Nox2-derived radicals contribute to neurovascular
and behavioral dysfunction in mice overexpressing
the amyloid precursor protein
Laibaik Park*, Ping Zhou*, Rose Pitstick†, Carmen Capone*, Josef Anrather*, Erin H. Norris‡, Linda Younkin§,
Steven Younkin§, George Carlson†, Bruce S. McEwen‡¶, and Costantino Iadecola*¶
*Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10065;‡Harold and
Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY 10065;§Mayo Clinic Jacksonville, Jacksonville, FL
32224; and†McLaughlin Research Institute, Great Falls, MT 59405
Contributed by Bruce S. McEwen, December 7, 2007 (sent for review October 23, 2007)
(AD), but their role in the amyloid deposition and cognitive impair-
ing the Swedish mutation of the amyloid precursor protein (Tg2576)
as a model of AD to examine the role of reactive oxygen species
produced by NADPH oxidase in the cerebrovascular alterations, amy-
loid deposition, and behavioral deficits observed in these mice. We
found that 12- to 15-month-old Tg2576 mice lacking the catalytic
subunit Nox2 of NADPH oxidase do not develop oxidative stress,
cerebrovascular dysfunction, or behavioral deficits. These improve-
ments occurred without reductions in brain amyloid-? peptide (A?)
levels or amyloid plaques. The findings unveil a previously unrecog-
nized role of Nox2-derived radicals in the behavioral deficits of
Tg2576 mice and provide a link between the neurovascular dysfunc-
tion and cognitive decline associated with amyloid pathology.
Alzheimer’s disease ? cerebral blood flow ? tg2576
in the elderly (1). A? peptides are cleaved from the amyloid
precursor protein (APP) by two aspartyl proteases, termed ?- and
?-secretases, and form deposits in the brain parenchyma (amyloid
plaques) and around blood vessels (amyloid angiopathy) (2). The
mechanisms by which A? leads to cognitive impairment have not
been completely elucidated, although recent evidence suggests that
synaptic function and inducing neuronal death (2).
However, A? also exerts powerful effects on cerebral blood
vessels (3). In vitro and in vivo studies have demonstrated that A?
enhances vasoconstriction, impairs responses to vasodilators, and
reduces cerebral blood flow (CBF) (4, 5). In addition, transgenic
mice overexpressing APP and A? have major alterations in resting
CBF and in key cerebrovascular control mechanisms (5–9). For
hyperemia), a response that matches the brain’s energy demands
with its blood supply, and the ability of cerebral endothelial cells to
(7, 10). The vasoconstriction induced by A? may underlie the
marked reductions in CBF observed in the early stages of AD (11).
The harmful cerebrovascular effects of A?, in concert with epide-
miological and pathological findings linking AD with cerebrovas-
cular diseases (12–16), have suggested that A? has deleterious
actions both on neurons and cerebral blood vessels, which may act
synergistically to induce brain dysfunction in AD (3, 17).
The cerebrovascular alterations observed in mice overexpressing
APP are associated with vascular oxidative stress and are counter-
acted by free radical scavengers (6, 18, 19), implicating reactive
oxygen species (ROS) in the dysfunction. A major source of ROS
in brain and blood vessels is the superoxide-producing enzyme
he amyloid-? peptide (A?) is central to the pathogenesis of
the catalytic subunit of NADPH oxidase, counteracts the oxidative
stress and the vascular dysfunction induced by A?, pointing to
NADPH oxidase as the source of the ROS (21). However, these
studies were performed in 3- to 4-month-old Tg2576 mice, an age
when amyloid plaques and behavioral deficits are not yet present
deficits associated with APP overexpression could not be assessed.
We used aged Tg2576 mice lacking Nox2 to determine whether
ROS derived from NADPH oxidase contribute to the cerebrovas-
cular dysfunction, amyloid deposition, and behavioral deficits in-
duced by APP overexpression. We found that genetic inactivation
of Nox2 reduces oxidative stress and rescues both the vascular and
behavioral alterations observed in 12- to 15-month-old Tg2576
mice. These improvements occurred in the absence of a reduction
in amyloid plaques. Thus, the cerebrovascular dysfunction induced
by Nox2-derived radicals may have a role in the neuronal dysfunc-
tion underlying the cognitive impairment in Tg2576 mice.
Nox2 Deletion Rescues the Cerebrovascular Dysfunction in Aged
Tg2576 Mice. We studied crosses between Tg2576 mice (22) and
mice deficient in the Nox2 catalytic subunit of NADPH oxidase
(24). First, we compared young (3- to 4-month-old) and aged (12-
amyloid deposition on the neurovascular dysfunction. We used a
in the whisker barrel cortex by mechanical stimulation of the facial
whiskers (functional hyperemia) (7). To study the ability of endo-
thelial cells to regulate CBF, we topically applied acetylcholine
(ACh), bradykinin, or the calcium ionophore A23187 to the
neocortex, a well established approach to test endothelium-
dependent relaxation of brain vessels (25). ACh increases CBF by
activating endothelial nitric oxide synthase via endothelial musca-
rinic receptors (26, 27), bradykinin acts through endothelial bra-
dykinin receptors and cyclooxygenase-1 products (28–30), and
A23187 increases CBF in a receptor independent manner via
cyclooxygenase-1 products (25, 30). Functional hyperemia, and the
increase in CBF produced by ACh or bradykinin were attenuated
by aging in WT mice, but not in Nox2-null mice (Fig. 1 A–C; P ?
0.05; analysis of variance; n ? 5 per group), attesting to the
Author contributions: L.P., P.Z., J.A., G.C., B.S.M., and C.I. designed research; L.P., R.P., C.C.,
L.Y., and S.Y. performed research; P.Z., J.A., E.H.N., L.Y., S.Y., G.C., and B.S.M. contributed
new reagents/analytic tools; L.P., S.Y., B.S.M., G.C., and C.I. analyzed data; and L.P., G.C.,
B.S.M., and C.I. wrote the paper.
The authors declare no conflict of interest.
¶To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
January 29, 2008 ?
vol. 105 ?
no. 4 ?
of aging (31). Aging did not attenuate the CBF response to A23187
in WT mice [supporting information (SI) Fig. 6A]. Functional
hyperemia and CBF responses to ACh and bradykinin were atten-
uated both in young and aged Tg2576 mice, the attenuation being
more marked in aged Tg2576 mice (Fig. 1 A–C; P ? 0.05; n ? 5 per
group). However, CBF responses were not reduced in young and
aged Tg2576 mice crossed with Nox2-null mice (Tg2576/Nox2?/?)
by hypercapnia or by the smooth muscle relaxant adenosine was
indicating that smooth muscle reactivity was intact. These obser-
vations establish that the cerebrovascular dysfunction induced by
APP overexpression is worse in aged Tg2576 mice but that inacti-
vation of Nox2 rescues the dysfunction in full.
we sought to determine whether the rescue of the cerebrovascular
alterations observed in aged Tg2576/Nox2?/?mice was related to
a reduction in oxidative stress. To this end, we examined ROS
production, using hydroethidine microfluorography (21) in WT,
Tg2576, and Tg2576/Nox2?/?mice (12–15 months of age). Cell
specific markers were used to identify the cell type producing ROS
(see Methods). In Tg2576 mice, the ROS signal was markedly
increased in neurons and endothelial cells (Fig. 2). Although
microglia and astrocytes were increased in the brain of Tg2576
mice, these cells were not a major source of ROS (SI Fig. 7). The
lack of ROS increase in astrocytes and microglia was not due to the
ROS detection technique, because superfusion with the protein
kinase C activator phorbol myristate acetate (PMA) was able to
increase ROS in these cells (SI Fig. 7B g–i and p–r). The increase
in ROS signal was blocked by the free radical scavenger manga-
nic(I-II)meso-tetrakis(4-benzoic acid) porphyrin (MnTBAP), at-
testing to the validity of the method (Fig. 2C). Superfusion with the
version, blocked the increase in ROS (Fig. 2C). Furthermore, the
increased ROS production was not observed in Tg2576/Nox2?/?
mice (Fig. 2C). Therefore, the rescue of neurovascular dysfunction
vascular and neuronal oxidative stress.
A Free Radical Scavenger or a Peptide Inhibitor of NADPH Oxidase
Ameliorates Neurovascular Dysfunction in Aged Tg2576 Mice.Inthese
studies, we examined whether the neurovascular dysfunction ob-
be ameliorated by agents that counteract oxidative stress. Super-
fusion of the neocortex with MnTBAP for 30 min did not affect
resting CBF (SI Fig. 8A; P ? 0.05; n ? 5 per group), but it
completely reversed the attenuation of the increase in CBF pro-
duced by whisker stimulation, ACh, bradykinin, and A23187 (Fig.
3 A–C and SI Fig. 8B; P ? 0.05 from young WT mice). MnTBAP
did not affect CBF responses to adenosine or hypercapnia (Fig. 3D
and SI Fig. 8C; P ? 0.05), attesting to the selectivity of the effect.
Then, we used the NADPH oxidase peptide inhibitor gp91ds-tat to
examine whether NADPH oxidase was the source of the ROS
mice. CBF responses to whisker stimulation (A) and topical neocortical appli-
cation of acetylcholine (10 ?M) (B), bradykinin (50 ?M) (C), or adenosine (400
?M) (D) in young and aged WT, Nox2?/?, Tg2576, or Tg2576/Nox2?/?mice.*,
P ? 0.05 from young WT; #, P ? 0.05 from young Tg2576; analysis of variance
and Tukey’s test; n ? 5 per group.
Nox2 inactivation rescues the cerebrovascular dysfunction in Tg2576
in 12–15-month-old Tg2576 mice. (A) Wild-type mice. Neurons immunola-
beled with NeuN (a) are colocalized with the ROS (HE) signal (b), as shown by
colocalized with the ROS signal (e and f). (Scale bar: 50 ?m.) (B) Tg2576 mice.
NeuN-positive neurons (a) are colocalized with the ROS signal (b and c).
CD31-positive endothelial cells (d) are also colocalized with the ROS signal (e
by the ROS scavenger MnTBAP and a NADPH peptide inhibitor (gp91-ds) but
from WT; analysis of variance and Tukey’s test; n ? 5 per group.
Nox2 inactivation attenuates neuronal and vascular oxidative stress
www.pnas.org?cgi?doi?10.1073?pnas.0711568105Park et al.
mediating the dysfunction. Superfusion with gp91ds-tat, but not its
scrambled control, reversed the attenuation in cerebrovascular
responses observed in Tg2576 mice (Fig. 3 E–H and SI Fig. 8 D–F;
P ? 0.05 from young WT mice). Therefore, the neurovascular
dysfunction in aged Tg2576 can be corrected by counteracting
Nox2 Deletion Does Not Affect Plaque Load or Brain A? in Aged
Tg2576 Mice. To determine whether the attenuation in ROS pro-
A? accumulation in the brain of aged Tg2576/Nox2?/?mice, we
measured brain A? in the Tg2576 and Tg2576/Nox2?/?mice used
levels did not differ between Tg2576 and Tg2576/Nox2?/?mice
(Fig. 4 A and B; P ? 0.05; n ? 12 per group). Similarly, plaque
number per square millimeter and the area occupied by plaques
(plaque load) in neocortex and hippocampus were comparable in
Tg2576 and Tg2576/Nox2?/?mice (Fig. 4 C–F; P ? 0.05; n ? 6 per
group). Amyloid angiopathy was occasionally observed in 12-
month-old Tg2576 mice, but was more frequent in older mice (SI
Fig. 9). Plaque load and brain A? levels did not differ between
Tg2576 and Tg2576/Nox2?/?mice even at 24 months of age when
plaques and brain A? levels were more abundant (SI Fig. 10).
Therefore, the rescue of the cerebrovascular dysfunction observed
in Tg2576/Nox2?/?mice occurs without a reduction in the amyloid
Nox2 Deletion Improves Behavioral Performance in Young and Aged
Tg2576 Mice. Finally, we used a two-trial spatial-memory task in a
Y maze (32, 33) to determine whether the reduced neurovascular
dysfunction and oxidative stress in Tg2576/Nox2?/?mice are asso-
ciated with improved cognitive performance. We chose this test
because it is less stressful and more consistent with the natural
water maze (33), and has been used in experiments involving
Tg2576 (34). Furthermore, the Y maze test does not involve
learning new rules and takes advantage of the natural tendency of
rodents to explore new environments (32). During their first trial
the third arm was blocked. During the second trial (retrieval)
performed 30 min later, access to the closed arm was allowed, so
mice were able to explore all three arms. Novel arm entries, time
spent in the novel arm, total arm visits, and amount of defecation
(an index of anxiety) were recorded. Although young Tg2576 mice
showed a preference for the novel arm comparable to that of WT
mice, aged Tg2576 mice visited the novel arm less often (Fig. 5A;
P ? 0.05, ?2test; n ? 10–15 per group). Furthermore, both young
and aged Tg2576 mice spent less time exploring the novel arm than
WT or Nox2?/?mice (Fig. 5B; P ? 0.05; analysis of variance). The
total number of arm visits and the amount of defecation were
increased in Tg2576 mice (Fig. 5 C and D; P ? 0.05), reflecting
increased locomotor activity and anxiety, respectively. These alter-
mice (34). However, in Tg2576/Nox2?/?mice, novel arm entries
and time spent in the novel arm were not reduced (Fig. 5 A and B;
P ? 0.05 from WT), whereas the increases in total arm visits and
are rescued by a 30-min neocortical application of MnTBAP (A–C) or gp91ds-tat (gp91ds) (E–G) but not by a scrambled peptide (sgp91) (E–G). Responses to
adenosine are not attenuated in Tg2576 mice (D and H).*, P ? 0.05 from vehicle; analysis of variance and Tukey’s test; n ? 5 per group. V, vehicle.
Short-term application of ROS scavengers counteracts the cerebrovascular dysfunction in 12- to 15-month-old Tg2576 mice. Cerebrovascular responses
Tg2576 mice lacking Nox2. (A and B) SDS-soluble (A) and -insoluble (B) A? are
not different in Tg2576 and Tg2576/Nox2?/?mice (P ? 0.05; analysis of
variance and Tukey’s test; n ? 12 per group). (C and D) The amyloid load is
comparable between Tg2576 (C) and Tg2576/Nox2?/?mice (D). (E and F) The
number of plaques (E) and plaque load (F) do not differ between Tg2576 and
Tg2576/Nox2?/?mice (P ? 0.05; n ? 6 per group).
Park et al.
January 29, 2008 ?
vol. 105 ?
no. 4 ?
defecation were attenuated (Fig. 5 C and D; P ? 0.05 from WT).
Therefore, Tg2576 mice lacking Nox2 are protected from the
behavioral dysfunction induced by APP overexpression.
We have demonstrated that Tg2576 mice have an age-dependent
dysfunction in the mechanisms regulating CBF associated with
oxidative stress and behavioral deficits. Oxidative stress, neurovas-
cular dysfunction, and behavioral deficits are not present in aged
Tg2576 mice lacking Nox2, implicating Nox2-derived ROS in the
mechanisms of the dysfunction. The improvement in CBF regula-
tion and behavior conferred by Nox2 inactivation occurs without
the cerebrovascular dysfunction in aged Tg2576 mice can be
counteracted by short-term treatment with a ROS scavenger or a
peptide inhibitor of NADPH oxidase. These observations unveil a
behavioral deficits induced by APP overexpression and A?.
Studies have demonstrated that APP overexpressing mice have
structural and functional abnormalities in cerebral blood vessels,
studies were conducted in mice at an age when the amyloid
deposition and behavioral deficits were not present (6). Although
these investigations provided evidence that A?-induced vascular
dysfunction is an early pathogenic event and is mediated by
Nox2-derived ROS (21), they were unable to provide insight into
the relationships among neurovascular dysfunction, amyloid dep-
osition, and behavioral deficits. Other investigators examined the
effect of APP overexpression on the morphology of cerebral
arteries and cerebral blood volume in mice at an age when amyloid
deposition was thought to be present (35, 36). However, these
investigations did not attempt to determine the mechanisms of the
effect and its impact on the amyloid deposition and behavioral
deficits. Therefore, the evidence provided here establishes a link
between Nox2-dependent oxidative stress, neurovascular dysfunc-
tion, and behavioral deficits in Tg2576 mice.
The vascular responses to whisker stimulation, ACh, and brady-
kinin were attenuated more in aged than in young Tg2576 mice.
This effect cannot result from a generalized vasoparalysis, because
the CBF responses to adenosine were not attenuated in aged
Tg2576 mice, suggesting that smooth muscle function was pre-
served. Considering that aging attenuates CBF responses also in
WT mice via Nox2-derived ROS (31), it is more likely that aging
and the associated increase in brain A? levels acted in concert to
enhance oxidative stress and the vascular dysfunction. The obser-
vation that vascular responses are normalized in Tg2576 mice
lacking Nox2 suggests that Nox2-dependent ROS production is the
final common pathway for the vascular alterations induced both by
aging and A?. However, a contribution from the alterations in
smooth muscle contractility induced by myocardin in Tg2576 mice
cannot be ruled out (37).
One of the key findings of the present study is that restoration of
improvement of the behavioral deficits. Studies have reported that
attenuation of oxidative or nitrosative stress ameliorates the cog-
nitive impairment in mice overexpressing APP, but the improve-
ment was usually associated with a reduction in amyloid plaques
(38–40). However, in the present study, the behavioral improve-
ment occurred without reductions in brain A? levels or amyloid
burden. Thus, amyloid deposition can be dissociated from the
behavioral deficits. This finding has two major implications. First,
plaques do not predict the brain dysfunction underlying the cog-
nitive deficits (41). Increasing evidence suggests that smaller A?
aggregates are potent mediators of synaptotoxicity and neuronal
death and best correlate with behavioral deficits (2). Second, this
finding suggests that Nox2-derived radicals, although contributing
APP overexpression, do not have a role in A? processing and
amyloid plaque formation. In contrast, enhancing mitochondrial
ROS production by inactivation of one allele of mitochondrial
superoxide dismutase increases the amyloid burden in mice over-
expressing human APP (42, 43). Conversely, the free radical
scavenger curcumin reduced amyloid load in mice overexpressing
APP (40, 44). Thus, not all sources of ROS contribute equally to
plaque formation. These observations, collectively, suggest a pre-
viously unrecognized specificity in the sources of ROS promoting
A? deposition. However, our findings do not provide insights into
the roles of Nox2-derived ROS in the amyloid angiopathy and its
contribution to the cerebrovascular and behavioral dysfunction
observed in Tg2576 mice.
An important question concerns whether there is a causal link
between the rescue of vascular function and the enhancement in
behavioral performance observed in Tg2576 mice lacking Nox2.
Although Nox2 inactivation reduces neuronal oxidative stress,
which may improve neuronal function and behavior, our data
suggest that vascular responses mediated by direct actions on
cerebral blood vessels also are rescued. Thus, the restoration of
responses to the endothelium-dependent vasodilators ACh, brady-
kinin, and A23187 cannot be attributed to an improvement in
neuronal function because these responses are independent of
neurons (25). Inasmuch as vascular dysregulation limits the supply
Nox2 inactivation should ameliorate neuronal function, which is
compromised by oxidative stress. Therefore, it is likely that im-
improvement of behavioral performance.
We have also observed that short-term administration of a free
radical scavenger or of a NADPH oxidase peptide inhibitor coun-
teracts oxidative stress and neurovascular dysfunction in aged
Tg2576 mice. This finding suggests that the vascular dysfunction is
reversible, even in aged mice with long-lasting oxidative stress,
amyloid deposition, and behavioral deficits. Therefore, vascular
oxidative stress does not produce permanent vascular damage, but
it induces a dysfunctional state that can be rescued by transient
antioxidant treatment. Treatment with agents that scavenge ROS
have been reported to ameliorate behavioral deficits in Tg2576
mice (40). Our data suggest that improvement of vascular function
may have a role in the improvement in cognition as well.
tested in a Y maze. The reduction in novel arm entry (A), time spent in the
novel arm (B), and the increase in total arm visits (C) and defecation (D)
observed in Tg2576 mice are not seen in Tg2576/Nox2?/?mice.*, P ? 0.05, ?2
Nox2 inactivation rescues the behavioral deficits in Tg2576 mice
www.pnas.org?cgi?doi?10.1073?pnas.0711568105 Park et al.
In conclusion, we have demonstrated that Nox2 inactivation
reduces vascular and neuronal oxidative stress, improves the neu-
rovascular dysfunction, and ameliorates the behavioral deficits in
aged Tg2576 mice. These improvements are independent of reduc-
tions of brain A? levels and amyloid burden. Furthermore, tran-
sient antioxidant treatment ameliorates the vascular dysfunction,
indicating that the deleterious effects of oxidative stress on brain
vessels are not permanent and are amenable to reversal by thera-
peutic interventions. The findings demonstrate that Nox2 derived
ROS are involved both in the vascular dysfunction induced by APP
overexpression and in the associated behavioral decline. Vascular
and behavioral improvements take place without a reduction in
brain A? load and amyloid plaques, indicating that Nox2-derived
ROS are not involved in the mechanisms of plaque formation, and
suggesting an unforeseen specificity in the ROS sources involved in
A? processing and deposition.
Mice. All procedures were approved by the Institutional Animal Care and Use
in male mice of ages ranging from 3 to 15 months. Methods for crossing APP
mice with Nox2?/?mice were identical to those described in ref. 21. Briefly,
breeder pairs of B6.129-Cybbtm1Din/J mice lacking the Nox2 subunit of NADPH
Swedish mutant (K670N M671I) human APP695driven by hamster prion pro-
tein promoter (22). The Tg2576 transgene array was maintained on a mixed
genotyping the APP transgene array as described in ref. 7. To produce Tg2576
mice with or without Nox2, Nox2?/?females were crossed to Tg2576 males;
because the gene encoding Nox2 (Cybb) is on the X chromosome, male
offspring do not express Nox2. To produce Tg2576-positive or -negative mice
expressing Nox2, C57BL/6J females were crossed to Tg2576 males; only male
offspring were used. CBF results did not differ between the offspring of
B6/SJL-Tg2576 or 129S6.Tg2576 males.
General Surgical Procedures. Mice were anesthetized with isoflurane (main-
tenance 2%), intubated, and artificially ventilated (SAR-830; CWE) (6, 7, 21).
The femoral vessels were cannulated for recording of arterial pressure and
blood sample collection. Rectal temperature was maintained at 37°C. After
surgery, anesthesia was maintained with urethane (750 mg/kg; i.p.) and
chloralose (50 mg/kg; i.p.) (6, 7, 21).
CBF and Cerebrovascular Reactivity. A craniotomy (2 ? 2 mm) was performed
to expose the somatosensory cortex, the dura was removed, and the site was
superfused with a modified Ringer solution (37°C; pH 7.3–7.4) (6, 7, 21). CBF
was monitored at the site of superfusion with a laser–Doppler probe (Vasa-
medic) positioned stereotaxically on the brain surface and connected to a
computer. CBF was expressed as percentage increase relative to the resting
Detection of Reactive Oxygen Species. ROS production was assessed by using
hydroethidine (HE) as a marker (21). HE (2 ?M; Molecular Probes) was super-
cut through cortex underlying the cranial window, using a cryostat; collected
at 100-?m intervals; and examined under a fluorescence microscope (Nikon).
per animal) were expressed as relative fluorescence units (RFU) (21).
Immunocytochemistry and Confocal Microscopy. Mice were anesthetized with
5% isoflurane and perfused transcardially with 4% paraformaldehyde (PFA).
?m thick). To identify HE-positive cells, sections were processed for immuno-
histochemistry with NeuN (1:100; Sigma), GFAP (1:1,000; Sigma), CD31 (1:100;
BD Biosciences), or CD11b (1:100; BD Parmingen). Sections were then incu-
bated with cyanine dye (Cy5)-conjugated goat anti-mouse IgG (for NeuN,
GFAP, and CD11b; Jackson ImmunoResearch) and goat anti-rat IgG (for CD31;
Jackson ImmunoResearch) secondary antibodies. The specificity of the label-
ing was established by omitting the primary antibody or by preabsorption
with the antigen. The images were sequentially acquired by using a confocal
laser-scanning microscope (Leica).
Measurement of A?. A? was measured by using an ELISA-based assay, as de-
scribed in refs. 6, 7, and 21. Briefly, the left hemispheres from the mice used for
CBF studies were sonicated in 1% SDS with protease inhibitors and centrifuged.
70% formic acid and centrifuged as above. The formic acid extract was neutral-
concentrations (picomoles per gram of brain tissue) were determined in super-
natant (SDS-soluble) and the formic acid extract of the pellet (SDS-insoluble),
using the BAN-50/BA-27 and BAN-50/BC005 sandwich ELISA as described
(6, 7, 21).
Determination of A? Load. A? load was determined as described in ref. 45.
studies (n ? 5 per group) was postfixed in 4% PFA. Coronal sections (20 ?M
thick) were cut in a cryostat at 400-?m intervals from a region located 1.94 to
window. Ten sets of 13–14 sections were collected. One of the sets was
randomly selected for immunocytochemistry, using an A? antibody (4G8;
Sigma). Grayscale images (magnification: ?1) of the cortex and hippocampus
were digitized with a camera (Q Imaging; Barnaby). The number of plaques
The A? load was determined in a blinded manner from the area occupied by
Institutes of Health Image software.
Behavioral Assessment by the Y Maze. Behavioral assessment was performed
by using the Y maze as described in ref. 33. Briefly, the Y maze consisted of
three identical arms made of transparent plastic joined in the middle to form
a ‘‘Y’’ (20 cm high, 10 cm wide, and 30 cm long). The mice were handled daily
and allowed to acclimate to the apparatus for a week before testing. Mice
were placed into one of the arms of the maze (start arm) and allowed to
explore only two of the arms for 5 min (training trial). The third arm, which
remained closed, was randomly chosen in each trial. The closed arm was
opened in the test trial, serving as the novel arm. After a 30-min intertrial
interval, the mice were returned to the same start arm and were allowed to
explore all three arms for 5 min (test trial). Sessions were video recorded and
replayed for determination of the parameters of interest by an observer
blinded to the genotype of the mice.
Experimental Protocol. CBF recordings were started after arterial pressure and
blood gases were in a steady state (SI Tables 1–3).
Functional hyperemia and CBF responses to ACh, bradykinin, A23187, and adenosine.
For testing functional hyperemia, the increase in CBF produced by gently
ACh (10 ?M; Sigma), bradykinin (50 ?M; Sigma), or the calcium ionophore
A23187 (3 ?M; Sigma) was topically superfused for 3–5 min, and the evoked
CBF increase was recorded. The CBF response to adenosine (400 ?M; Sigma)
was also tested. The increase in CBF induced by hypercapnia (pCO2to 50–60
mmHg) was examined by introducing 5% CO2in the circuit of ventilator.
Effect of MnTBAP. Responses to functional hyperemia, ACh, bradykinin,
A23187, adenosine, and hypercapnia were tested before and after superfu-
sion of the cranial window with the ROS scavenger MnTBAP for 30 min (100
?M; Porphyrin Products).
Effect of gp91ds-tat. As described in ref. 21, the NADPH oxidase assembly
peptide inhibitor gp91ds-tat (YGRKKRRQRRRCSTRIRRQL-NH2) and its scram-
bled control (sgp91ds-tat) (YGRKKRRQRRRCLRITRQSR-NH2) (Bio?Synthesis)
min after superfusion of gp91ds-tat (1 ?M) or sgp91ds-tat (1 ?M).
Effects of MnTBAP, gp91ds-tat, and PMA on ROS production. The cranial window
was superfused with Ringer’s solution, containing HE (2 ?M) alone (vehicle)
for 60 min or for 30 min followed by HE plus MnTBAP (100 ?M), gp91ds-tat (1
?M), or scrambled control peptide (1 ?M) for another 30 min. In aged Tg2576
mice, the effect of PMA (5 ?M; Sigma) was assessed to test the ability of
astrocytes or microglia to generate ROS. At the end of superfusion, brains
were removed and processed for ROS assessment.
Data Analysis. Data are expressed as means ? SEM. Two-group comparisons
were analyzed by the two-tailed t test. Multiple comparisons were evaluated
by the analysis of variance and Tukey’s test or the Newman–Keul test. Differ-
ences in novel arm entries were analyzed by the ?2test (X2). Differences were
considered statistically significant for P ? 0.05.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grant NS37853.
Park et al.
January 29, 2008 ?
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no. 4 ?
1. Kelley BJ, Petersen RC (2007) Alzheimer’s disease and mild cognitive impairment. Download full-text
Neurol Clin 25:577–609.
2. Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons
from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8:101–112.
3. Iadecola C (2004) Neurovascular regulation in the normal brain and in Alzheimer’s
disease. Nat Rev Neurosci 5:347–360.
4. Thomas T, Thomas G, McLendon C, Sutton T, Mullan M (1996) ?-Amyloid-mediated
vasoactivity and vascular endothelial damage. Nature 380:168–171.
Physiol Heart Circ Physiol 281:H2417–H2424.
6. Iadecola C, et al. (1999) SOD1 rescues cerebral endothelial dysfunction in mice over-
expressing amyloid precursor protein. Nat Neurosci 2:157–161.
7. Niwa K, et al. (2000) A? 1–40-related reduction in functional hyperemia in mouse
neocortex during somatosensory activation. Proc Natl Acad Sci USA 97:9735–9740.
8. Niwa K, et al. (2002) Cerebrovascular autoregulation is profoundly impaired in mice
overexpressing amyloid precursor protein. Am J Physiol Heart Circ Physiol 283:H315–
9. Niwa K, Kazama K, Younkin SG, Carlson GA, Iadecola C (2002) Alterations in Cerebral
Blood Flow and Glucose Utilization in Mice Overexpressing the Amyloid Precursor
Protein. Neurobiol Dis 9:61–68.
10. Takano T, Han X, Deane R, Zlokovic B, Nedergaard M (2007) Two-photon imaging of
astrocytic Ca2?signaling and the microvasculature in experimental mice models of
Alzheimer’s disease. Ann NY Acad Sci 1097:40–50.
11. Johnson KA, Albert MS (2000) Perfusion abnormalities in prodromal AD. Neurobiol
12. Beach TG, et al. (2007) Circle of Willis atherosclerosis: association with Alzheimer’s
disease, neuritic plaques and neurofibrillary tangles. Acta Neuropathol (Berlin)
13. Snowdon DA, et al. (1997) Brain infarction and the clinical expression of Alzheimer
disease. The Nun Study. J Am Med Assoc 277:813–817.
14. Skoog I, Gustafson D (2002) Hypertension and related factors in the etiology of
Alzheimer’s disease. Ann NY Acad Sci 977:29–36.
15. Ruitenberg A, et al. (2005) Cerebral hypoperfusion and clinical onset of dementia: The
Rotterdam Study. Ann Neurol 57:789–794.
16. Silvestrini M, et al. (2006) Cerebrovascular reactivity and cognitive decline in patients
with Alzheimer disease. Stroke 37:1010–1015.
17. Zlokovic BV (2005) Neurovascular mechanisms of Alzheimer’s neurodegeneration.
Trends Neurosci 28:202–208.
18. Park L, et al. (2004) A?-induced vascular oxidative stress and attenuation of functional
hyperemia in mouse somatosensory cortex. J Cereb Blood Flow Metab 24:334–342.
19. Tong XK, Nicolakakis N, Kocharyan A, Hamel E (2005) Vascular remodeling versus
amyloid beta-induced oxidative stress in the cerebrovascular dysfunctions associated
with Alzheimer’s disease. J Neurosci 25:11165–11174.
20. Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases:
Physiology and pathophysiology. Physiol Rev 87:245–313.
21. Park L, et al. (2005) NADPH oxidase-derived reactive oxygen species mediate the
cerebrovascular dysfunction induced by the amyloid ? peptide. J Neurosci 25:1769–
22. Hsiao K, et al. (1996) Correlative memory deficits, A? elevation, and amyloid plaques
in transgenic mice. Science 274:99–102.
23. Kawarabayashi T, et al. (2001) Age-dependent changes in brain CSF, plasma amyloid
24. Pollock JD, et al. (1995) Mouse model of X-linked chronic granulomatous disease, an
inherited defect in phagocyte superoxide production. Nat Genet 9:202–209.
25. Faraci FM, Heistad DD (1998) Regulation of the cerebral circulation: Role of endothe-
lium and potassium channels. Physiol Rev 78:53–97.
26. Rosenblum WI, McDonald M, Wormley B (1989) Calcium ionophore and acetylcholine
dilate arterioles on the mouse brain by different mechanisms. Stroke 20:1391–1395.
27. Sobey CG, Faraci FM (1997) Effects of a novel inhibitor of guanylyl cyclase on dilator
responses of mouse cerebral arterioles. Stroke 28:837–843.
28. Sobey CG, Heistad DD, Faraci FM (1997) Mechanisms of bradykinin-induced cerebral
vasodilatation in rats. Evidence that reactive oxygen species activate K? channels.
29. Mayhan WG (1996) Role of activation of bradykinin B2 receptors in disruption of the
blood–brain barrier during acute hypertension. Brain Res 738:337–341.
30. Niwa K, Haensel C, Ross ME, Iadecola C (2001) Cyclooxygenase-1 participates in selected
vasodilator responses of the cerebral circulation. Circ Res 88:600–608.
species mediate neurovascular dysregulation in the aging mouse brain. J Cereb Blood
Flow Metab 27:1908–1918.
to novelty and spatial memory using a two-trial recognition task in mice. Neurobiol
Learn Mem 73:31–48.
33. Sarnyai Z, et al. (2000) Impaired hippocampal-dependent learning and functional
abnormalities in the hippocampus in mice lacking serotonin(1A) receptors. Proc Natl
Acad Sci USA 97:14731–14736.
34. King DL, Arendash GW (2002) Behavioral characterization of the Tg2576 transgenic
model of Alzheimer’s disease through 19 months. Physiol Behav 75:627–642.
35. Beckmann N, et al. (2003) Age-dependent cerebrovascular abnormalities and blood
flow disturbances in APP23 mice modeling Alzheimer’s disease. J Neurosci 23:8453–
amyloid precursor protein 23 transgenic mouse model of Alzheimer’s disease. J Neu-
37. Chow N, et al. (2007) Serum response factor and myocardin mediate arterial hyper-
contractility and cerebral blood flow dysregulation in Alzheimer’s phenotype. Proc
Natl Acad Sci USA 104:823–828.
ablation of inducible nitric oxide synthase. J Exp Med 202:1163–1169.
39. Liang X, et al. (2005) Deletion of the prostaglandin E2 EP2 receptor reduces oxidative
damage and amyloid burden in a model of Alzheimer’s disease. J Neurosci 25:10180–
40. Cole GM, et al. (2004) NSAID and antioxidant prevention of Alzheimer’s disease: Lessons
from in vitro and animal models. Ann N Y Acad Sci 1035:68–84.
41. Holcomb LA, et al. (1999) Behavioral changes in transgenic mice expressing both
amyloid precursor protein and presenilin-1 mutations: Lack of association with amy-
loid deposits. Behav Genet 29:177–185.
42. Esposito L, et al. (2006) Reduction in mitochondrial superoxide dismutase modulates
Alzheimer’s disease-like pathology and accelerates the onset of behavioral changes in
human amyloid precursor protein transgenic mice. J Neurosci 26:5167–5179.
43. Li F, et al. (2004) Increased plaque burden in brains of APP mutant MnSOD heterozy-
gous knockout mice. J Neurochem 89:1308–1312.
44. Lim GP, et al. (2001) The curry spice curcumin reduces oxidative damage and amyloid
pathology in an Alzheimer transgenic mouse. J Neurosci 21:8370–8377.
amyloid deposits and its relationship with cognitive status in aging and Alzheimer’s
disease. Neuroscience 112:75–91.
46. Rey FE, Cifuentes ME, Kiarash A, Quinn MT, Pagano PJ (2001) Novel competitive
inhibitor of NAD (P)H oxidase assembly attenuates vascular O (2)(-) and systolic blood
pressure in mice. Circ Res 89:408–414.
www.pnas.org?cgi?doi?10.1073?pnas.0711568105Park et al.