with elevated levels of brain amyloid. This led to the suggestion that the pathological effects of apoE4 are mediated by cross-talk
the A?-mediated pathological effects of apoE4 are unknown. We have shown recently that inhibition of the A?-degrading enzyme
mice. We presently used the neprilysin inhibition paradigm to analyze the neuropathological and cognitive effects that are induced by
CA1 neurons and of entorhinal and septal neurons, which is accompanied by the accumulation of intracellular A? and apoE and with
lysosomal activation. Furthermore, these neuropathological effects are associated isoform specifically with the occurrence of pro-
nounced cognitive deficits in the ApoE4 mice. These findings provide the first in vivo evidence regarding the cellular mechanisms
underlying the pathological cross talk between apoE4 and A?, as well as a novel model system of neurodegeneration in vivo that is
Alzheimer’s disease (AD) is associated with several molecular
hallmarks and genetic risk factors. These include amyloid ? pep-
tide (A?) and hyperphosphorylated tau, which are the major
constituents of senile plaques and neurofibrilary tangles, respec-
ters and Beyreuther, 2006), and the allele E4 of apolipoprotein E
(apoE4), which is the most prevalent genetic risk factor for AD
(Corder et al., 1993; Saunders et al., 1993; Roses, 2006). His-
osition, hippocampal atrophy, and neuronal loss, as well as with
enhanced brain inflammation and impaired neuronal plasticity
(Schmechel et al., 1993; Arendt et al., 1997; Mori et al., 2002).
The finding that brain amyloid levels are specifically elevated
in apoE4-positive AD patients led to the suggestion that the
pathological effects of apoE4 are mediated by cross-talk interac-
tions with A?. Accordingly, in vitro studies revealed that apoE4
dan et al., 1998). In addition, amyloid ? protein precursor
(APP) ? apoE double transgenic mice that express apoE4 have
higher levels of A? deposits than the corresponding apoE3 ?
APP mice (Brendza et al., 2002; DeMattos, 2004; Holtzman,
The amyloid cascade is initiated by production of brain A?.
This is followed by the formation of distinct A? oligomers that
induce neuronal impairments and subsequent cell death (Hardy
that are affected by apoE4 and the cellular and molecular mech-
anisms that underlie the resulting pathological effects are not
known. Because the increased A? deposition in apoE4 ? APP
transgenic mice is apparent only in old mice (Holtzman et al.,
neuritic degeneration and neuronal loss (Holtzman et al., 2000;
duce the pathological interactions between apoE4 and A? in
infusion of thiorphan, which inhibits the A?-degrading enzyme
4690 • TheJournalofNeuroscience,April30,2008 • 28(18):4690–4701
tion, and that similar treatments in apoE3 and apoE4 transgenic
mice result in marked and isoform-specific enhancement of the
nucleation and aggregation of A? in apoE4 mice (Dolev and
within 1 week after the initiation of treatment.
In the present study, we used the neprilysin inhibition para-
digm to analyze the brain neuropathological processes and cog-
after activation of the amyloid cascade. Because apoE4 exacer-
bates the endosomal and lysosomal dysfunctions that occur in
ical effects of apoE4 correlate spatially and temporally with the
accumulation of intraneuronal A? and with the induction of
Transgenic mice. APOE target replacement mice were created by gene
targeting, as described previously (Sullivan et al., 1997). Construction of
these mice differs from other apoE transgenic mice in that the human
APOE gene was used to replace the mouse apoE gene. The two lines of
apoE target replacement mice contain chimeric genes consisting of
ing), the 5? half of mouse intron 1 continuous with the 3? half of human
intron 1, followed by human exons (and introns) 2–4 (Sullivan et al.,
Mice were back-crossed to C57BL/6J for eight generations and were ho-
et al., 2003). All experiments with mice were performed on age-matched
Tel-Aviv University Animal Care Committee, and every effort was made
to reduce animal stress and to minimize animal usage.
Implantation of Alzet miniosmotic pumps. Alzet (Palo Alto, CA) min-
iosmotic pumps (model 2004), which deliver their contents at a rate of
ylene catheter to a stainless steel cannula (Brain Infusion kit; Alzet) and
loaded either with 0.5 mM thiorphan (Sigma, St. Louis, MO) in artificial
CSF containing 1 mM ascorbic acid or with a similar solution without
thiorphan. Mice were anesthetized by intraperitoneal injection of ket-
amine (120 mg/kg), their skulls were carefully exposed, and a small hole
was drilled with a 25 gauge needle above the lateral ventricle (1 mm
taneously on the mouse’s back, and the cut skin over the skull was su-
tured. An antibiotic (1% oxytetracycline) was added to the drinking
water for 10 d.
Immunohistochemistry and immunofluorescence staining. Mice were
in 0.1 M phosphate buffer, pH 7.4, and then placed in 30% sucrose for
48 h. Frozen coronal sections (30 ?m) were then cut on a sliding mic-
rotome and collected serially. The free-floating sections were immuno-
ing primary antibodies (Abs): biotinylated anti-A? monoclonal Ab
(mAb) 4G8 (1:200; Signet, Dedham, MA); rabbit anti-A?42 (1:500;
Chemicon, Temecula, CA); rabbit anti-A?40 (1:500; Chemicon); goat
anti-apoE (1:5000; Calbiochem, La Jolla, CA); biotinylated mouse anti-
microtubule-associated protein-2 (MAP-2) (1:1000; Chemicon); rabbit
anti-synaptophysin (1:200, SYP H-93; Santa Cruz Biotechnology, Santa
Cruz, CA); and rabbit anti-cathepsin D (1:500; Calbiochem). Accord-
ingly, sections were washed with 10 mM PBS, pH 7.4, after which the
primary antibody, diluted in PBS with 0.1% Triton X-100 (PBST) and
with 2% of the appropriate serum, was applied overnight at 4°C. After
having been rinsed in PBST, sections were incubated for 1 h at room
temperature in the secondary antibody (Vector Laboratories, Burlin-
game, CA) and then diluted 1:200 in PBST that contained 2% of the
appropriate serum (this step was omitted when biotinylated first Abs
were used). After several additional rinses in PBST, sections were incu-
bated for 0.5 h in avidin-biotin-horseradish peroxidase complex (ABC-
Elite; Vector Laboratories) in PBST. After rinses in PBST, sections were
placed for up to 10 min in diaminobenzidine chromagen solution (Vec-
tor Laboratories). The reaction was monitored visually and stopped by
that they were preincubated before adding the first antibody with 70%
formic acid for 7 min. To minimize variability, sections from all animals
were stained simultaneously. For hematoxylin staining (Mayer’s Hema-
toxylin, #S3309; Dako Cytomation, Carpinteria, CA), free-floating sec-
tions were first mounted on slides and then stained according to the
Immunofluorescence staining. Coexpression of A? with MAP-2/apoE/
cathepsin D and synaptophysin was evaluated by double and single im-
blocked by incubation with 0.1% Triton X-100 and 10% normal donkey
serum in PBS for 1 h at room temperature. The primary antibodies were
then dissolved in 0.1% Triton X-100 and 2% normal donkey serum in
PBS, and finally incubated with the sections for 48 h at 4°C. Next, the
bound primary antibodies were visualized by incubating the sections for
1 h at room temperature with Alexa-fluor 488/633-conjugated donkey
anti-rabbit (1:1000; Invitrogen, Eugene, OR), Alexa-fluor 546-
conjugated donkey anti-goat (1:1000; Invitrogen), or streptavidin-
Alexa-fluor 488/633 (1:1000; Invitrogen), depending on the appropriate
slides, and fluorescence was visualized using a confocal scanning laser
microscope (LSM 510; Zeiss, Oberkochen, Germany). Images (1024 ?
1024 pixels) were obtained by a 63? water-immersion lens averaging
eight scans per slice. All images were processed using Adobe Photoshop
7.0 (Adobe Systems, San Jose, CA).
Image analysis. The peroxidase-immunostained sections were viewed
and photographed with a 40? objective and a Nikon DS-5M camera
staining as the percentage area stained were determined with the aid of
the Image-Pro Plus system (version 5.1; Media Cybernetics, Silver
brightness, the images were not manipulated.
The image analysis was based on our previous studies indicating that
(Dolev and Michaelson, 2004). Accordingly, the kinetic study, in which
the levels of cellular A? and the neuronal pathology (NeuN) were as-
mm bregma, which were evenly spaced at 480 ?m and of which CA1
domains were traced using a mouse brain atlas (Franklin and Paxinos,
brain area specificity of the immunostaining was assessed using sections
from mice that were treated with thiorphan for 2 weeks. Accordingly,
and visual cortex all at bregma ?2.5 and from the medial septum and
motor cortex at bregma ?0.6, and their intensities of staining were then
analyzed as described above. In addition, volumetric estimation of the
levels of A? and NeuN staining of the entire hippocampal CA1 subfield
was performed 2 weeks after the thiorphan treatment. Accordingly, the
stained area of the CA1 subfield of each of the sections was calculated
the next section. Summation of all the volumes of the positively stained
slabs resulted in a total volumetric estimation of the entire stained CA1
subfield. This was calculated as (total CA1 area [?m2] ? percentage of
the CA1 area that was stained ? 480 ?m). Analysis of CA1 volume
revealed no changes between mouse groups and no side effects of
al. (2006). For determining apoE, samples were blotted and immunore-
acted with goat anti-apoE (1:5000; Calbiochem). For A? analysis, sam-
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