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Journal of Alzheimer’s Disease 53 (2016) 1443–1458
DOI 10.3233/JAD-160182
IOS Press
1443
Reversal of ApoE4-Driven Brain Pathology
by Vascular Endothelial Growth Factor
Treatment
Shiran Salomon-Zimria, Micaela Johanna Glatb, Yael Barhumb, Ishai Luza, Anat Boehm-Cagana,
Ori Liraza, Tali Ben-Zurb, Daniel Offenband Daniel M. Michaelsona,∗
aDepartment of Neurobiology, The George S. Wise Faculty of Life Sciences, The Sagol School of Neuroscience,
Tel Aviv University, Tel Aviv, Israel
bSackler School of Medicine, Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
Handling Associate Editor: Debomoy Lahiri
Accepted 10 May 2016
Abstract. Apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for Alzheimer’s disease (AD), is associated with
increased neurodegeneration and vascular impairments. Vascular endothelial growth factor (VEGF), originally described as
a key angiogenic factor, has recently been shown to play a crucial role in the nervous system. The objective of this research
is to examine the role of VEGF in mediating the apoE4-driven pathologies. We show that hippocampal VEGF levels are
lower in apoE4 targeted replacement mice compared to the corresponding apoE3 mice. This effect was accompanied by a
specific decrease in both VEGF receptor-2 and HIF1-␣. We next set to examine whether upregulation of VEGF can reverse
apoE4-driven pathologies, namely the accumulation of hyperphosphorylated tau (AT8) and A42, and reduced levels of the
pre-synaptic marker, VGluT1, and of the ApoE receptor, ApoER2. This was first performed utilizing intra-hippocampal
injection of VEGF-expressing-lentivirus (LV-VEGF). This revealed that LV-VEGF treatment reversed the apoE4-driven
cognitive deficits and synaptic pathologies. The levels of A42 and AT8, however, were increased in apoE3 mice, masking any
potential effects of this treatment on the apoE4 mice. Follow-up experiments utilizing VEGF-expressing adeno-associated-
virus (AAV-VEGF), which expresses VEGF specifically under the GFAP astrocytic promoter, prevented this effects on
apoE3 mice, and reversed the apoE4-related increase in A42 and AT8. Taken together, these results suggest that apoE4-
driven pathologies are mediated by a VEGF-dependent pathway, resulting in cognitive impairments and brain pathology.
These animal model findings suggest that the VEGF system is a promising target for the treatment of apoE4 carriers in AD.
Keywords: Alzheimer’s disease, apolipoprotein E4, behavior, hippocampus, lentivirus,Morris water maze, object recognition,
targeted replacement mice, vascular endothelial growth factor
INTRODUCTION
Alzheimer’s disease (AD), the most common form
of dementia in the elderly, is characterized by cog-
nitive decline and by the occurrence of brain senile
∗Correspondence to: D.M. Michaelson, Department of Neuro-
biology, The George S. Wise Faculty of Life Sciences, Tel Aviv
University, Ramat Aviv 6997801, Tel Aviv, Israel. Tel.: +972 3
6409624; Fax: +972 6406356; E-mail: dmichael@post.tau.ac.il.
plaques and neurofibrillary tangles, as well as synapse
and neuronal loss in the brain [1–3]. Genetic stud-
ies of familial AD revealed that mutations in APP,
PSEN1, and PSEN2 results in elevated levels of A
[4, 5]. Further sporadic AD genetic studies revealed
allelic segregation of the apolipoprotein E (apoE)
gene to families and individuals with a higher risk
of late onset AD and to sporadic AD [6–8]. There
are three major alleles of apoE, termed E2, E3, and
ISSN 1387-2877/16/$35.00 © 2016 – IOS Press and the authors. All rights reserved
1444 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
E4, of which apoE4 is a risk factor for AD. ApoE4 is
the most prevalent genetic risk factor for AD; about
half of AD patients express apoE4, which increases
the risk for AD by lowering the age of onset of
the disease by 7 to 9 years per allele copy. Interest-
ingly the prevalence of apoE4 is somewhat higher
in North America and northern Europe (∼60%) and
lower in Asian and Mediterranean countries (∼40%)
[7, 9, 10] and is also higher in females than in males
[11]. ApoE4 is associated with distinct brain patholo-
gies. These include impaired neurite outgrowth and
synaptogenesis [12], impairments in plastic neuronal
remodeling [13], and increased neurodegeneration
[14]. In neuropsychological tests, apoE4 has been
associated with cognitive decline in subjects with
cognitive impairment, but not in cognitively normal
individuals [15]. ApoE4 carriers were also found
to have more pronounced vascular pathologies than
non-apoE4 carriers [16]. Vascular endothelial growth
factor-A (referred to here as VEGF) is the founding
member of a family of homodimeric glycoproteins.
Emerging evidence suggests that VEGF plays impor-
tant roles in neurogenesis and neuroprotection and
that it affects neuronal and synaptic plasticity and
potentiation [17, 18]. Specifically, VEGF was found
to stimulate axonal outgrowth and improve the sur-
vival of cultured superior cervical and dorsal root
ganglion neurons, thus enhancing the survival of
mesencephalic neurons, and protecting mouse hip-
pocampal neurons from death induced by serum
withdrawal [19]. In addition, it was found to reduce
the hypoxic death of cerebral cortical neurons, and
to protect cultured hippocampal and cortical neurons
from apoptosis [20]. VEGF is also a key angiogenic
factor that regulates neovascularization in different
tissues. It is a heparin-binding growth factor spe-
cific for vascular endothelial cells, and is able to
induce angiogenesis in vivo [21]. VEGF is regulated
by hypoxia-inducible factor 1␣(HIF-1 ␣) which is
involved in cell survival [22] and induced angiogen-
esis [23]. Although significant interactions between
VEGF and the APOE4 allele in both AD and MCI
patients have been reported [24, 25], the specific
mechanism underlying this crosstalk and its role in
mediating the effects of apoE4 are still unknown and
remain to be determined.
We have previously shown that young (4-month-
old) apoE4-targeted replacement (TR) mice exhibit
reduced levels of the presynaptic glutamatergic vesic-
ular transporter VGlut1 in hippocampal neurons [26],
elevated levels of the neurogenesis marker dou-
blecortin (DCX) [27], and reduced levels of apoE
receptor 2 (apoER2) [28] as well as the accumu-
lation of A42 and of hyperphosphorylated tau in
hippocampal neurons [26]. These biochemical find-
ings were accompanied by cognitive impairment, as
shown by numerous hippocampal-related behavioral
tests (e.g., novel object recognition test, the Morris
water maze, and the fear conditioning test) [29].
In view of the major role of VEGF in both the neu-
ronal and vascular systems and its association with
AD pathology, we presently investigated the extent
to which apoE4 affects the levels and expression
of VEGF and VEGF-related molecules in targeted
replacement mice which express apoE4 and the
possible role of VEGF in mediating the patholog-
ical effects of apoE4. This revealed that the levels
of VEGF and of VEGF-receptor 2 (VEGFR2) and
HIF1␣in the hippocampus were lower in the apoE4
TR mice than in corresponding mice, which express
the AD benign isoform, apoE3. Further experiments
revealed that the apoE4-related downregulation of the
VEGF system can be reversed by intra-hippocampal
injection of VEGF expressing viruses and that impor-
tantly this treatment results in reversal of the apoE4
driven brain pathology and cognitive impairments.
MATERIALS AND METHODS
Mice
ApoE-TR mice, in which the endogenous mouse
apoE was replaced by either human apoE3 or apoE4,
were created by gene targeting [30], and were pur-
chased from Taconic (Germantown, NY). Mice were
back-crossed to wild-type C57BL/6J mice (Harlan
2BL/610) for ten generations and were homozygous
for the apoE3 (3/3) or apoE4 (4/4) alleles. These
mice are referred to here as apoE3 and apoE4 mice,
respectively. The apoE genotype of the mice was con-
firmed by PCR analysis, as described previously [31,
32]. Wild-type C57BL/6J male mice were used for
comparison of VEGF levels in human-apoE apoE3
and mouse-apoE. All experiments were performed
on age-matched male animals (4 months of age), and
were approved by the Tel Aviv University animal
care committee. Every effort was made to reduce ani-
mal stress and to minimize animal usage. Following
treatment, the mice were anesthetized with ketamine
and xylazine and were perfused transcardially with
phosphate buffer saline (PBS). Their brains were
then removed and halved, and each hemisphere was
further processed for either biochemical or histolog-
ical analysis, as outlined below. Results presented
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1445
correspond to five cohorts. The first two cohorts were
used in the histological and biochemical studies of the
effects of the apoE genotype on VEGF family in na¨
ıve
non-treated apoE4 and apoE3 mice. The next two
cohorts were used respectively for the histological
and behavioral assessment of the effects of the VEGF
expressing lentivirus (LV-VEGF), whereas the mice
of the last cohort were subjected to histological anal-
ysis following treatment with the VEGF expressing
adeno associated virus (AAV-VEGF). All experi-
ments were performed with age-matched young male
TR mice. The virus experiments (cohorts 3–5) each
contained 6 groups: 2 genotypes (apoE3 or apoE4) x
3 treatments (na¨
ıve mice and mice treated either with
a sham GFP labeled virus or with the VEGF express-
ing virus). The groups will be address here as na¨
ıve
and LV-GFP or AAV-GFP for the control groups and
LV-VEGF or AAV-VEGF for the treated groups. Each
group consisted of 8–12 mice.
Lentivirus preparation
The human VEGF gene (mouse is one amino
acid shorter [33]) was amplified from a pBlue-
script plasmid purchased from Harvard Institute
of Proteomics, Boston, MA, USA, and cloned
into pLenti6/R4R2/V5-DEST (Invitrogen) using the
ViralPower Promoterless Lentiviral Gateway Kit
(Invitrogen). For the lentiviral production, the VEGF
vector or a pLL3.7-CMV-EGFP control plasmid
were co-transfected with the packaging plasmids
pLP1, pLP2, and pLP/VSVG into the 293T producer
cell line using Lipofectamine 2000 (Invitrogen).
The supernatant was collected 48 and 72 h post
transfection and was subsequently deposited using
ultracentrifugation at 25,000 RPM for 2 h. The
virus-containing pellet was aspirated using HBSS,
aliquoted, and stored at –80◦C until use. Lentiviral
titer was determined using the Lenti-X p24 Rapid
Titer Kit, following the manufacturer’s recommended
procedure (Clontech Laboratories). The titer was esti-
mated to be 108.
Adeno-associated virus preparation
VEGF and GFP coding sequences were cloned
into the AAV2-GFAP-WPRE backbone, kindly pro-
vided by the Diesseroth lab. Concentrated AAV2/1
mosaic particles were produced by the Tel Aviv Uni-
versity’s vector core facility. Briefly, HEK293 cells
were transfected with the AAV2-GFAP-GFP/VEGF-
WPRE vector plasmid along with AdenoHelper,
Rep-Cap1 and Rep-Cap2 plasmids. Seventy-two
hours post transfection the cells were harvested, lysed
using three freeze-thaw cycles and incubated with
Benzonase for 90 min. The lysate was purified using
a heparin-agarose binding column and subsequently
concentrated and desalinated using an Amicon filter.
Intracerebral administration of viral vectors
Four-month-old apoE3 and apoE4-TR mice were
anesthetized with a mixture of ketamine-xylazine
and placed in a stereotactic apparatus (model 940;
David Kopf). Subsequently, 1 L of the viral prepa-
ration was injected bilaterally into the CA3 region
of the hippocampus using the following coordinates:
±2.3 mm medial/lateral, –2.1 mm anterior/posterior,
and –2.2 mm dorsal/ventral from the bregma. The
preparation was injected with a speed of 0.5 L/min
over a period of 2 min utilizing a Hamilton 10-L
syringe and a 26 gage needle. The mice were stitched
and then returned to their cages.
Behavioral testing
The behavioral tests were initiated 20 days after the
lentivirus injection. The mice were first subjected to
the novel object recognition test for 3 days and then,
after a 4-day interval, to the Morris water maze for 5
days.
Novel object recognition test
This was performed as previously described [29].
In brief, the mice were first placed in an arena
(60 ×60 cm with 50 cm walls) in the absence of
objects, after which two identical objects were added
(control test). Either 2 h (short-term memory test,
STM) or 24 h later (long-term memory test, LTM),
the mice were re-introduced to the arena in which
one of the objects was replaced by a novel one. The
behavior of the mice was then monitored utilizing the
EthoVision XT 11 program for 5min, and the time
and frequency that the mice visited each of the objects
were measured. The results are presented as the ratio
of the number of visits to the novel object relative to
the total visits to both new and old objects.
Morris water maze
This was performed as previously described [29].
Accordingly, the mice were placed in a 140cm circu-
lar pool with the water rendered opaque with milk
powder and a 10 cm circular platform, submerged
1 cm below the surface of the water, was placed at
1446 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
a fixed position. The mice were subjected to 4 tri-
als per day for 5 days, such that for each trial the
mice were placed in one of equally spaced locations
along the perimeter of the pool. The inter-trial inter-
val was 30 min and the location of the platform was
unchanged between days. The mice were introduced
to the arena from four random locations, whose order
was unchanged between days. The performance of
the mice was monitored by measuring the time they
took to reach the platform. Measurements of the time
to reach the platform were performed using the Etho-
Vision XT 11 program.
Immunohistochemistry and immunofluorescence
confocal microscopy
One brain hemisphere was fixed overnight with
4% paraformaldehyde 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 microtome, collected serially, placed in
200 l of cryoprotectant (containing glycerin, ethy-
lene glycol, and 0.1 M sodium-phosphate buffer,
pH 7.4), and stored at –20◦C until use. Immuno-
histochemistry and innunofluorescence analysis was
performed as previously described [26]. VEGF was
visualized utilizing an anti-VEGF antibody directed
against the human VEGF-A165, which is the most
abundant isoform of VEGF-A and which cross reacts
strongly with the corresponding mouse VEGF-A164
isoform. The pathological effects of apoE4 were
monitored utilizing the following primary antibodies
(Abs): mouse anti-neuN (1:500; Chemicon), rab-
bit anti-VEGF (1:1000, Calbiochem), guinea-pig
anti-vesicular glutamatergic transporter 1 (VGluT1;
1:2000; Millipore), rabbit anti-Collagen IV (1:1000,
Abcam), rabbit anti-apoER2 (␣CT kindly provided
by J. Herz lab; 1:1000), rabbit anti-doublecortin
(DCX; 1:200; Santa Cruz), rabbit anti-A42 (1:500;
Chemicon, Temecula, CA), rabbit anti-202/205 phos-
phorylated tau (AT8; 1:200, Innogenetics). All the
groups were stained together and the results pre-
sented correspond to the mean ±SEM of the percent
area stained normalized relative to the young control
apoE3 mice.
The immunostained sections were viewed using a
Zeiss light microscope (Axioskop, Oberkochen, Ger-
many) interfaced with a CCD video camera (Kodak
Megaplus, Rochester, NY, USA). Pictures of stained
brains were obtained at X10 magnification. The
staining was analyzed and quantified using the Image-
Pro plus system for image analysis (v. 5.1, Media
Cybernetics, Silver Spring, MD, USA). The images
were analyzed by marking the area of interest and
setting a threshold for all sections subjected to the
same staining. The stained area above the threshold
relative to the total area was then determined for each
section. All the groups were stained together and the
results presented correspond to the mean ±SEM of
the percent area stained normalized relative to the
young control apoE3 mice.
Sections stained for immunofluorescence were
visualized using a confocal scanning laser micro-
scope (Zeiss, LSM 510). Images (1024 ×1024 pixels,
12 bit) were acquired by averaging eight scans. Con-
trol experiments revealed no staining in sections
lacking the first Ab. The intensities of immunoflu-
orescence staining were calculated utilizing the
Image-Pro Plus system (version 5.1, Media Cyber-
netics) as previously described [26]. All images for
each immunostaining were obtained under identi-
cal conditions, and their quantitative analyses were
performed with no further handling. Moderate adjust-
ments for contrast and brightness were performed
similarly on all the presented images of the different
mouse groups. The images were analyzed by setting
a threshold for all sections having a specific labeling.
The area of staining over the threshold relative to the
total area of interest was determined and averaged for
each mouse and each group, and was normalized to
the apoE3 na¨
ıve group.
Immunoblot analysis
Immunoblot analysis was performed as previously
described [34, 35]. The following Abs were used:
rabbit anti-VEGF (1:1000; Calbiochem), rabbit anti-
VEGFR2 (1:500, Cell Signaling), rabbit HIF-1␣
(1:100, Santa Cruz), mouse anti-VGluT1 (1:1000;
Millipore), rabbit anti-Collagen IV (1:1000, Abcam),
goat anti-apoE (1:10000; Chemicon), and mouse
anti-GAPDH (1:1000; Abcam). The immunoblot
bands were visualized utilizing the ECL chemi-
luminescent substrate (Pierce), after which their
intensity was quantified using EZQuantGel software
(EZQuant, Tel Aviv, Israel). All results were normal-
ized to na¨
ıve-apoE3 group and were employed by
GAPDH as gel-loading control.
qRT-PCR analysis
TaqMan qRT-PCR analysis was performed as
previously described [28]. Assays were con-
ducted according to the manufacturer’s specifications
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1447
(Applied Biosystems). VEGF, VEGFR2, VEGFR1,
HIF1␣, and HIF2␣gene expression levels were deter-
mined utilizing TaqMan RT-PCR specific primers
(Applied Biosystems). Analysis and quantification
were conducted using 7300 system software and com-
pared to the expression of the housekeeping HPRT-1
gene.
Statistical analysis
The results of the na¨
ıve mice experiment, which
consisted of non-treated apoE3 and apoE4 mice (n=
9–15 mice /group), were normalized relative to apoE3
and were analyzed utilizing Student’s t-test. The
experimental design of the viral constructs treated
mice consisted of two genotypes (apoE3 and apoE4)
and three treatments (na¨
ıve, VEGF expressing virus,
and sham GFP labeled virus). The results for each
of the viruses experiments (e.g., lentivirus and
adenovirus associated virus; n= 8–11 mice/group)
were analyzed using STATISTICA software (Ver-
sion 8.0 StatSoft, Inc., Tulsa, USA). Specifically,
2-way ANOVA was performed for all tested group
together followed by the appropriate post hoc analysis
(marked here in asterisks). When significant effects
of treatment were obtained this was followed by 1-
way ANOVA on the relevant for the specific effect of
the VEGF treatment on apoE4 mice compared to the
corresponded sham treated group.
RESULTS
The effects of apoE4 on VEGF levels in the CNS
of young na¨ıve TR mice
The extent to which apoE4 affects the levels of
VEGF in the hippocampus was first examined histo-
logically. As can be seen in Fig. 1A, apoE4 mice
exhibited lower levels of VEGF than the corre-
sponding apoE3 mice. These results were obtained
both in the CA1 and CA3 regions (results presented
for CA1, p< 0.05 by Student’s t-test), however, no
effect was observed in the dentate gyrus (DG) sub-
region of the hippocampus. Moreover, these results
were verified utilizing an immunoblot assay of the
total level of VEGF in the hippocampus. As can be
seen in Fig. 1B, the levels of VEGF were lower
in the apoE4 mice compared to the apoE3 mice
(p< 0.01 by Student’s t-test). These effects were
accompanied by a similar reduction in mRNA lev-
els, as presented in Fig. 1C (p< 0.05 by Student’s
t-test), suggesting that the apoE4-related decrease in
VEGF is driven by gene expression. Interestingly,
comparison of hippocampal VEGF protein levels in
C57BL/6J Wild type mice expressing mouse apoE
and apoE3 and apoE4 targeted replacement mice
expressing human apoE utilizing immunoblot anal-
ysis showed similar VEGF levels for both apoE3
TR mice and the C57BL/6J Wild type mice which
were higher than the corresponding appoE4 TR mice
(not shown).
Examination of VEGF receptors showed lower
levels of VEGF receptor 2 (VEGF-R2) both in pro-
tein and mRNA levels (Fig. 1D and E, respectively,
p< 0.05 by Student’s t-test). In contrast, the levels of
VEGF receptor 1 were not significantly affected by
apoE4 in both the protein and mRNA levels (data not
shown).
The apoE4-induced reduction in VEGF levels was
accompanied by reduced levels of HIF1␣both pro-
tein and mRNA levels (Fig. 1F and G, respectively;
p< 0.05 by Student’s t-test), however, no effect was
observed on the HIF2␣levels in the hippocampus (not
shown), suggesting that the effects of VEGF in the
apoE4 mice are associated with specific stress-related
conditions.
The effect of LV-VEGF treatment on VEGF
expression in young na¨ıve mice
We next examined whether the apoE4-related
decrease in VEGF is associated with other patho-
logical effects of apoE4, and whether it can be
counteracted by a lentivirus-expressing VEGF con-
struct (LV-VEGF). This was pursued, as described in
Materials and Methods, by intra-hippocampal injec-
tion into the CA3 sub-region of VEGF or the GFP
construct as a control, (Fig. 2A). As depicted in
Fig. 2B, this resulted in a specific elevation of VEGF
levels in CA3 sub-region of the LV-VEGF treated
apoE4 group compared with the matched control LV-
GFP group. Quantification of the results revealed
p< 0.05 for the post-hoc analysis of the specific effect
of LV-VEGF on apoE4 mice compared with the
matched control LV-GFP group. Similar results were
obtained for the CA1 sub-region (data not shown).
These findings were reinforced by immunoblot anal-
ysis, as depicted in Fig. 1C. Quantification of the
results revealed p< 0.05 for the post-hoc analysis
of the specific effect of LV-VEGF on apoE4 mice
compared with the matched control LV-GFP group.
Complementary measurement of VEGF mRNA lev-
els showed similar effect to those obtained in na¨
ıve
mice (data not shown).
1448 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
Fig. 1. Theeffect of apoE4 on VEGF expression in na¨
ıve CNS of young na¨
ıve mice. A) VEGF immunohistochemistry of the CA1 hippocampal
sub-region. Representative sections are depicted on the left panel. Quantification is shown on the right panel. ∗p<0.05 by Student’s t-test.
B) Immunoblot analysis of hippocampal VEGF. Representative immunoblots are depicted on the left panel. Quantification is shown on the
right panel. ∗∗p< 0.01 by Student’s t-test. C) VEGF-A hippocampal qRT-PCR measurements. ∗p< 0.05 by Student’s t-test. D) Immunoblot
analysis of VEGF-R2. Representative sections are depicted on the left panel. Quantification is shown on the right panel. ∗p< 0.05 by Student’s
t-test. E) qRT-PCR hippocampal measurements. ∗p< 0.05 by Student’s t-test. F) Immunoblot analysis of HIF-1␣. Representative sections
are depicted on the left panel. Quantification is shown on the right panel. ∗p< 0.05 by Student’s t-test. G) HIF-1␣qRT-PCR hippocampal
measurements. ∗∗∗p< 0.001 by Student’s t-test. ApoE3 mice are depicted in white bars, whereas apoE4 mice are depicted in black bars. All
results represent the mean ±SEM; n=8–15 per group.
The effects of the elevation of VEGF were also
associated with a corresponding effect on the VEGF-
R2 protein level, as shown in Fig. 2D. Quantification
of these results revealed p< 0.001 for the post-hoc
analysis of the specific effect of LV-VEGF on apoE4
mice compared with the matched control LV-GFP
group. A corresponding mRNA analysis revealed
similar results to those obtained in na¨
ıve mice (data
not shown).
Examination of the apoE4-driven effect on
hypoxia-inducible factors showed that expectedly,
the levels of HIF-1␣, which is upstream to VEGF
in the VEGF cascade, were not affected by the LV-
VEGF treatment; however, the total HIF-1␣levels
specifically decreased in apoE3 following the injec-
tion procedure (Fig. 2E). Measurements of mRNA
levels showed a similar effect (not shown). The lev-
els of the corresponding HIF 2␣, however, were not
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1449
Fig. 2. Elevating VEGF levels via lentivirus treatment. A) The lentivirus injection paradigm. Representative sections of staining for NeuN
in na¨
ıve and GFP-injected mice. B) VEGF immunohistology staining of the hippocampal CA3 sub-region of na¨
ıve, LV-GFP, and LV-VEGF
treated mice. p< 0.05 for genotype and p< 0.01 for the effects of treatment by 2-way ANOVA; p<0.05 for further analysis of the effect of
treatment utilizing 1-way ANOVA and ∗p< 0.05 for the post-hoc analysis of the specific effect of LV-VEGF on apoE4 mice compared with
the matched control LV-GFP group. C) Immunoblot analysis of hippocampal VEGF in na¨
ıve, LV-GFP, and VEGF treated mice. p< 0.05 for
genotype and p< 0.001 for the effect of treatment by 2-way ANOVA; p< 0.05 for further analysis of the effect of treatment utilizing 1-way
ANOVA and ∗p< 0.05 for the post-hoc analysis of the specific effect of LV-VEGF on apoE4 mice compared with the matched control LV-GFP
group. D) Immunoblot analysis of VEGF-R2 in na¨
ıve, LV-GFP, and LV-VEGF treated mice p< 0.05 for the genotype and p< 0.001 for the
effects of treatment by 2-way ANOVA; p< 0.01 for further analysis of the effect of treatment utilizing 1-way ANOVA and ∗∗∗ p<0.001 for
the post-hoc analysis of the specific effect of LV-VEGF on apoE4 mice compared with the matched control LV-GFP group. E) Immunoblot
analysis of HIF-1␣in na¨
ıve, LV-GFP, and LV-VEGF treated mice. p< 0.05 for genotype effects by 2-way ANOVA. All representative sections
and blots are depicted in the left panel whereas quantification is shown on the right panel. ApoE3 mice are depicted in white bars, whereas
apoE4 mice are depicted in black bars. All results represent the mean ±SEM; n=6–10 per group.
affected by neither genotype nor treatment (data not
shown).
The effects of LV-VEGF treatment
on apoE4-driven cognitive deficits
ApoE4 mice were shown to be cognitively
impaired at the age of 4 months in two hippocampal-
dependent learning and memory tests; the Morris
water maze and the novel object recognition tests
[29]. The extent to which LV-VEGF treatment can
counteract these apoE4-driven behavioral deficits
was next examined. The behavioral results thus
obtained are depicted in Fig. 3. As can be seen in
Fig. 3A, in the Morris water maze test all mouse
groups (e.g., na¨
ıve, LV-GFP, and LV-VEGF-treated
mice) improved their performance over time and
reached similar plateau levels on day 5, as measured
by the latency to reach the hidden platform. How-
ever, both na¨
ıve and LV-GFP treated apoE4 mice
showed a deficit in the learning curve. These apoE4-
dependent deficits were counteracted by the VEGF
treatment. None of the treatments affected the apoE3
mice. Quantification of the results by 2-way ANOVA
revealed p< 0.001 for post hoc analysis of the spe-
cific effect of LV-VEGF treatment on apoE4 mice
compared with the matched control LV-GFP group
(Fig. 3A). Additional examination of the cognitive
performance was pursued utilizing the novel object
recognition test in which, as previously described
[29], the apoE3 mice paid more visits to the novel
object compared to the familiar one. In contrast, the
apoE4 mice paid the same number of visits to the
familiar and novel objects, indicating a deficit in
the memory of the familiar object. This deficit was
abolished by LV-VEGF treatment (Fig. 3B). Quantifi-
cation of the results revealed p< 0.01 for the post-hoc
analysis of the specific effect of LV-VEGF treatment
on apoE4 mice compared with the matched control
LV-GFP group.
1450 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
Fig. 3. The effects of LV-VEGF treatment on apoE4-driven behavioral deficits. A) Morris water maze. The latency of time to reach the
hidden platform was measured utilizing the ANOVA multiple comparison. p< 0.05 for the effect of genotype X treatment by 2-way ANOVA
and ∗∗∗p< 0.001 for the post-hoc analysis of the specific effect of LV-VEGF on apoE4 mice compared with the matched control LV-GFP
group on day 3. Squares and correspond to control apoE3 and apoE4 mice, respectively, whereas triangles, and correspond to
LV-GFP apoE3 and apoE4 mice, dots ◦and •correspond to LV-VEGF apoE3 and apoE4 mice, respectively. n= 9–12 per group. B) Novel
object recognition test. The ratio of the number of visits to the novel object to the sum of visits to both old and novel objects was measured.
∗∗p< 0.001 for the post-hoc analysis of the specific effect of LV-VEGF on apoE4 mice compared with the matched control LV-GFP group.
ApoE3 mice are depicted in white bars, whereas apoE4 mice are depicted in black bars. Mean ±SEM; n=9–12 per group.
The effect of LV-VEGF treatment
on apoE4-driven brain pathology
The extent to which up-regulation of VEGF utiliz-
ing lentivirus injection reversed apoE4-related brain
pathology was next examined. The results of this
battery of measurements will be divided into three
groups of parameters:
Synaptic and neuronal pathology parameters
We first focused on the apoE4-induced deficit in
the pre-synaptic marker VGluT1 [26]. Immunohis-
tocemical examinations of the CA3 hippocampal
sub-region, in which the pathology of apoE4 is most
pronounced. As can been seen, and consistent with
previous findings [26] the level of VGluT1 were lower
in apoE4 mice than those observed in the corre-
sponding apoE3 in both control groups (e.g., na¨
ıve
and LV-GFP apoE4 mice). Examination of the effect
of LV-VEGF on VGluT1 revealed that LV-VEGF
treatment markedly elevated VGluT1 levels in the
apoE4 mice, thus reversing the pathological effects
of apoE4. The upper panel of Fig. 4A depicts the
immunohistological analysis of these results, which
revealed p< 0.05 for the post-hoc analysis of the spe-
cific effect of LV-VEGF treatment on apoE4 mice
compared with the matched control LV-GFP group.
This was confirmed by complementary immunoblot
analysis (Fig. 4B, lower panel). Quantification of
these results revealed p< 0.001 for the specific effect
of LV-VEGF treatment on apoE4 mice compared with
the matched control LV-GFP group.
Neurogenesis in the hippocampus, as measured by
the marker Doublecortin (DCX), is upregulated in
apoE4 mice compared with apoE3 [27]. We there-
fore examined the effects of LV-VEGF treatment on
this parameter (Fig. 4C). This revealed, in accordance
with previous findings [27], that the levels of DCX
were higher in apoE4 mice than those observed in
the corresponding apoE3 in na¨
ıve treated groups.
The sham treatment (LV-GFP) decreased the lev-
els of DCX in apoE4 mice compared to the apoE3
mice, whereas upregulation of VEGF, utilizing LV-
VEGF treatment, resulted in specific elevation of
DCX in the apoE4 mice up to the levels of the cor-
responding apoE3 mice, thus abolishing the apoE
genotype-related differences. Quantification of the
results revealed the significant effect of p< 0.001 for
post-hoc analysis of the specific effect of LV-VEGF
treatment on apoE4 mice compared with the matched
control LV-GFP group.
We next examined the effects of LV-VEGF treat-
ment on the levels of apoER2, which serves as both
an apoE receptor and a modulator of synaptic trans-
mission. As previously shown [28], the levels of
apoER2 are lower in apoE4 mice compared to the cor-
responding apoE3 mice for both na¨
ıve and LV-GFP
control groups. However, this effect was reversed by
LV-VEGF treatment, as can be seen in the immuno-
histology staining depicted in Fig. 4C. Quantification
of these results revealed p< 0.05 for the post-hoc
analysis of the specific effect of LV-VEGF treatment
on apoE4 mice compared with the matched control
LV-GFP group.
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1451
Fig. 4. The effects of LV-VEGF treatment on apoE4-driven synaptic and neuronal parameters. A) Upper panel: VGluT1 immunohistological
staining of CA3 hippocampal subregions in na¨
ıve, LV-GFP, and LV-VEGF treated mice. p< 0.05 for genotype X treatment by 2-way ANOVA
and ∗p< 0.05 for post-hoc analysis for the specific effect of LV-VEGF treatment on apoE4 mice compared with the matched control LV-
GFP group. Lower panel: VGluT1 immunoreactivity measurements in na¨
ıve, LV-GFP, and LV-VEGF treated mice. p< 0.05 for genotype X
treatment by 2-way ANOVA and ∗∗∗ p< 0.001 for post-hoc analysis of the specific effect of LV-VEGF treatment on apoE4 mice compared
with the matched control LV-GFP group. B) DCX immunohistology staining of the CA3 hippocampal subregion in na¨
ıve, LV-GFP, and
LV-VEGF treated mice. p<0.05 for genotype X treatment by 2-way ANOVA. C) ApoER2 immunohistology staining of CA3 hippocampal
subregions in na¨
ıve, LV-GFP, and LV-VEGF treated mice. ∗p< 0.05 for genotype X treatment by 2-way ANOVA and p< 0.05 for post-hoc
analysis of the specific effect of LV-VEGF treatment on apoE4 mice compared with the matched control LV-GFP group. All representative
sections and blots are depicted in the left panels whereas quantification is shown on the right panel. ApoE3 mice are depicted in white bars,
whereas apoE4 mice are depicted in black bars. All results represent the mean ±SEM; n=6–10 per group.
Brain vascularization
ApoE4 is associated with vascular pathology [36],
and VEGF is known as a vascular permeability factor
which induces angiogenic activity, and vascular sur-
vival activity [17]. We therefore next examined the
effects of LV-VEGF on the pan vascular marker col-
lagen IV. Immunohistochemical examination of the
CA3 hippocampal sub-region in na¨
ıve mice revealed
no significant apoE4-driven effects (Fig. 5A). Sim-
ilar effects were observed in other hippocampal
sub-regions (not shown). This was reinforced by
corresponding immunoblot analysis (Fig. 5B). More-
over, following LV-VEGF treatment, there was no
significant effect on collagen IV levels, suggesting
that increasing the levels of VEGF via LV-VEGF does
not affect vascularization in mature mice.
The effect of LV-VEGF treatment
on apoE4-driven brain pathology
The extent to which LV-VEGF can reverse the
apoE4-driven accumulation of A42 and hyper-
phosphorylated tau in CA3 neurons, which is the
hippocampal subfield in which these pathologies
were most pronounced [26], was next examined.
This was executed immunohistochemically in order
to examine a specific hippocampal location, in which
the apoE4-driven effects were most robust. As can
be seen in Fig. 6A, and consistently with previous
findings [26], the levels of immunohistochemically
determined A42 were higher in the apoE4 mice
than in the apoE3 mice for both control groups (e.g.,
na¨
ıve and LV-GFP groups). Similar effects obtained
for the levels of tau phosphorylation using the AT8
mAb (Fig. 6B). Examination of the LV-VEGF treated
group revealed that in contrast to the effect of LV-
VEGF treatment in correcting the apoE4-related
cognitive, neuronal, and apoE-receptor pathologies
by elevating the levels of apoE4 and rendering
them to the levels of the corresponding apoE3, the
apoE4-driven effect on both A42 (Fig. 5A) and tau
phosphorylation (AT8; Fig. 5B), showed no signifi-
cant effect on apoE4, but elevated the levels of apoE3.
ApoE levels are known to be downregulated by
apoE4 [37]. As can be seen in Fig. 6C, and in
accordance with previous findings [26], apoE levels
were lower in the apoE4 mice than in the observed
1452 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
Fig. 5. The effects of LV-VEGF treatment on vascularization. A) Collagen IV immunohistological staining of the CA3 hippocampal subregion
in na¨
ıve, LV-GFP, and LV-VEGF treated mice. Representative sections are depicted in the left panel. Quantification of the results is shown
in the right panel. B) Collagen IV immunoreactivity measurements. Representative blots are depicted in the left panel. ApoE3 mice are
depicted in white bars, whereas apoE4 mice are depicted in black bars. All results represent the mean ±SEM; n=7–10 per group.
corresponding apoE3 mice in all the mice groups
suggesting that upregulation of VEGF levels had no
effect on the levels of apoE.
The effect of AAV-VEGF treatment
on apoE4-driven brain pathology
The expression of VEGF in the lentivirus con-
structs is driven by the strong CMV promoter whose
expression is and cell type specific [38]. We therefore
turned to an adeno-associated virus (AAV), which
expresses VEGF under the regulation of the GFAP
promoter, and examined the extent to which the
pathological effects of apoE4 on Aand tau hyper-
phosphorylation can be reversed by this cell type
specific treatment. The treatment with AAV-VEGF,
like that with LV-VEGF reversed the decrease in
VEGF of the hippocampus of the apoE4 mice but had
no effect on the corresponding apoE3 mice (Fig. 7A).
Quantification of these results revealed p< 0.01 for
the effect of genotype by 2-way ANOVA. However,
in contrast to the LV-VEGF treatment (Fig. 6A), the
AAV-VEGF treatment lowered the levels of A42 in
apoE4 mice and rendered them similar to those of
the apoE3 mice (Fig. 7B). Quantification of these
results revealed p< 0.05 for the post-hoc analysis of
the specific effect of AAV-VEGF treatment on apoE4
mice compared with the matched control AAV-GFP
group. Furthermore, measurements of tau phosphory-
lation (AT8; Fig. 7C) revealed a decrease and partial
reversal of the hyperphosphorylation of the AAV-
VEGF treated apoE4 mice. Quantification of these
results revealed p< 0.05 for the effect of genotype
by 2-way ANOVA. Additional experiments revealed
that the pathological effects of apoE4 on VGluT1,
apoER2, and DCX which were reversed by LV-VEGF
(Fig. 3A-C) were also reversed by AAV-VEGF (not
shown).
DISCUSSION
ApoE4, the most prevalent genetic risk factor
for AD, is associated with numerous brain patholo-
gies which include impaired neurite outgrowth and
synaptogenesis [12], impairments in plastic neuronal
remodeling [13], and increased neurodegeneration
[14]. ApoE4 is also associated with more pronounced
vascular risk factors than non-carriers [16]. In view
of the important role of VEGF in both the neuronal
and vascular systems, and of its suggested thera-
peutic potential in AD [39], we presently examined
the possibility that VEGF plays a role in meditat-
ing the brain’s pathological effects of apoE4 and
the associated cognitive deficits. This was performed
in vivo by examining young apoE3 and apoE4-TR
mice in two successive stages. The first stage inves-
tigated the extent to which apoE4-related brain and
cognitive pathologies are associated with changes in
VEGF and key factors of the VEGF cascade. This was
then followed by assessment of the extent to which
upregulation of VEGF levels and expression in the
hippocampus of the apoE4 mice by viral adminis-
tration of VEGF can reverse the brain and cognitive
pathological effects of apoE4.
This revealed that the protein levels of VEGF and
its receptor, VEGF-R2, which is implicated in medi-
ating the neuronal effects of VEGF [40] and the
corresponding messenger RNA levels, are lower in
the hippocampi of apoE4-TR mice than in the cor-
responding apoE3 mice. This was associated with a
reduction in the protein and mRNA levels of HIF1␣,
the transcription factor that regulates the expres-
sion of VEGF in the hippocampus of the apoE4
mice relative to that of the apoE3 mice. Injection
of lentivirus-expressing VEGF into the hippocam-
pus elevated the levels of VEGF and VEGF-R2 in
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1453
Fig. 6. The effects of LV-VEGF treatment on apoE4-driven AD hallmark parameters. A) Aimmunohistological staining of CA3 hippocam-
pal subregions in na¨
ıve, LV-GFP, and LV-VEGF treated mice. p< 0.05 for the effect of the genotype and p<0.01 for the effect of treatment
by 2-way ANOVA. Further analysis of the effect of treatment showed p< 0.01 by 1-way ANOVA. B) AT8 immunohistological staining of
CA3 hippocampal subregions in na¨
ıve, LV-GFP, and LV-VEGF treated mice. p< 0.05 for the effect of genotype by 2-way ANOVA. C) ApoE
immunoreactivity measurements. p< 0.05 for the effect of genotype X treatment by 2-way ANOVA. ∗∗∗ p< 0.001 for post-hoc analysis of the
effects of LV-VEGF treatment in apoE4 mice shown for the corresponding apoE3 mice in all groups. All representative sections and blots
are depicted in the left panel whereas quantification is shown on the right panel. ApoE3 mice are depicted in white bars, whereas apoE4
mice are depicted in black bars. All results represent the mean ±SEM; n=6–10 per group.
1454 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
Fig. 7. The effects of AAV-VEGF treatment on VEGF levels and on apoE4-driven AD hallmark parameters. ApoE3 and apoE4 mice were
treated with AA-VEGF and its sham construct AA-GFP as described in Materials and Methods. A) VEGF immunohistology staining of the
hippocampal CA3 sub-region of na¨
ıve, AAV-GFP, and AAV-VEGF treated mice. p< 0.01 for the effect of genotype by 2-way ANOVA. B)
Aimmunohistological staining of CA3 hippocampal subregions in na¨
ıve, AAV-GFP, and AAV-VEGF treated mice. p< 0.05 for the effect
of genotype X treatment by 2-way ANOVA and p< 0.01 for the effect of treatment by 2-way ANOVA. C) AT8 immunohistological staining
of CA3 hippocampal subregions in na¨
ıve, LV-GFP, and LV-VEGF treated mice. p< 0.05 for the effect of the genotype by 2-way ANOVA. All
representative sections and blots are depicted in the left panel whereas quantification is shown on the right panel. ApoE3 mice are depicted
in white bars, whereas apoE4 mice are depicted in black bars. All results represent the mean ±SEM; n=6–10 per group.
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1455
the apoE4 mice and rendered them similar to those
of the apoE3 mice, whose corresponding VEGF and
VEGF-R2 levels were not affected by the VEGF
treatment. In contrast, the apoE4-driven reduction of
HIF1␣was not affected by the LV-VEGF treatment;
however, total HIF1␣levels were reduced following
the injection procedure specifically in apoE3 mice.
Examination of the effects of this treatment on the
apoE4-driven cognitive and brain pathology revealed
that the cognitive deficits of the apoE4 mice in the
novel object recognition and in the Morris water maze
tests were reversed by LV-VEGF treatment and that
this was associated with reversal of the apoE4-driven
reduction in the levels of VGluT1 and apoER2 in
hippocampal neurons and a corresponding effect on
DCX-positive neurons and neurogenesis. In contrast,
the apoE4-driven accumulation of Aand hyper-
phosphorylated tau was not reversed by the LV-VEGF
treatment but was associated with increased lev-
els of Aand hyperphosphorylated in hippocampal
neurons of the apoE3 mice (Fig. 6). Additional exper-
iments utilizing an adeno associated virus construct
which expresses VFGF under the regulation of the
astrocytic promotor GFAP revealed that the increased
expression of VEGF via this, more specific, con-
struct had no effect on Aand hyperphosphorylated
in hippocampal neurons of the apoE3 mice and that it
abolished the apoE4 driven accumulation of Aand
partially reversed the corresponding increased levels
of tau hyperphosphorylation (Fig. 7B and C, respec-
tively). Taken together, these findings suggest that the
cognitive, neuronal, A, and tau related pathological
effects of apoE4 are driven via a VEGF-dependent
mechanism which can be reversed by upregulation
of the expression of VEGF. Importantly, there were
no significant differences in the overall vascular den-
sity in the hippocampus of non-treated apoE4 and
apoE3 mice, as assessed utilizing collagen IV as a
marker, which were not affected by the viral upregu-
lation of the VEGF levels (Fig. 5). These findings are
consistent with previous observations showing that
in the adult brains blood vessels are relatively inde-
pendent of VEGF [17] and that under steady-state
conditions VEGF does not play an important role in
the maintenance of mature vessels [41].
Next, we will discuss the mechanism underly-
ing the effects of apoE4 on the levels of VEGF
and the putative mechanisms that mediate the VEGF
dependent mechanisms. HIF is an essential transcrip-
tion factor that protects from hypoxic damage and
is known to be neuroprotective [42–44]. It is com-
posed of constituently expressed HIF1␣ subunits
and the HIF-1␣subunit, which is regulated at multiple
levels [45, 46]. Elevated levels of HIF are neuropro-
tective and are associated with increased expression
of VEGF and of other genes [45]. Although the
exact mechanisms by which apoE4 reduces the
levels of HIF-1␣remain to be determined, the
apoE4-associated decrease in HIF-1␣and the related
reduction in VEGF levels suggest that the apoE4-
driven effect on VEGF is mediated by HIF-1␣and
that this leads to the apoE4-driven pathology. It is
interesting to note that the control construct, namely,
LV-GFP, reduces the levels of HIF-1␣in the apoE3
mouse group without affecting the HIF-1␣-related
pathology. The underlying mechanism is unknown
and remains to be determined.
The finding that the apoE4-driven decrease in
VEGF in the hippocampus results in reduced levels of
the presynaptic glutamate receptor is consistent with
previous observations that excitatory synaptic trans-
mitters in the hippocampus are enhanced by VEGF
[47] and that the NMDA glutamate receptors are
activated by VEGF [48]. Furthermore, since VEGF
induces phosphorylation and activation of Dab1,
which is an adaptor protein associated with apoER2
and the NMDA receptor [49], the low levels of VEGF
in the apoE4 mice could account for the reduction in
the apoER2 levels of these mice. Moreover, previous
publication showed a robust increase in VEGF-R2
- ApoER2 interaction following VEGF treatment
[49]. These results, together with our current findings
showing elevated levels of VEGF-R2 and ApoER2
following LV-VEGF treatment suggest that VEGF
and apoER2 acts via a common mechanism.
We have previously shown that neurogenesis in
the hippocampus of apoE4 mice is elevated, presum-
ably as a compensatory response, relative to apoE3
mice and that stressing the mice reduces the levels
of neurogenesis (shown by the marker DCX) in the
apoE4 mice to levels below the corresponding lev-
els in apoE3 mice, which were not affected by this
stress [40]. It is thus possible that the presently shown
decrease in neurogenesis in the apoE4 mice, follow-
ing treatment with LV-GFP, which had no effect on
the VEGF levels (Fig. 2A, B), is due to the gen-
eral response to injection of the viral construct into
the hippocampus. The finding that the injection of
LV-VEGF elevates both the VEGF and neurogene-
sis levels in apoE4 mice to levels similar to those of
the LV-GFP apoE3 mice, which were not affected by
the treatment, is in accordance with previous find-
ings that neurogenesis is activated by VEGF [20, 39,
50–52]. Note that under steady-state conditions, the
1456 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
vascular density of the adult brain is relatively inde-
pendent of VEGF [17] and therefore apoE4 had no
significant effect on the overall hippocampal vascular
density (see Fig. 5). It is expected, however, that fol-
lowing injury or under more plastic conditions where
the vasculature and the associated angiogenesis are
dependent of VEGF, apoE4 will also be associated
with a more pronounced vascular phenotype.
The findings that the Aand tau hyperphosphoryla-
tion are reversed by the AAV-VEGF treatment (Fig. 7)
and not by the corresponding LV–VEGF treatment
(Fig. 6) related to the observation that LV-VEGF treat-
ment increases the levels of these AD pathological
markers in the apoE3 mice whereas the AAV-VEGF
did not. This outcome may be due to the fact that the
VEGF in the AAV-VEGF construct is expressedunder
the regulation of the GFAP promoter specifically in
astrocytes whereas in the corresponding LV-VEGF
construct, VEGF is expressed under the powerful
CMC promoter in a non-cell specific manner.
In conclusion, these findings show that apoE4-
driven brain pathology and cognitive impairments
in young apoE4 TR mice are associated with down
regulation of the VEGF system and can be reversed
by upregulation of the expression of VEGF in the
hippocampus. Examination of the long term VEGF
treatment and the extents to which such treatment can
also reverse the pathological effects of apoE4 in aged
mice remain to be determined. These animal model
findings suggest that VEGF is a promising target for
treatment of apoE4 carriers in AD.
ACKNOWLEDGMENTS
We thank Alex Smolar for his technical assis-
tance. This research was supported in part by grants
from the Legacy Heritage Bio-Medical Program of
the Israel Science Foundation (grant No. 1575/14),
from the Joseph K. and Inez Eichenbaum Foundation,
from the Harold and Eleanore Foonberg Foundation,
and from the Joseph Sagol fellowship program for
brain research. DMM is the incumbent of the Myriam
Lebach Chair in Molecular Neurodegeneration.
Authors’ disclosures available online (http://j-
alz.com/manuscript-disclosures/16-0182r1).
REFERENCES
[1] Masters CL, Simms G, Weinman NA, Multhaup G,
McDonald BL, Beyreuther K (1985) Amyloid plaque core
protein in Alzheimer disease and Down syndrome. Proc
Natl Acad SciUSA82, 4245-4249.
[2] Alzheimer A, Stelzmann RA, Schnitzlein HN, Murtagh FR
(1995) An English translation of Alzheimer’s 1907 paper,
“Uber eine eigenartige Erkankung der Hirnrinde". Clin Anat
8, 429-431.
[3] Masliah E, Crews L, Hansen L (2006) Synaptic remodeling
during aging and in Alzheimer’s disease. J Alzheimers Dis
9, 91-99.
[4] Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima
J, Pettingell WH, Yu CE, Jondro PD, Schmidt SD, Wang K,
et al. (1995) Candidate gene for the chromosome 1 familial
Alzheimer’s disease locus. Science 269, 973-977.
[5] Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque
G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar
L, Foncin JF, Bruni AC, Montesi MP, Sorbi S, Rainero
I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A,
Sanseau P, Polinsky RJ, Wasco W, Da Silva HA, Haines
JL, Perkicak-Vance MA, Tanzi RE, Roses AD, Fraser PE,
Rommens JM, St George-Hyslop PH (1995) Cloning of
a gene bearing missense mutations in early-onset familial
Alzheimer’s disease. Nature 375, 754-760.
[6] Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE,
Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-
Vance MA (1993) Gene dose of apolipoprotein E type 4
allele and the risk of Alzheimer’s disease in late onset fam-
ilies. Science 261, 921-923.
[7] Roses AD (1996) Apolipoprotein E alleles as risk factors in
Alzheimer’s disease. Annu Rev Med 47, 387-400.
[8] Saunders AM, Strittmatter WJ, Schmechel D, George-
Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BL, Gusella
JF, Crapper-MacLachlan DR, Alberts MJ, Hulette C,
Crain B, Goldgaber D, Roses AD (1993) Association of
apolipoprotein E allele epsilon 4 with late-onset familial and
sporadic Alzheimer’s disease. Neurology 43, 1467-1472.
[9] Ward A, Crean S, Mercaldi CJ, Collins JM, Boyd D, Cook
MN, Arrighi HM (2012) Prevalence of apolipoprotein E4
genotype and homozygotes (APOE e4/4) among patients
diagnosed with Alzheimer’s disease: A systematic review
and meta-analysis. Neuroepidemiology 38, 1-17.
[10] Crean S, Ward A, Mercaldi CJ, Collins JM, Cook MN,
Baker NL, Arrighi HM (2011) Apolipoprotein E epsilon4
prevalence in Alzheimer’s disease patients varies across
global populations: A systematic literature review and meta-
analysis. Dement Geriatr Cogn Disord 31, 20-30.
[11] Genin E, Hannequin D, Wallon D, Sleegers K, Hiltunen
M, Combarros O, Bullido MJ, Engelborghs S, De Deyn P,
Berr C, Pasquier F, Dubois B, Tognoni G, Fievet N, Brouw-
ers N, Bettens K, Arosio B, Coto E, Del Zompo M, Mateo
I, Epelbaum J, Frank-Garcia A, Helisalmi S, Porcellini E,
Pilotto A, Forti P, Ferri R, Scarpini E, Siciliano G, Sol-
frizzi V, Sorbi S, Spalletta G, Valdivieso F, Vepsalainen
S, Alvarez V, Bosco P, Mancuso M, Panza F, Nacmias B,
Bossu P, Hanon O, Piccardi P, Annoni G, Seripa D, Galim-
berti D, Licastro F, Soininen H, Dartigues JF, Kamboh MI,
Van Broeckhoven C, Lambert JC, Amouyel P, Campion D
(2011) APOE and Alzheimer disease: A major gene with
semi-dominant inheritance. Mol Psychiatry 16, 903-907.
[12] Mahley RW, Huang Y (2012) Apolipoprotein e sets the
stage: Response to injury triggers neuropathology. Neuron
76, 871-885.
[13] Kutner KC, Erlanger DM, Tsai J, Jordan B, Relkin NR
(2000) Lower cognitive performance of older football play-
ers possessing apolipoprotein E epsilon4. Neurosurgery 47,
651-657; discussion 657-658.
[14] Arendt T, Schindler C, Bruckner MK, Eschrich K, Bigl V,
Zedlick D, Marcova L (1997) Plastic neuronal remodeling
S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology 1457
is impaired in patients with Alzheimer’s disease carrying
apolipoprotein epsilon 4 allele. J Neurosci 17, 516-529.
[15] Dik MG, Jonker C, Bouter LM, Geerlings MI, van Kamp GJ,
Deeg DJ (2000) APOE-epsilon4 is associated with memory
decline in cognitively impaired elderly. Neurology 54, 1492-
1497.
[16] Peila R, Rodriguez BL, Launer LJ (2002) Type 2 dia-
betes, APOE gene, and the risk for dementia and related
pathologies: The Honolulu-Asia Aging Study. Diabetes 51,
1256-1262.
[17] Licht T, Keshet E (2013) Delineating multiple functions of
VEGF-A in the adult brain. Cell Mol Life Sci 70, 1727-1737.
[18] Ruiz de Almodovar C, Lambrechts D, Mazzone M,
Carmeliet P (2009) Role and therapeutic potential of VEGF
in the nervous system. Physiol Rev 89, 607-648.
[19] Jin KL, Mao XO, Greenberg DA (2000) Vascular endothe-
lial growth factor: Direct neuroprotective effect in in vitro
ischemia. Proc Natl Acad SciUSA97, 10242-10247.
[20] Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA (2002)
Vascular endothelial growth factor (VEGF) stimulates neu-
rogenesis in vitro and in vivo.Proc Natl Acad SciUSA99,
11946-11950.
[21] Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara
N (1989) Vascular endothelial growth factor is a secreted
angiogenic mitogen. Science 246, 1306-1319.
[22] Feast A, Martinian L, Liu J, Catarino CB, Thom M, Sisodiya
SM (2012) Investigation of hypoxia-inducible factor-1alpha
in hippocampal sclerosis: A postmortem study. Epilepsia
53, 1349-1359.
[23] Krock BL, Skuli N, Simon MC (2011) Hypoxia-induced
angiogenesis: Good and evil. Genes Cancer 2, 1117-1133.
[24] Combarros O, Cortina-Borja M, Smith AD, Lehmann DJ
(2009) Epistasis in sporadic Alzheimer’s disease. Neurobiol
Aging 30, 1333-1349.
[25] Chiappelli M, Borroni B, Archetti S, Calabrese E, Corsi
MM, Franceschi M, Padovani A, Licastro F (2006) VEGF
gene and phenotype relation with Alzheimer’s disease and
mild cognitive impairment. Rejuvenation Res 9, 485-493.
[26] Liraz O, Boehm-Cagan A, Michaelson DM (2013) ApoE4
induces Abeta42, tau, and neuronal pathology in the hip-
pocampus of young targeted replacement apoE4 mice. Mol
Neurodegener 8, 16.
[27] Shiran Salomon-Zimri1 OL, Michaelson DM (2015)
Behavioral testing affects the phenotypic expression of
APOE 3 and APOE 4 in targeted replacement mice and
reduces the differences between them. Alzheimers Dement
(Amst) 1, 127-135.
[28] Gilat-Frenkel M, Boehm-Cagan A, Liraz O, Xian X, Herz J,
Michaelson DM (2014) Involvementof the Apoer2 and Lrp1
receptors in mediating the pathological effects of ApoE4 in
vivo.Curr Alzheimer Res 11, 549-557.
[29] Salomon-Zimri S, Boehm-Cagan A, Liraz O, Michaelson
DM (2014) Hippocampus-related cognitive impairments in
young apoE4 targeted replacement mice. Neurodegener Dis
13, 86-92.
[30] Sullivan PM, Mezdour H, Aratani Y, Knouff C, Najib
J, Reddick RL, Quarfordt SH, Maeda N (1997) Targeted
replacement of the mouse apolipoprotein E gene with
the common human APOE3 allele enhances diet-induced
hypercholesterolemia and atherosclerosis. J Biol Chem 272,
17972-17980.
[31] Levi O, Jongen-Relo AL, Feldon J, Roses AD, Michaelson
DM (2003) ApoE4 impairs hippocampal plasticity isoform-
specifically and blocks the environmental stimulation of
synaptogenesis and memory. Neurobiol Dis 13, 273-282.
[32] Belinson H, Michaelson DM (2009) ApoE4-dependent
Abeta-mediated neurodegeneration is associated with
inflammatory activation in the hippocampus but not the
septum. J Neural Transm 116, 1427-1434.
[33] Cross MJ, Dixelius J, Matsumoto T, Claesson-Welsh L
(2003) VEGF-receptor signal transduction. TrendsBiochem
Sci 28, 488-494.
[34] Haas A, Liraz O, Michaelson DM (2012) The effects of
apolipoproteins E3 and E4 on the transforming growth
factor-beta system in targeted replacement mice. Neurode-
gener Dis 10, 41-45.
[35] Kariv-Inbal Z, Yacobson S, Berkecz R, Peter M, Janaky T,
Lutjohann D, Broersen LM, Hartmann T, Michaelson DM
(2012) The isoform-specific pathological effects of apoE4 in
vivo are prevented by a fish oil (DHA) diet and are modified
by cholesterol. J Alzheimers Dis 28, 667-683.
[36] Gavett BE, John SE, Gurnani AS, Bussell CA, Saurman JL
(2015) The role of Alzheimer’s and cerebrovascular pathol-
ogy in mediating the effects of age, race, and apolipoprotein
E genotype on dementia severity in pathologically-
confirmed Alzheimer’s disease. J Alzheimers Dis 49,
531-545.
[37] Sullivan PM, Han B, Liu F, Mace BE, Ervin JF, Wu S,
Koger D, Paul S, Bales KR (2011) Reduced levels of human
apoE4 protein in an animal model of cognitive impairment.
Neurobiol Aging 32, 791-801.
[38] Glat MJ, Benninger F, Barhum Y, Ben-Zur T, Kogan E,
Steiner I, Yaffe D, Offen D (2016) Ectopic muscle expres-
sion of neurotrophic factors improves recovery after nerve
injury. J Mol Neurosci 58, 39-45.
[39] Herran E, Perez-Gonzalez R, Igartua M, Pedraz JL, Carro
E, Hernandez RM (2015) Enhanced hippocampal neuroge-
nesis in APP/PS1 mouse model of Alzheimer’s disease after
implantation of VEGF-loaded PLGA nanospheres. Curr
Alzheimer Res 12, 932-940.
[40] Rosenstein JM, Krum JM, Ruhrberg C (2010) VEGF in the
nervous system. Organogenesis 6, 107-114.
[41] Benjamin LE, Keshet E (1997) Conditional switching
of vascular endothelial growth factor (VEGF) expres-
sion in tumors: Induction of endothelial cell shedding
and regression of hemangioblastoma-like vessels by
VEGF withdrawal. Proc Natl Acad Sci U S A 94,
8761-8766.
[42] Chai X, Kong W, Liu L, Yu W, Zhang Z, Sun Y (2014)
A viral vector expressing hypoxia-inducible factor 1 alpha
inhibits hippocampal neuronal apoptosis. Neural Regen Res
9, 1145-1153.
[43] Ogunshola OO, Antoniou X (2009) Contribution of hypoxia
to Alzheimer’s disease: Is HIF-1alpha a mediator of neu-
rodegeneration? Cell Mol Life Sci 66, 3555-3563.
[44] Sun YM, Hao HQ, Kong WN, Liu LY, Chai XQ (2010)
Effect of intracerebroventricular injection of rAAV-HIF-
1alpha on hippocampal neuronal apoptosis in a rat model
of Alzheimer disease. Nan Fang Yi Ke Da Xue Xue Bao 30,
2711-2714.
[45] Giaccia A, Siim BG, Johnson RS (2003) HIF-1 as a target
for drug development. Nat Rev Drug Discov 2, 803-811.
[46] Xiao H, Gu Z, Wang G, Zhao T (2013) The possible mech-
anisms underlying the impairment of HIF-1alpha pathway
signaling in hyperglycemia and the beneficial effects of cer-
tain therapies. Int J Med Sci 10, 1412-1421.
[47] Huang YF, Yang CH, Huang CC, Tai MH, Hsu
KS (2010) Pharmacological and genetic accumulation
of hypoxia-inducible factor-1alpha enhances excitatory
synaptic transmission in hippocampal neurons through the
1458 S. Salomon-Zimri et al. / VEGF Treatment for ApoE4-Driven Pathology
production of vascular endothelial growth factor. J Neurosci
30, 6080-6093.
[48] Howell KR, Hoda MN, Pillai A (2013) VEGF activates
NR2B phosphorylation through Dab1 pathway. Neurosci
Lett 552, 30-34.
[49] Sharma V, Dixit D, Koul N, Mehta VS, Sen E (2011) Ras reg-
ulates interleukin-1beta-induced HIF-1alpha transcriptional
activity in glioblastoma. J Mol Med (Berl) 89, 123-136.
[50] Schanzer A, Wachs FP, Wilhelm D, Acker T, Cooper-Kuhn
C, Beck H, Winkler J, Aigner L, Plate KH, Kuhn HG (2004)
Direct stimulation of adult neural stem cells in vitro and
neurogenesis in vivo by vascular endothelial growth factor.
Brain Pathol 14, 237-248.
[51] Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D,
During MJ (2004) VEGF links hippocampal activity with
neurogenesis, learning and memory. Nat Genet 36, 827-835.
[52] Licht T, Goshen I, Avital A, Kreisel T, Zubedat S, Eavri R,
Segal M, Yirmiya R, Keshet E (2011) Reversible modula-
tions of neuronal plasticity by VEGF. Proc Natl Acad Sci
USA108, 5081-5086.
