Departamentosde1Farmacologiaand2Cie ˆnciasFisiolo ´gicas,CentrodeCie ˆnciasBiolo ´gicas,and3DepartamentodeAnatomiaPatolo ´gica,Hospital
Universita ´rio–UniversidadeFederaldeSantaCatarina,88049-900,Floriano ´polis,SantaCatarina,Brazil,and4FaculdadedeOdontologia,Pontifı ´cia
Increased brain deposition of amyloid ? protein (A?) and cognitive deficits are classical signals of Alzheimer’s disease (AD) that have
been highly associated with inflammatory alterations. The present work was designed to determine the correlation between tumor
means of both in vivo and in vitro approaches. The intracerebroventricular injection of A?1–40in mice resulted in marked deficits of
dysfunction and glial cell activation. The pharmacological blockage of either TNF-? or iNOS reduced the cognitive deficit evoked by
in TNF-? expression and oxidative alterations in prefrontal cortex and hippocampus. Likewise, A?1–40led to activation of both JNK
(c-Jun-NH2-terminal kinase)/c-Jun and nuclear factor-?B, resulting in iNOS upregulation in both brain structures. The anti-TNF-?
antibody reduced all of the molecular and biochemical alterations promoted by A?1–40. These results provide new insights in mouse
Alzheimer’s disease (AD) is a progressive neurodegenerative dis-
order associated with memory loss, spatial disorientation, and
brain have revealed the presence of classical hallmarks including
neurofibrillary tangles and senile plaques. The deposition of
tions is assumed to initiate a pathological cascade that results in
synaptic dysfunction, synaptic loss, and neuronal death (Walsh
and Selkoe, 2004).
which is characterized by astrogliosis, microgliosis, cytokine ele-
vation, and changes in acute phase proteins (Walsh and Selkoe,
2004; Wyss-Coray, 2006). Tumor necrosis factor-? (TNF-?) is a
cytokine thought to play a central role in the self-propagation of
neuroinflammation (Perry et al., 2001). TNF-? regulates many
cellular processes, including inflammation, differentiation, and
cell death through activation of TNF receptor 1 (TNFR1) or
TNFR2 (Wajant et al., 2003). The transduction pathways acti-
vated by TNF-? include mitogen-activated protein kinases
transcriptional factors such as activator protein-1 (AP-1) and
nuclear factor-?B (NF-?B) (Wajant et al., 2003). Regarding the
CNS, microglia and astrocytes are believed to be the primary
sources of TNF-?. Evidence indicates the presence of increased
levels of TNF-? in the brain and plasma of AD patients and an
et al., 1991; Li et al., 2004). In addition, TNF-? has been impli-
cated recently as a critical mediator of long-term potentiation
(LTP) reduction by A? (Wang et al., 2005). However, the mech-
anisms through which TNF-? promotes its pathological actions
in AD are poorly understood.
nitive impairment observed in AD. Inducible nitric oxide syn-
ThisworkwassupportedbygrantsfromtheConselhoNacionaldeDesenvolvimentoCientı ´ficoeTecnolo ´gico,the
Coordenaca ˜odeAperfeic ¸oamentodePessoaldeNı ´velSuperior,theProgramadeApoioaosNu ´cleosdeExcele ˆncia,
andtheFundac ¸a ˜odeApoioaPesquisadoEstadodeSantaCatarina,allfromBrazil.
eraldeSantaCatarina,CampusUniversita ´rio,Trindade,BlocoD,CaixaPostal476,88049-900,Floriano ´polis,Santa
5394 • TheJournalofNeuroscience,May16,2007 • 27(20):5394–5404
thase (iNOS) generates nitric oxide (NO) and NO-derived reac-
tive nitrogen species such as peroxynitrite. Accumulation of
trosylation, DNA oxidative damage, and neuronal disruption,
al., 2001). Interestingly, these events seem to be modulated by
ner with cytokines to induce neuronal damage via reactive oxy-
gen species (ROS)- and NO-dependent pathways (Goodwin et
al., 1995; Meda et al., 1995).
Numerous animal models have been used to evaluate the role
mimics the progression of AD was developed using an intracere-
broventricular injection of A? in mice (for review, see Van Dam
and De Deyn, 2006). Using this model, we assessed the role of
TNF-? and iNOS in A?-induced early impairment of learning
and memory. Our data indicate that TNF-? production is one of
the earliest events induced by A?1–40, representing an important
signal for iNOS expression. The cross talk between TNF-? and
iNOS is probably mediated by activation of two major intracel-
?B. The present results implicate TNF-? and iNOS as important
mediators of A?-induced cognitive impairment.
Subjects. Experiments were conducted using male Swiss, C57BL/6,
TNFR1 knock-out, and iNOS knock-out mice (20–30 g) kept in a con-
trolled room temperature (22 ? 2°C) and humidity (60–80%) under a
12 h light/dark cycle (lights on 6:00 A.M.). TNFR1 (Rothe et al., 1993)
the present study followed the “Principles of Laboratory Animal Care”
were approved by the Animal Ethics Committee of the Universidade
Federal de Santa Catarina.
Drug treatment protocol. Human A?1–40(Tocris, Ellisville, MO) and
A?40–1(Bachem, Torrance, CA) were dissolved in sterile PBS, pH 7.4, at
1 mg/ml and were incubated at 37°C for 4 d, as described previously (El
Khoury et al., 1996). Control mice received sterile PBS (vehicle). The
aggregated form of amyloid fragments (400 pmol per mice) and vehicle
ously (Laursen and Belknap, 1986). As a positive control for molecular
studies, some animals received an intracerebroventricular injection of
2.5 ?g of lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:
B4; Sigma-Aldrich, Sao Paulo, Brazil). Briefly, each mouse was given an
injection at bregma with a 5 ?l Hamilton microsyringe fitted with a 26
gauge needle that was inserted to a depth of 2.4 mm. The injection vol-
ume was 3 ?l. Mice exhibited normal behavior within 1 min after injec-
tion. Accurate placement of the injection or needle track was verified at
previous literature data (Yan et al., 2001).
Some animals were treated with anti-TNF-? antibody (AbTNF-?; 10
?g, i.c.v., per mouse; R & D Systems, Minneapolis, MN) 15 min before
A?1–40injection. The AbTNF-? dose was determined in pilot experi-
ments (results not shown). The iNOS inhibitor aminoguanidine (AG;
100 mg/kg, i.p.; Sigma-Aldrich) was administered 1 h before an intrace-
rebroventricular injection of A?1–40and throughout consecutive days
until the day of the experiment. In addition, some animals were pre-
treated with the selective inhibitor of JNK, SP600126 (anthra[1-9-
cd]pyrazol-6(2H)-one; 50 mg/kg; i.p.; 1 h before; Tocris), or the NF-?B
blocker pyrrolidine dithiocarbamate (PDTC; 100 mg/kg, i.p., 1 h before;
performed as described previously (Morris et al., 1982). The experimen-
tal apparatus consisted of a circular water tank (diameter, 97 cm; height,
60 cm) containing water at 23 ? 2°C. The target platform (10 ? 10 cm)
was submerged 1 cm below the water surface and placed at the midpoint
from the center and the wall of the pool. The pool was located in a test
ously (Prediger et al., 2007). The acquisition training session was per-
formed 7 d after A?1–40injection and consisted of 10 consecutive trials
to swim freely to the escape platform. If an animal did not find the
allowed to remain on the platform for 10 s after escaping to it, and it was
then removed from the tank for 20 s before being placed at the next
was performed 24 h after the training session (on day 8 after injection).
removed from the pool and each mouse was allowed to swim for 60 s in
was located on the training session) was recorded, and the percentage of
the total time was calculated.
activity, the animals were tested in the open-field paradigm. The appa-
ratus, made of wood covered with impermeable Formica, had a black
floor of 30 ? 30 cm (divided by white lines into nine squares of 10 ? 10
cm) and transparent walls, 15 cm high. Each mouse was placed in the
four paws and the rearing behavior were registered for 5 min.
Histology. Eight days after intracerebroventricular injection of aggre-
gated A?1–40, mice were perfused transcardially with PBS solution con-
taining 4% paraformaldeyde (w/v). Brain was removed and kept over-
the lateral ventricle areas. Sections were stained with 0.5% cresyl violet
reagent (Sigma-Aldrich) according to standard procedures. Stained cells
CA3 subfields and cortical layer with a microscope (Eclipse 50I; Nikon,
Melville, NY) using a counting grid at 400? magnification. The light-
microscopic characteristics of the A?1–40aggregates were studied using
the alkaline Congo red technique according to standard procedures.
Immunohistochemistry. Immunohistochemistry detection of apopto-
tic cell death, immunoreactivity of the astrocyte marker glial fibrillary
acidic protein (GFAP), and synaptic changes were assessed on paraffin
tissue sections 1 and 8 d after A?1–40intracerebroventricular injection,
using the polyclonal rabbit anti-caspase-3 (1:200; Cell Signaling Tech-
nology, Beverly, MA), monoclonal mouse anti-GFAP (1:300; Dako Cy-
tigen retrieval was performed by immersion of the slides in a water bath
at 95–98°C in 10 mM trisodium citrate buffer, pH 6.0, for 45 min. The
normal serum diluted in PBS. After overnight incubation at 4°C with
primary antibodies, the slides were washed with PBS and incubated with
the secondary antibody Envision plus (Dako Cytomation), ready to use,
for 1 h at room temperature. The sections were washed in PBS, and the
visualization was completed by using 3,3?-diaminobenzidine (Dako Cy-
tomation) in chromogen solution and counterstained lightly with Har-
50i; Nikon) and digital sight camera (DS-5M-L1; Nikon). Control and
experimental tissues were placed on the same slide and processed under
the same conditions. Settings for image acquisition were identical for
control and experimental tissues. For each mouse, we obtained four
section) of the hippocampal CA1, CA2, and CA3 subregions. Digitized,
sity of synaptophysisn staining was calculated for each image using NIH
ImageJ 1.36b imaging software (NIH, Bethesda, MD). For each mouse,
were averaged. This approach for the assessment of synaptic degenera-
tion has been validated in various experimental models of neurodegen-
Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficitJ.Neurosci.,May16,2007 • 27(20):5394–5404 • 5395
determined on visual inspection in the same
brain areas using a counting grid at 400?
Enzymatic studies. Mice were anesthetized
with pentobarbital (50 mg/kg, i.p.) and per-
fused transcardially with ice-cold 0.9% NaCl
(10 ml/10 g body weight) to remove the free
sources in the brain. The prefrontal cortex and
HEPES, pH 7.0, and centrifuged at 20,000 ? g
for 30 min at 4°C. The enzyme activities were
determined in the supernatant in a Varian
(Palo Alto, CA) Cary 50 spectrophotometer.
Glutathione reductase (GR) and glutathione
peroxidase (GPx) activity was determined as
described previously (Prediger et al., 2007).
Briefly, GR reduces oxidized glutathione
(GSSG) to glutathione (GSH) in expending
NADPH, the disappearance of which can be
sured indirectly by the NADPH consumption at 340 nm. The GPx uses
GSH to reduce the tert-butylhydroperoxide-producing GSSG, which is
readily reduced to GSH by excess GR, consuming NADPH. The disap-
pearance of NADPH in this reaction reflects the GPx activity. The en-
pus were isolated and homogenized in cooled 0.5 M perchloric acid. The
total of reduced (GSH) and oxidized (GSSG) forms, was determined by
the GR–5?,5?-dithio-bis(2-nitrobenzoic acid) recycling assay as de-
scribed previously (Prediger et al., 2007).
1 mM phenylmethylsulphonyl fluoride, 5 ?g/ml leupeptin, 5 ?g/ml pep-
statin A, 10 ?g/ml aprotinin, 1 mM sodium orthovanadate, 10 mM
?-glycerophosphate, 50 mM sodium fluoride, and 0.5 mM dithiothreitol
(all from Sigma-Aldrich). The homogenates were chilled on ice for 15
min and vigorously shaken for 15 min in the presence of 0.1% Triton
X-100. The nuclear fraction was precipitated by centrifugation at
?l of high-salt extraction buffer (20 mM HEPES, pH 7.4, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 25% v/v glycerol, 1 mM phenylmethylsul-
tinin, and 0.5 mM dithiothreitol) and incubated under continuous shak-
at 10,000 ? g, and the supernatant was aliquoted and stored at ?70°C.
Protein concentration was determined using the Bio-Rad protein assay
Western blot analysis. Equal protein amounts were separated on an
mobilon P; Millipore, Bedford, MA). The membranes were saturated by
incubation with 10% nonfat dry milk solution and incubated overnight
with one of the following antibodies: p65 NF-?B (sc-372, 1:1000), c-jun
(sc-45, 1:1000), iNOS (sc-7271, 1:200), ?-actin (sc-1615, 1:2000), JNK1
(sc-571, 1:1000), phosphorylated JNK (sc-6254, 1:1000) (all from Santa
Cruz Biotechnology, Santa Cruz, CA), or lamin A/C (catalog #2032; Cell
Signaling Technology). After washing, the membranes were incubated
alkaline phosphatase. The immunocomplexes were visualized using the
ECL chemiluminescence detection system (GE Healthcare, Sao Paulo,
color development substrate (Promega, Madison, WI). Band density
measurements were made using the Scion (Frederick, MD) Image soft-
RNA preparation and reverse transcription-PCR. Total RNA was ex-
tracted from ?100 mg of prefrontal cortex and hippocampus using
Trizol reagent (Invitrogen, Sao Paulo, Brazil) according to the manufac-
turer’s instructions. The concentration of total RNA was determined by
of the preparations was checked using gel electrophoresis. One micro-
gram of total RNA was reverse transcribed using oligo(dT) and 200 U of
reverse transcriptase (Invitrogen) in 20 ?l of PCR buffer containing (in
at 70°C, and 5 min at 4°C. Specific primers (Invitrogen) were used for
TNF-? (sense, TCTCATCAGTTCTATGGCCC; antisense, GGGAGTA-
antisense, CTTCTGGTCGATGTCATGA), and ?-actin (sense, TCCT-
TCGTTGCCGGTCCACA; antisense, CGTCTCCGGAGTCCATCACA).
?-Actin cDNA was used for standardization of the amount of RNA. Five
microliters of reverse transcription (RT) aliquots were mixed in a 20 mM
?-actin. Aliquots of 25 ?l were analyzed by PAGE and stained with silver
salts. Band density measurements for TNF-?, iNOS, and ?-actin mRNAs
Software. All Western blot and RT-PCR experiments were scanned to
acquire digital images using a Genius ColorPage Scanner (KYE Systems,
5396 • J.Neurosci.,May16,2007 • 27(20):5394–5404Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficit
ware package (Adobe Systems, Mountain View, CA), complying with
strict standards (Rossner and Yamada, 2004).
Statistical analysis. All values are expressed as means ? SEM (n is the
number of mice included in each analysis). Differences between groups
in total GSH levels and GR and GPx activities
were analyzed using an unpaired Student’s t
test. Statistical analysis for the rest of the data
were performed using two- or three-way
ANOVA with strain (or pretreatment), treat-
ment, or the number of trials (repeated mea-
sure) as independent variables. After signifi-
cant ANOVAs, multiple post hoc comparisons
were performed using the Newman–Keuls test.
p ? 0.05. All tests were performed using the Sta-
Learning and memory functions are vul-
nerable to several pathological processes,
including AD (Budson and Price, 2005).
volved in A?-induced cognitive decline,
the Morris water maze task was used. We
tial information as indicative of learning
and memory functions. As expected, in-
tracerebroventricular injection of A?1–40
in Swiss mice resulted in a significant de-
cline in both learning and memory, as in-
dicated by longer latencies (Fig. 1A) and
reduced target quadrant preference (Fig.
1B) during the probe trial. No cognitive
deficits were observed in Swiss mice
treated with the inverse A?40–1(Fig. 1).
The effects of A?1–40administration on
water maze performance are not directly
related to motor impairment, because
no alterations of the swimming speed in
the water maze (data not shown), or the
total squares crossed and rearing behav-
ior in the open-field arena, were ob-
served (Table 1).
cline, pharmacological and genetic ap-
proaches were applied. Swiss mice were
treated with the specific antibody against
mouse TNF-? (AbTNF-?), 15 min before
A?1–40, or the preferential iNOS inhibitor
AG, 1 h before A?1–40, and throughout 7
consecutive days, until the test day (day 8
after injection). As shown in Figure 2, A
and E, both pharmacological treatments
resulted in a significant improvement of
spatial learning deficits induced by A?1–
40, with a significant reduction in the final
escape latency to find the platform in the
training session. Additionally, the statisti-
cal analysis of the probe test scores re-
vealed that either AbTNF-? or AG signif-
icantly prevented the memory impairment induced by A?1–40,
increasing the time spent in the correct quadrant (Fig. 2B,F).
Genetic deletion of TNFR1 or iNOS also significantly attenuated
knock-out (iNOS?/?; G, H) mice were significantly more resistant than wild-type C57BL/6 mice to the deleterious effect of
A?1–40in the spatial learning (TNFR1?/?: F(1,30)? 29.58, p ? 0.0001; iNOS?/?: F(1,26)? 81.46, p ? 0.0001) and spatial
retrieval (TNFR1?/?: F(1,30)? 15.45, p ? 0.001; iNOS?/?: F(1,26)? 28.46, p ? 0.0001). *p ? 0.05 compared with the
Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficit J.Neurosci.,May16,2007 • 27(20):5394–5404 • 5397
the spatial learning deficits induced by A?1–40, as indicated by a
test session, TNFR1?/?and iNOS?/?mice given injections of
A?1–40presented higher swimming scores in the correct quad-
rant compared with wild-type A?1–40-treated mice, indicating a
diminished sensitivity to memory deficits (Fig. 2D,H).
To evaluate the possible amyloid deposits and/or neuronal dam-
age after A?1–40intracerebroventricular injection, we performed
histological and immunohistochemistry analysis. Histological
examination was performed on cresyl violet- and on Congo red-
age in control animals, at or distant from the injection site. The
administration of A?1–40peptide also failed to produce any neu-
shown). Using Congo red staining, control brain sections or
of amyloid deposits (data not shown).
To further evaluate the neuronal integrity, we performed im-
munohistochemistry analysis for the presynaptic protein synap-
tophysin and the pro-apoptotic protein caspase-3 in the parietal
cortex and the hippocampal CA1, CA2, and CA3 subregions 1
and 8 d after A?1–40intracerebroventricular injection. No mod-
ification in the synaptophysin levels was found 1 d after A?1–40
treatment (results not shown). However, a significant reduction
after intracerebroventricular injection, suggesting a decrease in
synaptic density (Fig. 3 and supplemental Fig. 1, available at www.
jneurosci.org as supplemental material). On the other hand, no
day 1 or 8, indicating that neuronal apoptosis was not activated,
at least not up to 8 d after A?1–40treatment (data not shown).
Also, either AbTNF-? or AG (Fig. 3A–D) significantly prevented
A?1–40-induced decrease in synaptophysin levels 8 d after A?1–40
treatment. Consistent with these findings, comparable results were
Astrogliosis is one of the earliest pathological hallmarks of AD
and may occur in response to the increasing number of degener-
ating neurons and synapses, or to the accumulation of A?. The
at day 1 after A?1–40intracerebroventricular injection, mainly in
the hippocampus, as demonstrated by a rise in the number of
GFAP-positive cells (supplemental Fig. 2, available at www.
in the GFAP immunoreactivity was found in the parietal cortex,
whereas the GFAP detection was reduced 8 d after A?1–40treat-
ment in the hippocampus (data not shown). The involvement of
Results show that treatment with AbTNF-? or AG failed to re-
supplemental material). These data were also confirmed by ex-
periments using knock-out mice. TNFR1?/?or iNOS?/?mice
failed to prevent GFAP immunodetection compared with wild-
type (C57BL/6) A?-treated mice (supplemental Fig. 2, available
at www.jneurosci.org as supplemental material). These data in-
dicate that astrogliosis present a role in the initial inflammatory
response induced by A?1–40in the AD model. In addition, it
suggests that astrocyte activation may be involved in initial
firm this hypothesis.
Next, we attempted to outline the temporal sequence of intracel-
lular events after A?1–40intracerebroventricular injection. The
aggregated A?1–40(400 pmol per mouse) intracerebroventricular injection. A–H, Relative
tivity was used as a measure of synaptic density. A, C, E, G, Pretreatment with the specific
antibody against mouse TNF-? (AbTNF-?; 10 ?g, i.c.v., per mouse) or with the preferential
iNOS inhibitor AG (100 mg/kg, i.p., once per day) prevented the A?1–40-induced synaptic
(F(3,8)? 3.17; p ? 0.08; B) and parietal cortex (F(3,8)? 4.00; p ? 0.05; H). The values
5398 • J.Neurosci.,May16,2007 • 27(20):5394–5404 Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficit
and prefrontal cortex at different time points after A?1–40injec-
time-dependent induction of TNF-? mRNA was observed in
A?1–40-treated mice (Fig. 4A,B). The increase in TNF-? mRNA
levels was detected after 15 min of A?1–40treatment, remaining
in both brain structures. Similarly, the time-course analysis re-
vealed that iNOS mRNA expression was minimal under basal
1–3 h and being reduced 24 h after A?1–40
treatment (Fig. 4A,B). Moreover, West-
ern blot analysis indicated that A?1–40in-
tracerebroventricular injection led to a
little alteration of iNOS expression was
verified up to 60 min, but a marked in-
changes might indicate a correlation be-
mRNA/protein expression (Fig. 4E,F).
Thus, we examined the effects of TNF-?
signaling blockage on A?1–40-induced
iNOS expression. The treatment with
AbTNF-? almost completely inhibited
iNOS protein expression, without affect-
ing the control protein ?-actin (Fig.
treated mice with the reverse peptide
A?40–1and found that it had no signifi-
cant effect on the TNF-? mRNA (results
not shown) or iNOS expression (Fig.
4C,D). In our model, TNF-? production
appears to be one of the earliest events in-
duced by A?, representing a signal for
iNOS protein expression.
Previous evidence suggests a causal link-
age among A?, iNOS expression, and cel-
lular redox state. NO generated by iNOS
reacts with superoxide anion (O2
ducing peroxynitrite (ONOO?) that ex-
erts cytotoxic effects (Beckman et al.,
A?1–40-induced iNOS expression is ac-
companied by oxidative alterations, in a
quantification of NO is more related with
our data, it is well accepted that GSH pa-
rameters are associated with cellular oxi-
dative state, and this might be indirectly
duction. Hence, we evaluated the GSH
status and GSH-dependent enzyme activ-
ities after A?1–40treatment. As shown in
supplemental Figure 3 (available at www.
jneurosci.org as supplemental material),
total GSH levels were reduced on days 1 and 7 after intracere-
broventricular injection in the hippocampus and prefrontal cor-
tex of A?1–40-treated Swiss mice, respectively. In addition, GR
and GPx activities were significantly increased by A?1–40in both
after 7 d (supplemental Fig. 3, available at www.jneurosci.org as
supplemental material). As expected, AbTNF-? treatment re-
verted all the oxidative alterations induced by A?1–40in the hip-
pocampus and prefrontal cortex (Fig. 5). These results establish
?-actin levels remained constant. C, D, A?1–40induced a time-dependent and prolonged iNOS protein expression in the hip-
pocampus (C) and prefrontal cortex (D). Western blot analysis revealed that pretreatment with the specific antibody against
by arbitrarily setting the densitometry from the maximal responsive group and are expressed as the mean of three to four
Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficitJ.Neurosci.,May16,2007 • 27(20):5394–5404 • 5399
Recent evidence suggests that JNK and its target c-Jun play an
possible requirement of JNK and c-Jun for A?-mediated effects
in our AD paradigm. As shown in Figure 6, A and B, a low basal
level of activated JNK was detected in the hippocampus and pre-
frontal cortex. However, an evident increase in JNK phosphory-
lation was found between 15 and 30 min. This reached a peak at
6 h and lasted for up to 7 d after A?1–40treatment in both brain
in c-Jun translocation to the nucleus (Fig. 6C,D), which suggests
were treated with the JNK inhibitor SP600125 (1 h before A?1–
40), and the hippocampus and prefrontal cortex were isolated for
TNF-? signaling effectors include JNK and c-Jun that might
also contribute to iNOS promoter activity (Eberhardt et al.,
1996). Pretreatment with AbTNF-? completely blocked A?1–40-
caused JNK phosphorylation (Fig. 6A,B) and c-Jun nuclear
of SP600125-treated mice revealed a partial reduction in the
iNOS expression (see Fig. 8). These data indicate a sequence of
events subsequent to A?1–40intracerebroventricular injection
that involves TNF-? production, JNK phosphorylation, c-Jun
translocation, and iNOS expression.
In resting cells, NF-?B dimmers are found in the cytoplasm as-
sociated with I?B proteins. Pathological conditions (including
AD) result in NF-?B translocation to the nucleus where it regu-
lates the transcription of several genes (Ghosh and Karin, 2002).
iNOS expression, we first assessed the temporal profile of NF-?B
activation after A?1–40treatment. Immunodetection of p65 sub-
unit performed in the cytosolic and nuclear homogenates from
hippocampus and prefrontal cortex confirmed the activation of
NF-?B. As shown in Figure 7, A and B, p65 was found to be
increased in nuclear extracts after 15 and 30 min of A?1–40treat-
ment, respectively. After 7 d, nuclear p65 levels returned toward
baseline in both brain areas. Next, we determined to what extent
NF-?B activation caused by A?1–40was dependent on the
TNF-?. The AbTNF-? almost completely blocked the transloca-
tion of NF-?B induced by A?1–40(Fig. 7A,B). To further verify
how NF-?B activation could participate in the iNOS expression
induced by A?1–40, Swiss mice were treated with the NF-?B in-
hibitor PDTC, at a dose that was sufficiently effective to prevent
p65 translocation to the nucleus (Fig. 7C,D). PDTC was given
intraperitoneally 60 min before A?1–40injection, and the hip-
pocampus and the prefrontal cortex were isolated 24 h later for
Western blot analysis. Data in Figure 8, A and B, show that
PDTC-treated mice presented a significant reduction in A?-
induced iNOS expression in both brain regions. Collectively,
these results suggest that A?1–40-induced iNOS expression is de-
pendent, at least in part, on TNF-?-driven NF-?B activation.
Evidence suggests that inflammatory reaction induced by A? in
the CNS involves the release of damaging factors, such as cyto-
kines, NO, and ROS, that promote the activation of intracellular
pathways, contributing to the progression of AD (Walsh and
Selkoe, 2004; Wyss-Coray, 2006). In the present study, we dem-
at a picomolar dose, induces a neuroinflammatory response that
is linked to a decline in learning and memory functions. Present
results are suggestive of glial activation and synaptic loss after
A?1–40injection; however, assessment of apoptotic protein
caspase-3 and cresyl violet staining indicated that programmed
cell death seems not likely involved. Of high interest, the cogni-
nal death (Walsh and Selkoe, 2004). In addition, by use of phar-
macological and molecular tools, we have shown a connecting
link between A?, TNF-?, iNOS, oxidative stress, and the cogni-
(AbTNF-?; 10 ?g, i.c.v., per mouse) 15 min before the administration of the aggregated
A?1–40(400 pmol per mouse) or vehicle (PBS). A–F, Total GSH levels (GSH total; A, B) and
frontal cortex of Swiss mice at 24 h after A?1–40administration. The values represent the
and prefrontal cortex (B) of A?1–40-treated mice. C–F, AbTNF-? treatment prevented the
increase in GR and GPx activities in the hippocampus (C, E) (F(3,16)? 2.05, p ? 0.05 and
5400 • J.Neurosci.,May16,2007 • 27(20):5394–5404Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficit
tive decline. Our results suggest that TNF-?, probably through
TNFR1 activation, exerts an essential role in A?-mediated iNOS
TNF-? is a multifunctional cytokine
that triggers a wide range of cellular re-
sponses. In the CNS, TNF-? (through ac-
tivation of TNFR1) disrupts learning and
memory and regulates neuronal death.
TNF-? has also been shown to participate
in A?-induced inhibition of LTP, a form
of synaptic plasticity closely associated
with learning and memory, and it is prob-
ably dependent on mGluR5 (metabo-
tropic glutamate receptor 5) and p38
MAPK (Wang et al., 2005). Moreover, the
activation of programmed cell death
seems to rely on the TNF-? signaling
death domain (DD) that binds to adaptor
TRADD (TNFR-associated DD), which
ing apoptosis (Varfolomeev and Ash-
kenazi, 2004). In this context, it has been
suggested that A?1–40binds directly to
TNFR1, resulting in neuronal apoptosis
(Li et al., 2004). Our data clearly demon-
strate that A?1–40induced the expression
of TNF-? in the hippocampus and pre-
the cognitive functions. Despite mRNA,
TNF-? levels started to reduce before 6 h;
it is reasonable to expect that TNF-? pro-
tein remains elevated for a longer period.
In fact, Song et al. (2001) have shown that
in mice results in a rapid onset and time-
dependent TNF-? protein expression,
values only after 72 h. A similar effect
might be expected in our experimental
paradigm. Notably, pharmacological and
genetic inhibition of TNF-? signaling re-
duced the synaptic loss and improved the
treated mice even 8 d after treatment.
These findings clearly place TNF-? as one
of the earliest mediators in the neurode-
generative process induced by A?1–40.
However, additional studies are necessary
to determine whether or not TNF-? ef-
fects depend on the participation of other
inflammatory proteins (e.g., IL-1?, IL-6,
The signal transduction cascades elic-
ited by TNF-? culminate in the activation
of key transcriptional regulators, includ-
ing NF-?B (Beg et al., 1993) and AP-1
(Angel and Karin, 1991). In most cell
types, NF-?B complexes are found in the
cytoplasm associated with I?Bs, which
prevent their nuclear localization. Cell
stimulation and/or injury leads to the
rapid degradation of I?B, allowing NF-?B translocation to the
nucleus, where it regulates the transcription of several genes
(Ghosh and Karin, 2002). Functional NF-?B dimers are present
in essentially all cell types in the CNS, including neurons, astro-
specific antibody against mouse TNF-? (AbTNF-?; 10 ?g, i.c.v., per mouse) prevented A?1–40-induced JNK activation and
Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficit J.Neurosci.,May16,2007 • 27(20):5394–5404 • 5401
cytes, microglia, and oligodendrocytes
(O’Neill and Kaltschmidt, 1997). In AD
greater in neurons and astrocytes sur-
rounding amyloid plaques (Terai et al.,
1996; Kaltschmidt et al., 1997). Addition-
ally, it has been reported that A? stimula-
tion leads to NF-?B activation in cultured
neurons and glia (Akama et al., 1998).
Corroborating these findings, we demon-
strate that A? promotes NF-?B activation
in the hippocampus and prefrontal cortex
through a TNF-? dependent pathway.
AP-1 is a transcriptional factor com-
cytokines (Angel and Karin, 1991). Phos-
phorylation and activation of c-Jun is cat-
alyzed by the MAPK family members
JNKs. The activation of both JNK and
c-Jun has been described in cultured neu-
rons after A? exposure, and their inhibi-
tion attenuates A? toxicity (Bozyczko-
Coyne et al., 2001; Morishima et al., 2001;
Troy et al., 2001). JNK activation has also
been implicated in A?-mediated LTP de-
cline, indicating a possible role for learn-
ing and memory (Wang et al., 2004a). In
the present study, we demonstrate that
tivation of JNK and its target c-Jun, via
TNF-?. These results are consistent with
son et al., 1996; Zhu et al., 2001).
In the CNS, iNOS is not commonly
found in healthy tissues, but it can be ex-
pressed after brain insult in astrocytes,
neurons, and endothelial cells, where it
triggers the production of high amounts
of NO (Vallance and Leiper, 2002). Overproduction of NO may
lead to neuronal damage and death. The predominant mecha-
nism by which NO promotes neuronal toxicity implicates the
reaction of NO with superoxide anion to generate the cytotoxic
substance peroxynitrite (Beckman et al., 1990; Vallance and
Leiper, 2002). Under physiological conditions, highly reactive
ing GPxs (Thannickal and Fanburg, 2000). However, in patho-
logical conditions, excessively accumulated reactive species in-
duce several cellular dysfunctions (Thannickal and Fanburg,
2000). The present data revealed that intracerebroventricular
A?1–40injection induced a marked iNOS expression, a phenom-
enon accompanied by a reduction in GSH levels, suggesting a
possible increase in the production of reactive species, including
NO. The iNOS inhibition or the iNOS genetic deletion signifi-
NO synthesis have been deeply associated with AD pathology. In
1997; Luth et al., 2002). A recent report showed that genetic
administration. Top, Pretreatment with the selective inhibitor of JNK, SP600126 (50 mg/kg,
i.p.), or NF-?B blocker PDTC (100 mg/kg, i.p.) 1 h before A?1–40treatment reduced iNOS
expression in the hippocampus (F(3,8)? 40.00; p ? 0.0001) and prefrontal cortex (F(3,8)?
12.68; p ? 0.01). Bottom, Graphs showing quantification of iNOS protein normalized by
?-actin protein (loading control). The values represent the mean ? SEM. **p ? 0.01 com-
pared with the vehicle-treated group;#p ? 0.05,##p ? 0.01 compared with the A?1–40-
5402 • J.Neurosci.,May16,2007 • 27(20):5394–5404Medeirosetal.•RoleofTNF-?andiNOSonA?CognitiveDeficit
deletion of iNOS substantially protected mice from A? toxicity
iNOS exerts an important role in A?-caused cognitive deficits in
rats and mice (Tran et al., 2001; Wang et al., 2004b).
Production of iNOS protein is tightly regulated at the tran-
scriptional level, but the upstream signaling events mediating
of iNOS gene revealed the presence of binding sites to several
transcriptional factors, such as AP-1, NF-?B, and TNF response
element (Eberhardt et al., 1996). Of high interest, in the present
study, we show for the first time that iNOS induction by A?1–40
mental Fig. 4, available at www.jneurosci.org as supplemental
represents an unusual enzyme that can be modulated by highly
Overall, our study suggests that TNF-? and iNOS signaling
pathways are linked and exert an important role in the cognitive
deficits observed in the earlier stages of AD. Additional charac-
terization of a candidate substrate(s) for both proteins would
help to clarify their role and potential as molecular targets for
therapeutic intervention in human AD.
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