ectopic axon terminals.
a, Disruption of the layer-specific termination of the
PHAL-labeled entorhinal axons in the vicinity of an amyloid plaque
(asterisk) is evidenced by the invasion of these fibers
(brown reaction product) into the inner molecular layer
(IML) of the dentate gyrus, which is labeled for
calretinin (blue-gray reaction product). Calretinin is
specific for the commissural fibers in the mouse and labels
specifically the IML (Liu et al., 1996). b,
Calretinin-labeled commissural fibers (brown) also form
dystrophic terminals (arrow) and deviate from their
normally specific termination in the IML when in vicinity of amyloid
(blue-gray). c, High magnification of
the amyloid plaque (blue-gray) shown in Figure
1b. PHAL-labeled entorhinal axons are in
brown and reveal a hyperinnervation of the thalamic
region around the amyloid plaque (see Fig. 1a for
control). Here, entorhinal axons curve around the amyloid and form
dystrophic boutons directly adjacent to the amyloid. Interestingly,
PHAL-labeled entorhinal axons also formed dystrophic boutons around
vascular amyloid deposits (arrow). GC,
Granule cell layer of the dentate gyrus. Scale bars: a,
10 µm; c, 50 µm; panels a and
b have the same magnification.
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Additional evidence for amyloid-induced axonal aberration of entorhinal
fibers was found in the thalamus. In control mice, very few entorhinal
fibers are normally found in this region (Fig. 1a). In
contrast, in all APP23 mice that had significant amyloid deposition in
the thalamus, a dense plexus of labeled fibers was observed (Figs.
1b, 3c). Entorhinal fibers surrounding the
amyloid plaques formed the characteristic swollen boutons that were
also observed in the hippocampus (Fig. 3c). Thus, the
normally weak entorhinal projection to the thalamus was not only found
to be appreciably increased but also abnormal (dystrophic) whenever amyloid deposition occurred in this region.
Another intriguing finding was the observation of PHAL-labeled fibers
projecting toward and abutting blood vessels containing vascular
amyloid (Fig. 3c). In the thalamus, an area heavily affected by vascular amyloid in APP23 mice (Calhoun et al., 1998; Jucker et al.,
1998), entorhinal fibers frequently formed dystrophic boutons adjacent
to the vascular amyloid deposits. In control mice, entorhinal fibers
are not usually associated with blood vessels, and if they are found in
the vicinity of a blood vessel, for example in the outer molecular
layer of the hippocampus, no dystrophic boutons are formed. This shows
that the deposition of vascular amyloid also leads to ectopic axon
terminals and abnormal axonal structures and is thus contributing to
the disruption of neuronal circuits.
To better understand the process that leads to the entorhinal fiber
disruption and ectopic terminal formation in the vicinity of amyloid
plaques, we studied markers for axonal growth that are believed to play
a role in AD (Geddes et al., 1985, 1986; Masliah et al., 1991b).
For this purpose we focused on compact amyloid plaques in the white
matter, which are frequently found in the alveus of the hippocampus in
aged APP23 mice. Entorhinal fibers travel through the alveus to reach
the hippocampus, but they have no known targets in this typically
asynaptic white matter tract. Yet, in the alveus, PHAL-labeled
entorhinal axons that lie in the vicinity of a plaque form numerous
swollen boutons around it (Fig.
4a). Immunostaining for GAP-43
(Benowitz et al., 1990) and A revealed amyloid-associated
GAP-43-positive boutons similar to the spheroid structures formed by
entorhinal fibers around plaques (Fig. 4b). Furthermore,
similar to that reported in AD (Masliah et al., 1991b), GAP-43
labeled more fibers throughout the neuropil of APP23 mice than in
controls (data not shown). Double-labeling for PHAL and GAP-43 revealed
that indeed many dystrophic entorhinal terminals were
GAP-43-immunopositive (Fig. 4d-f), supporting the
idea that entorhinal fibers found in ectopic locations are the result
of amyloid-associated aberrant sprouting.
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Figure 4.
Axonal growth associated with amyloid plaques in
the white matter. a, PHAL-labeled entorhinal axons form
dystrophic terminals around an amyloid plaque (asterisk)
in the alveus, b, Immunohistochemistry with an antibody
against GAP-43 reveals that throughout the neuropil and interestingly,
as shown here, within the alveus, many dystrophic boutons
(brown) associated with amyloid deposits (blue,
asterisk) were GAP-43 positive. c, Similar
dystrophic neurites were also identified around plaques
(asterisk), shown here within the alveus, when sections
were immunolabeled for the synaptic marker synaptophysin. d-f, Double
fluorescent immunolabeling for PHAL (d,
green) and GAP-43 (e, red) revealed that
a percentage of PHAL-labeled entorhinal terminals are positive for the
growth-associated marker GAP-43 (d-f, arrows). Scale
bars: a, 20 µm; b, 20 µm;
d, 5 µm; panels b and c,
and d-f have the same magnification.
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To assess whether the observed aberrant fibers form terminals,
immunostaining for the synaptic vesicle protein synaptophysin was
performed. Amyloid plaques, even those located in the white matter,
were typically engulfed by synaptophysin-positive terminals (Fig.
4c). Electron microscopy of white matter plaques confirmed that dystrophic entorhinal terminals form around amyloid deposits (Fig.
5a,b). These dystrophic
terminals are identical to those observed in the hippocampus, and some
of them show elements of presynaptic specialization, such as clustered
synaptic vesicles (Fig. 5c,d). These data suggest that
cerebral amyloid deposition can induce aberrant axonal growth of
entorhinal fibers, and also that it induces synaptic differentiation of
these axons in ectopic locations.
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Figure 5.
Ultrastructure of entorhinal dystrophic boutons in
the white matter. a, Light micrograph of an amyloid
plaque (asterisk) in the alveus of the hippocampus.
Three PHAL-labeled entorhinal boutons surround the plaque
(arrows). b, Electron micrograph of the
plaque illustrated in a. The amyloid core
(asterisk) is surrounded by numerous dystrophic
neurites. The three large entorhinal boutons illustrated in
a are indicated by arrows. One of the
unlabeled dystrophic neurites is myelinated (short
arrow). c, Serial section of the middle
PHAL-labeled bouton illustrated in b. The heavily
immunolabeled bouton is filled with multilamellar bodies.
d, Serial section of the rectangle
illustrated in c. This part of the dystrophic bouton
shows elements of synaptic specialization, i.e., clustered vesicles.
Scale bars: a, 10 µm; b, 5 µm;
c, 1 µm; d, 0.25 µm.
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DISCUSSION |
Although the fact that sprouting occurs in AD brain has been
recognized earlier (Geddes et al., 1985, 1986; Masliah et al., 1991b), the cause, magnitude, and aberrant nature of the
sprouting has remained unclear. In the present study, we examined the
effect of amyloid deposition on a well defined axonal projection in APP transgenic mice. Our results revealed that (1) aged APP23 mice show
axonal sprouting directly related to amyloid, demonstrating that
amyloid deposition and sprouting are intimately linked; (2) axons grow
into ectopic locations, and moreover sprout within the white matter and
toward vascular amyloid, indicating that amyloid deposition exerts a
neurotropic effect on axons not only in vitro (Koo et al.,
1993) but also in vivo; and (3) young mice that overexpress
APP do not exhibit plaques, dystrophic boutons, or axonal sprouting,
demonstrating that APP overexpression alone does not underlie the
aberrant sprouting process. Our data therefore suggest that the primary
factor underlying abnormal axonal sprouting is the deposition of amyloid.
The well defined and specific laminar organization of the
entorhinohippocampal projection, in conjunction with the amyloid deposition in this transgenic model, allowed us to show that not only
is amyloid central to the observed sprouting, but that such sprouting
is indeed aberrant. It may be argued that the observed sprouting is
induced by neurodegeneration or glia activation previously observed in
these mice (Calhoun et al., 1998; Stalder et al., 1999). However, if
such factors govern the observed sprouting, the sprouting of entorhinal
axon should respect the laminar borders of the DG as they do in
experimental lesion paradigms (Frotscher et al., 1997). It is the
unprecedented nature of the observed sprouting of entorhinal axons into
the inner molecular layer of the DG, around blood vessels and within
white matter, that strongly supports a primary role for cerebral
amyloidosis in the observed axonal sprouting. However, it is possible
that other constituents of amyloid deposits, such as perlecan, agrin,
and laminin (Snow et al., 1988; Perlmutter et al., 1991; Donahue et
al., 1999) also play an important role in the observed sprouting
response. It must also be considered that amyloid deposition is a
consequence rather then cause of the axonal sprouting in mice that
overexpress APP. Yet, the absence of aberrant axons or ectopic
terminals in young APP23 mice does not support such a conclusion. The
possibility that sprouting/dystrophic terminals release APP/A and
thus initiate/accelerate amyloid deposition in a feedforward manner
remains open.
The present study has focused on the entorhinohippocampal system, one
of many axonal projections in the brain. The vastness of the sprouting
and ectopic terminal formation observed in the present study was
unexpected. The labeling of amyloid-associated dystrophic terminals
throughout the brain using synaptophysin and GAP-43 immunostaining
indicates that axon terminal disruption and sprouting is not restricted
to the entorhinal-hippocampal projection and occur in all areas that
develop amyloidosis. Thus, our results suggest that both the magnitude
and significance of alterations to neuronal circuits in AD brain caused
by such dystrophic and ectopic axons have been greatly underestimated.
Finally, it should be noted that all PHAL-labeled entorhinal terminals,
even those identified as sprouting axon terminals, had dystrophic
morphologies. This suggests that axon growth in response to the
deposition of amyloid and the formation of abnormal presynaptic
terminals, ectopic or otherwise, are not likely to result in functional
or restorative neuronal connections. These morphological abnormalities
are likely the anatomical correlate of changes in electrophysiological
characteristics reported in APP transgenic mouse models with cerebral
amyloidosis (Chapman et al., 1999; Hsia et al., 1999).
In conclusion, our results demonstrate that the deposition of cerebral
amyloid induces dystrophic boutons, aberrant axonal growth, disrupted
fiber tracts, as well as the formation of ectopic terminals. Such
changes disrupt normal neuronal connectivity and are a likely candidate
for the morphological correlate of cognitive decline in AD.
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FOOTNOTES |
Received June 3, 1999; revised July 14, 1999; accepted July 14, 1999.
This study was supported by grants to M.J. from the Swiss National
Foundation and the VerUm Foundation (Munich, Germany) and by grants to
T.D. and M.F. from the Deutsche Forschungsgemeinschaft (SFB 505). We
thank A. Schneider and M. Winter (Institute of Anatomy, Freiburg,
Germany), E. Billy (FMI, Basel, Switzerland), C. Sturchler-Pierrat, D. Abramowski, and K.-H. Wiederhold (Novartis,
Basel, Switzerland) for excellent technical support, J. Geddes
(University of Kentucky, Lexington, KY), L. Walker (Parke-Davis, Ann
Arbor, MI), M. Tolnay, and A. Probst (Institute of Pathology, Basel,
Switzerland) for helpful comments on this manuscript, and P. Caroni
(FMI, Basel, Switzerland), L. Binder (Northwestern University Medical
School, Chicago, IL), and P. Greengard (Rockefeller University, New
York, NY) for antibody gifts.
A. L. Phinney and T. Deller contributed equally to this work.
Correspondence should be addressed to Dr. M. Jucker, Department of
Neuropathology, Institute of Pathology University of Basel, Schönbeinstrasse 40, CH-4003 Basel, Switzerland.
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REFERENCES |
-
Aigner L,
Arber S,
Kapfhammer JP,
Laux T,
Schneider C,
Botteri F,
Brenner HR,
Caroni P
(1995)
Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.
Cell
83:269-278[Web of Science][Medline].
-
Allen PB,
Ouimet CC,
Greengard P
(1997)
Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines.
Proc Natl Acad Sci USA
94:9956-9961[Abstract/Free Full Text].
-
Benowitz LI,
Rodriguez WR,
Neve RL
(1990)
The pattern of GAP-43 immunostaining changes in the rat hippocampal formation during reactive synaptogenesis.
Brain Res Mol Brain Res
8:17-23[Medline].
-
Braak H,
Braak E
(1991)
Neuropathological staging of Alzheimer-related changes.
Acta Neuropathol (Berl)
82:239-259[Medline].
-
Caceres A,
Payne MR,
Binder LI,
Steward O
(1983)
Immunocytochemical localization of actin and microtubule-associated protein MAP2 in dendritic spines.
Proc Natl Acad Sci USA
80:1738-1742[Abstract/Free Full Text].
-
Calhoun ME,
Wiederhold KH,
Abramowski D,
Phinney AL,
Probst A,
Sturchler-Pierrat C,
Staufenbiel M,
Sommer B,
Jucker M
(1998)
Neuron loss in APP transgenic mice.
Nature
395:755-756[Medline].
-
Chapman PF,
White GL,
Jones MW,
Cooper-Blacketer D,
Marshall VJ,
Irizarry M,
Younkin L,
Good MA,
Bliss TV,
Hyman BT,
Younkin SG,
Hsiao KK
(1999)
Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice.
Nat Neurosci
2:271-276.[Web of Science][Medline]
-
Deller T,
Martinez A,
Nitsch R,
Frotscher M
(1996)
A novel entorhinal projection to the rat dentate gyrus: direct innervation of proximal dendrites and cell bodies of granule cells and GABAergic neurons.
J Neurosci
16:3322-3333[Abstract/Free Full Text].
-
Deller T,
Drakew A,
Frotscher M
(1999)
Different primary target cells are important for fiber lamination in the fascia dentata: a lesson from reeler mutant mice.
Exp Neurol
156:239-253[Web of Science][Medline].
-
Donahue JE,
Berzin TM,
Rafii MS,
Glass DJ,
Yancopoulos GD,
Fallon JR,
Stopa EG
(1999)
Agrin in Alzheimer's disease: altered solubility and abnormal distribution within microvasculature and brain parenchyma.
Proc Natl Acad Sci USA
96:6468-6472[Abstract/Free Full Text].
-
Frotscher M,
Heimrich B,
Deller T
(1997)
Sprouting in the hippocampus is layer-specific.
Trends Neurosci
20:218-223[Web of Science][Medline].
-
Games D,
Adams D,
Alessandrini R,
Barbour R,
Berthelette P,
Blackwell C,
Carr T,
Clemens J,
Donaldson T,
Gillespie F,
Hagopian S,
Johnson-Wood K,
Khan K,
Lee M,
Liebowitz P,
Lieberburg I,
Little S,
Masliah E,
McConglogue L,
Montoya-Zavala M,
Mucke L,
Paganini L,
Penniman E,
Power M,
Schenk D,
Seubert P,
Snyder B,
Sorlano F,
Tan H,
Vitale J,
Wadsworth S,
Wolozin B,
Zhao J
(1995)
Alzheimer-type neuropathology in transgenic mice overexpressing V717F -amyloid precursor protein.
Nature
373:523-527[Medline].
-
Geddes JW,
Monaghan DT,
Cotman CW,
Lott IT,
Kim RC,
Chui HC
(1985)
Plasticity of hippocampal circuitry in Alzheimer's disease.
Science
230:1179-1181[Abstract/Free Full Text].
-
Geddes JW,
Anderson KJ,
Cotman CW
(1986)
Senile plaques as aberrant sprout-stimulating structures.
Exp Neurol
94:767-776[Web of Science][Medline].
-
Gerfen CR,
Sawchenko PE
(1984)
An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L).
Brain Res
290:219-238[Web of Science][Medline].
-
Geula C
(1998)
Abnormalities of neural circuitry in Alzheimer's disease: hippocampus and cortical cholinergic innervation.
Neurology
51:S18-29[Abstract/Free Full Text].
-
Hsia AY,
Masliah E,
McConlogue L,
Yu GQ,
Tatsuno G,
Hu K,
Kholodenko D,
Malenka RC,
Nicoll RA,
Mucke L
(1999)
Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models.
Proc Natl Acad Sci USA
96:3228-3233[Abstract/Free Full Text].
-
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].
-
Hyman BT,
Van Horsen GW,
Damasio AR,
Barnes CL
(1984)
Alzheimer's disease: cell-specific pathology isolates the hippocampal formation.
Science
225:1168-1170[Abstract/Free Full Text].
-
Jucker M,
Walker LC,
Schwarb P,
Hengemihle J,
Kuo H,
Snow AD,
Bamert F,
Ingram DK
(1994)
Age-related deposition of glia-associated fibrillar material in brains of C57BL/6 mice.
Neuroscience
60:875-889[Web of Science][Medline].
-
Jucker M,
Stalder M,
Tolney M,
Wiederhold KH,
Abramowski D,
Sommer B,
Staufenbiel M,
Calhoun ME
(1998)
Cerebral amyloid angiopathy in APP transgenic mice with amyloid plaque formation.
Soc Neurosci Abstr
24:1502.
-
Knowles RB,
Gomez-Isla T,
Hyman BT
(1998)
Abeta associated neuropil changes: correlation with neuronal loss and dementia.
J Neuropathol Exp Neurol
57:1122-1130[Web of Science][Medline].
-
Koo EH,
Park L,
Selkoe DJ
(1993)
Amyloid -protein as a substrate interacts with extracellular matrix to promote neurite outgrowth.
Proc Natl Acad Sci USA
90:4748-4752[Abstract/Free Full Text].
-
Kowall NW,
Kosik KS
(1987)
Axonal disruption and aberrant localization of tau protein characterize the neuropil pathology of Alzheimer's disease.
Ann Neurol
22:639-643[Web of Science][Medline].
-
Liu Y,
Fujise N,
Kosaka T
(1996)
Distribution of calretinin immunoreactivity in the mouse dentate gyrus. I. General description.
Exp Brain Res
108:389-403[Web of Science][Medline].
-
Masliah E,
Hansen L,
Albright T,
Mallory M,
Terry RD
(1991a)
Immunoelectron microscopic study of synaptic pathology in Alzheimer's disease.
Acta Neuropathol (Berl)
81:428-433[Medline].
-
Masliah E,
Mallory M,
Hansen L,
Alford M,
Albright T,
DeTeresa R,
Terry R,
Baudier J,
Saitoh T
(1991b)
Patterns of aberrant sprouting in Alzheimer's disease.
Neuron
6:729-739[Web of Science][Medline].
-
Perlmutter LS,
Barron E,
Saperia D,
Chui HC
(1991)
Association between vascular basement membrane components and the lesions of Alzheimer's disease.
J Neurosci Res
30:673-681[Medline].
-
Peters A,
Palay S,
Webster H
(1991)
In: The fine structure of the nervous system. New York: Oxford UP.
-
Phinney AL,
Calhoun ME,
Wolfer DP,
Lipp HP,
Zheng H,
Jucker M
(1999)
No hippocampal neuron or synaptic bouton loss in learning-impaired aged -amyloid precursor protein-null mice.
Neuroscience
90:1207-1216[Web of Science][Medline].
-
Selkoe DJ
(1991)
The molecular pathology of Alzheimer's disease.
Neuron
6:487-498[Web of Science][Medline].
-
Snow AD,
Mar H,
Nochlin D,
Kimata K,
Kato M,
Suzuki S,
Hassell J,
Wight TN
(1988)
The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer's disease.
Am J Pathol
133:456-463[Abstract].
-
Stalder M,
Phinney AL,
Probst A,
Sommer B,
Staufenbiel M,
Jucker M
(1999)
Association of microglia with amyloid plaques in brains of APP23 transgenic mice.
Am J Pathol
154:1673-1684[Abstract/Free Full Text].
-
Stanfield BB,
Caviness Jr VS,
Cowan WM
(1979)
The organization of certain afferents to the hippocampus and dentate gyrus in normal and reeler mice.
J Comp Neurol
185:461-483[Web of Science][Medline].
-
Sturchler-Pierrat C,
Abramowski D,
Duke M,
Wiederhold KH,
Mistl C,
Rothacher S,
Ledermann B,
Burki K,
Frey P,
Paganetti PA,
Waridel C,
Calhoun ME,
Jucker M,
Probst A,
Staufenbiel M,
Sommer B
(1997)
Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.
Proc Natl Acad Sci USA
94:13287-13292[Abstract/Free Full Text].
-
Wallenstein GV,
Eichenbaum H,
Hasselmo ME
(1998)
The hippocampus as an associator of discontiguous events.
Trends Neurosci
21:317-323[Web of Science][Medline].
-
Wisniewski H,
Terry D
(1973)
Reexamination of the pathogenesis of the senile plaque.
In: Progress in neuropathology (Zimmerman H,
ed), pp 1-26. New York: Grune and Stratton.
-
Yankner BA
(1996)
Mechanisms of neuronal degeneration in Alzheimer's disease.
Neuron
16:921-932[Web of Science][Medline].
Copyright © 1999 Society for Neuroscience 0270-6474/99/19198552-08$05.00/0
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[Full Text]
[PDF]
|
|
|
|
|
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[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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1619 - 1627.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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8717 - 8726.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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ABSTRACT
INTRODUCTION
