Perluigi 20et 20al 202006 20J 20Neuroscience 20Res 2084 20418 426

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					                                                                         Journal of Neuroscience Research 84:418–426 (2006)

In Vivo Protective Effects of Ferulic Acid
Ethyl Ester Against Amyloid-Beta Peptide
1–42-Induced Oxidative Stress
Marzia Perluigi,1,2 Gururaj Joshi,2,3 Rukhsana Sultana,2,3 Vittorio Calabrese,4
Carlo De Marco,1 Raffaella Coccia,1 Chiara Cini,1 and D. Allan Butterfield2,3,5*
 Department of Biochemical Sciences, University of Rome ‘‘La Sapienza,’’ Rome, Italy
 Department of Chemistry, University of Kentucky, Lexington Kentucky
 Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky
 Department of Chemistry, Section of Biochemistry, University of Catania, Catania, Italy
 Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky

Alzheimer’s disease (AD) is a neurodegenerative disor-              sis of this disorder (Butterfield, 2002; Mattson and Mattson,
der characterized by the deposition of amyloid-beta                 2002).
peptide (Ab), a peptide that as both oligomers and                         Loss of synaptic terminals in AD brain demonstrates a
fibrils is believed to play a central role in the develop-           higher correlation with decreased cognitive function than
ment and progress of AD by inducing oxidative stress                do cell death or plaque development, which has led to the
in brain. Therefore, treatment with antioxidants might,             hypothesis that disappearance of synapses is a key event in
in principle, prevent propagation of tissue damage and              early cognitive decline (Terry et al., 1991). The cellular
neurological dysfunction. The aim of the present study              location of initial amyloid-related damage is controversial,
was to investigate the in vivo protective effect of the             but a growing body of evidence suggests that intracellular
antioxidant compound ferulic acid ethyl ester (FAEE)                accumulation of Ab precedes plaque formation. However,
against Ab-induced oxidative damage on isolated syn-                the mechanisms of synapse loss in AD remain uncertain.
aptosomes. Gerbils were injected intraperitoneally (i.p.)           Nevertheless, the damage in these regions is directly corre-
with FAEE or with dimethylsulfoxide, and synaptosomes               lated with severity of dementia, oxidative stress, and depo-
were isolated from the brain. Synaptosomes isolated                 sition of Ab1–42. Therefore, the investigation of oxidative
from FAEE-injected gerbils and then treated ex vivo                 damage occurring in synaptosomes isolated from murine
with Ab1–42 showed a significant decrease in oxidative               brain has been shown to be a suitable experimental model
stress parameters: reactive oxygen species levels, pro-             with which to study the extent of Ab-induced toxicity
tein oxidation (protein carbonyl and 3-nitrotyrosine lev-           (Mattson et al., 1998; Lauderback et al., 2001).
els), and lipid peroxidation (4-hydroxy-2-nonenal levels).                 Increased production of reactive oxygen and nitro-
Consistent with these results, both FAEE and Ab1–42                 gen species such as superoxide radical anion and nitric ox-
increased levels of antioxidant defense systems, evi-               ide, together with an imbalance of antioxidant defenses,
denced by increased levels of heme oxygenase 1 and                  was observed in neuronal systems after Ab1–42 treatment
heat shock protein 72. FAEE led to decreased levels of              (Keller et al., 1997; Yatin et al., 1999; Varadarajan et al.,
inducible nitric oxide synthase. These results are dis-             2000; Butterfield, 2002). Previous studies from our labo-
cussed with potential therapeutic implications of FAEE,             ratory and others have reported that Ab1–42 induces in
a brain accessible, multifunctional antioxidant com-                vitro and in vivo reactive oxygen species (ROS) produc-
pound, for AD involving modulation of free radicals                 tion, protein oxidation, DNA and RNA oxidation, and
generated by Ab. V 2006 Wiley-Liss, Inc.
                     C                                              lipid peroxidation (Keller et al., 1997, 2000; Butterfield,
                                                                    2002; Butterfield et al., 2002b; Butterfield and Lauder-
Key words: ferulic acid ethyl ester; amyloid-beta peptide;          back, 2002; Drake et al., 2003; Mohmmad Abduel et al.,
Alzheimer’s disease; heme oxygenase-1; heat shock                   2004, 2006; Boyd-Kimball et al., 2005a).
proteins; oxidative stress
                                                                    *Correspondence to: Prof. D. Allan Butterfield, Department of Chemis-
      Alzheimer’s disease (AD) is a neurodegenerative dis-          try, Center of Membrane Sciences, and Sanders-Brown Center on Aging,
order characterized by a progressive cognitive decline re-          University of Kentucky, Lexington KY 40506-0055.
sulting from selective neuronal dysfunction, synaptic loss,         E-mail:
and neuronal cell death. AD is accompanied by the pres-             Received 29 November 2005; Revised 16 February 2006; Accepted 15
ence of extracellular amyloid plaques containing aggregated         March 2006
amyloid-beta peptide (Ab), a polypeptide of 39–43 amino             Published online 21 April 2006 in Wiley InterScience (www.
acids that is thought to play a major role in the pathogene- DOI: 10.1002/jnr.20879

' 2006 Wiley-Liss, Inc.
                                                                     In Vivo Neuroprotection Against Ab1–42 by FAEE             419

       Indeed, several studies performed to date have exam-        tosomes. All the experimental protocols were approved by the
ined whether dietary intake of several antioxidants, such as       University of Kentucky Animal Care and Use Committee. All
flavonoids, carotenoids, and vitamins, might prevent or             the animals were kept under 12-hr light/dark conditions at the
reduce the progression of AD (Butterfield et al., 2002a).           University of Kentucky Animal Faciltiy and fed with standard
For this class of molecules, we focused our attention on           Purina rodent laboratory chow ad libidum. The gerbils were
the phenol compound ferulic acid ethyl ester (FAEE). Fe-           injected i.p. with freshly prepared FAEE dissolved in DMSO
rulic acid (FA) is a substance found in most plants, espe-         (150 mg/kg body weight) 1 hr before sacrifice. The dose and
cially in the brans of grasses such as wheat, rice, and oats.      the time of FAEE were chosen according to previous data
Because of its phenolic nucleus and an extended side chain         obtained in our laboratory (data not shown) to achieve brain
conjugation, FA readily forms a resonance-stabilized phe-          accessibility of the compound with a lack of any toxic effects
noxy radical, which accounts for its antioxidant potential         (Joshi et al., 2006). Control animals were injected with DMSO
(Kanski et al., 2002). The esterification of the acid group         for the same period. The animals were euthanized with sodium
(FAEE) confers lipophilic properties upon the molecule,            pentobarbital.
thus increasing its antioxidant potential (Schroeter et al.,
2000; Kikuzaki et al., 2002; Scapagnini et al., 2004; Sultana      Synaptosomal Preparation
et al., 2005c). Previous studies from our laboratory and                  Synaptosomes were isolated from gerbils injected i.p. with
others have demonstrated in vitro its scavenging activities        DMSO (contol; CTR) or with FAEE in DMSO 1 hr after
toward hydroxyl radical, peroxy-nitrite, and oxidized low-         injection. The isolation of synaptosomes from whole brain was
density lipoprotein (oxLDL; Pannala et al., 1998; Schroeter        carried out according to the procedure described by Keller et al.
et al., 2000; Sultana et al., 2005c). Recent findings have          (2000). The brain was isolated immediately after decapitation
shown the ability of FAEE potently to induce hemeoxyge-            and placed in a 0.32 M sucrose isolation buffer containing
nase 1 (HO-1) and heat shock protein 72 (HSP72) expres-            4 lg/ml leupeptin, 4 lg/ml pepstatin, 5 lg/ml aprotinin,
sion in neuronal cell culture (Scapagnini et al., 2004; Joshi      20 lg/ml trypsin inhibitor, 0.2 mM phenylmethylsulfonyl fluo-
et al., 2005) and synaptosomal systems (Joshi et al., 2006).       ride (PMSF), 2 mM EDTA, 2 mM EGTA, 20 mM HEPES,
       Given the neuroprotective success of FAEE against           pH 7.4. Samples were homogenized with a Wheaton tissue ho-
Ab1–42 in vitro (Sultana et al., 2005c) and based on the           mogenizer and centrifuged at 1,500g for 10 min. The pellet was
mechanisms by which FAEE scavenges free radicals, the              discarded, and the supernatant was retained and centrifuged at
aim of the present study was to investigate the ability of         20,000g for 10 min. The resulting pellet was resuspended in
FAEE to provide in vivo neuroprotection against Ab-                1 ml of 0.32 sucrose buffer and layered onto discontinuous
induced oxidative stress. For this purpose, different pa-          sucrose density gradients of 10 ml each of 0.85 M, pH 8.0;
rameters of oxidative stress have been evaluated: ROS              1.0 M, pH 8.0; 1.18 M, pH 8.5; sucrose solutions, each con-
levels, protein oxidation, lipid peroxidation, and the role        taining 10 mM HEPES, 2 mM EDTA, and 2 mM EGTA. The
of heat shock response. The results are consistent with the        gradients were spun in a Beckman L7-55 ultracentrifuge at
hypothesis that FAEE is a potent brain-accessible antioxi-         82,550g for 1 hr at 48C. The purified synaptosomes were col-
dant that potentially could be beneficial in the treatment          lected at the 1/1.18 M sucrose interface and washed twice with
of AD and other oxidative stress-related disorders.                PBS for 10 min at 32,000g, yelding synaptosomes. Protein con-
                                                                   centrations of the purified synaptosomal membranes were deter-
                                                                   mined by the BCA assay (Pierce, Rockford, IL).
Materials                                                                 Synaptosomes from DMSO- and FAEE-injected gerbils
       FAEE and all other chemicals were purchased from            were incubated with and without 10 lM Ab1–42 for 6 hr at
Sigma-Aldrich (St. Louis, MO). The fluorescent indicator for        378C (time chosen based on prior studies; Boyd-Kimball et al.,
ROS measurement, 2,7-dichlorofluorescin diacetate (DCFH-            2005a). Therefore, the present study entails four groups for
DA), was obtained from Molecular Probes (Eugene, OR), and a        comparison: synaptosomes isolated from DMSO- and FAEE-
fresh 10 mM stock solution was prepared in ethanol. Fresh          injected gerbils not treated with 10 lM Ab1–42, both used as
FAEE (50 lM) was prepared by dissolution in dimethylsulfoxide      controls, and synaptosomes from DMSO- and FAEE-injected
(DMSO). The Oxyblot oxidized protein kit was obtained from         gerbils treated with 10 lM Ab1–42.
Intergen, Inc. (Purchase, NY). Ab1–42 (HPLC- and MS-certified
purity) was purchased from Anaspec, Inc. (San Jose, CA). For all   ROS Measurements
experiments, Ab was incubated for 24 hr in phosphate-buffered             The dichlorofluorescein (DCF) assay was used to measure
saline (PBS) at 378C before application to synaptosomes. Primary   the levels of ROS, according to the procedure previously
antibodies for 4-hydroxynonenal (HNE) and 3-nitrotyrosine          described by Wang and Joseph (1999). The cell-permeable
were obtained from Chemicon (Temecula, CA). Anti-HO-1,             dichlorofluorescin diacetate (DCFH-DA) crosses inside the syn-
anti-iNOS, and anti-HSP72 primary antibodies were purchased        aptosomal vescicle, where it is deesterified by cellular esterases
from Santa Cruz Biotechonology (Santa Cruz, CA).                   resulting in DCFH. DCFH in turn is converted upon oxidation
                                                                   to the highly fluorescent DCF. By measuring the fluorescence,
Animals                                                            we were able to quantify the levels of ROS. After incubation
       For the present study, 3-month-old male Mongolian ger-      with Ab1–42 for 6 hr, synaptosomes (1 mg/ml) were washed
bils, approximately 70 g in weight, were used to isolate synap-    with PBS and incubated with 10 lM of nonfluorescent

Journal of Neuroscience Research DOI 10.1002/jnr
420     Perluigi et al.

DCFH-DA for 30 min. Previous studies showed that fluores-
cence was due to intrasynaptosomal oxidative process rather
than to DCF exiting the synaptosomes to react with oxidant
(Joshi et al., 2005). Synaptosomes were spun at 3,000g in a tab-
letop Eppendorf centrifuge for 5 min at 48C. Synaptosomes
were resuspended in 500 ll of PBS and run in triplicate (100 ll
per well) in a black microtiter plate. The measurements were
performed on a Molecular Devices SpectraMax microtiter plate
reader with kex ¼ 495 nm and kem ¼ 530 nm. Data are given
as percentage of corresponding controls and are the mean of at
least six independent experiments.

Protein Carbonyl Measurement
                                                                     Fig. 1. Protective effects of FAEE against Ab1–42-induced ROS pro-
       Protein oxidation was determined by an oxidized protein       duction. ROS levels were determined by the DCF fluorescence
detection kit (Oxyblot; Chemicon). Briefly, 5 ll of synapto-          assay. Ctr, synaptosomes isolated from DMSO-injected gerbils with
somes (4 mg/ml) was incubated for 20 min with 12% sodium             no further treatment (n ¼ 6); FAEE, synaptosomes isolated from
dodecyl sulfate (SDS) and 2,4-dinitrophenylhydrazine (DNPH)          FAEE-injected gerbils with no further treatment (n ¼ 6); Ab1–42,
in 10% trifluoroacetic acid with vortexing every 5 min, then          synaptosomes isolated from DMSO-injected gerbils and treated with
neutralized with Oxyblot Neutralization solution. We blotted         10 lM Ab1–42 for 6 hr (n ¼ 6); Ab1–42 þ FAEE, synaptosomes iso-
250 ng of protein onto nitrocellulose paper by the slot blotting     lated from FAEE-injected gerbils and treated with 10 lM Ab1–42 for
technique. Membranes were incubated with blocking buffer for         6 hr (n ¼ 6). Data are mean 6 SEM of six independent experiments,
                                                                     expressed as percentage of control values. Statistical comparison was
60 min at 278C and incubated with rabbit antibodies to DNPH          via ANOVA test (n ¼ 6 for each group). *P < 0.05, Ab1–42 vs. con-
(diluted 1:150) for 90 min, then by anti-rabbit IgG coupled to       trol; **P < 0.01, Ab1–42 vs. Ab1–42 þ FAEE.
alkaline phosphatase (1:10,000) for 1 hr at 278C. After being
washed and developed with SigmaFast chromogen (Sigma),
blots were scanned into Adobe Photoshop (Adobe Systems,              ferred on nitrocellulose at 80 mA/gel for 2 hr. The blots were
Inc., Mountain View, CA) and quantitated with Scion Image            then blocked for 2 hr in 3% nonfat dry milk in PBS. Mem-
(PC version of Macintosh-compatible NIH Image).                      branes were next probed with primary antibody, anti-HSP72,
                                                                     anti-HO-1, and anti-iNOS (1:1,000), for 2 hr at room tem-
3-Nitrotyrosine Levels                                               perature. After three washes with PBS, membranes were incu-
       Nitrotyrosine content was determined by incubating the        bated for 1 hr with horseradish preroxidase-conjugated sec-
samples with Laemmli buffer (0.125 M Trizma base, pH 6.8,            ondary antibodies in PBS. The membranes were washed again
4% SDS, 20% glycerol) for 20 min. Samples (250 ng of protein)        three times with PBS, and the bands were visualized by using
were blotted onto nitrocellulose membranes, and immuno-              a chemiluminescence kit (Amersham, Piscataway, NJ).
chemical methods were performed. The rabbit anti-3-nitrotyro-
sine (3-NT) primary antibody was incubated 1:200 in blocking         Statistical Analysis
buffer [bovine serum albumin (BSA) 3% in TBS-T] for 2 hr.                  Analysis of variance (ANOVA) was used for comparison
The membranes were washed three times with TBS-T and                 among the groups, followed by Student’s t-tests for analysis of
incubated with alkaline phosphatase-conjugated goat anti-rabbit      significance. P < 0.05 was considered significant for compari-
secondary antibody (1:10,000). Densitometric analysis of bands       son between control and experimental results.
in images of the blots was used to calculate levels of 3-NT.
Lipid Peroxidation Measurement                                       FAEE Protects In Vivo Against Ab1–42-Induced
       4-Hydroxy-2-nonenal (HNE) levels were measured as a           ROS Production
marker of lipid peroxidation. The samples (5 ll) were incubated             ROS levels generated by Ab1–42 in our experimen-
with 10 ll Laemmli buffer for 20 min at room temperature, and        tal model were measured by DCF fluorescence. In the
250 ng of protein samples was loaded into each well on nitrocellu-   absence of Ab1–42, levels of ROS in synaptosomes from
lose membrane in a slot blot apparatus under vacuum. The mem-        DMSO-injected gerbils did not show any significant dif-
branes were incubated with anti-HNE rabbit polyclonal antibody       ference compared with the levels measured in synapto-
(1:5,000) for 2 hr, washed three times with TBS-T, and then incu-    somes from FAEE-injected gerbils (Fig. 1). Hence, both
bated with an anti-rabbit IgG alkaline phosphatase-conjugated sec-   these groups can be referred to as controls. Thus, FAEE
ondary antibody (1:10,000). Blots were developed with SigmaFast      itself at the concentration used does not reduce basal oxi-
tablets (BCIP/NBT), dried, and quantified in Scion Image.             dation levels. Synaptosomes isolated from DMSO-injected
                                                                     gerbils and treated with 10 lM Ab1–42 for 6 hr displayed
Western Blot                                                         an increased fluorescence, about 20% compared with con-
      Synaptosome samples (100 lg) were added with sample            trol synaptosomes (untreated; P < 0.05). Synaptosomes
loading buffer, denaturated for 5 min at 1008C, and then             isolated from FAEE-injected gerbils did not lead to Ab1–42-
loaded on 10% SDS-polyacrylamide gels. Proteins were trans-          induced ROS accumulation (P < 0.01). Thus, FAEE

                                                                                             Journal of Neuroscience Research DOI 10.1002/jnr
                                                                         In Vivo Neuroprotection Against Ab1–42 by FAEE               421

                                                                       Fig. 3. Protective effect of FAEE on Ab1–42-induced 3-NT forma-
Fig. 2. Protective effects of FAEE on Ab1–42-induced protein oxida-    tion. 3-NT levels were determined as described in Materials and
tion. Protein carbonyl content of synaptosomes was measured as         Methods. Ctr, synaptosomes isolated from saline- or FAEE-injected
described in Materials and Methods. Synaptosomes isolated from         gerbils with no further treatment; FAEE, synaptosomes isolated from
DMSO-injected gerbils and treated with 10 lM Ab1–42 demonstrate        FAEE-injected gerbils with no further treatment; Ab1–42, synapto-
a higher level of protein carbonyls than untreated controls (Ctr and   somes isolated from DMSO-injected gerbils and treated with 10 lM
FAEE). *P < 0.01, Ab1–42 vs. control. Synaptosomes isolated from       Ab1–42 for 6 hr; Ab1–42 þ FAEE, synaptosomes isolated from FAEE-
FAEE-injected gerbils were completely protected from Ab-induced        injected gerbils and treated with 10 lM Ab1–42 for 6 hr. Data are
oxidative modifications (Ab1–42 þ FAEE). **P < 0.005, Ab1–42 vs.        mean 6 SEM expressed as percentage of control values (n ¼ 6). *P <
Ab1–42 þ FAEE.                                                         0.05, Ab1–42 vs. control; **P < 0.01, Ab1–42 vs. Ab1–42 þ FAEE.

in vivo significantly prevents free radical formation in                      Free radical attack on phospholipid polyunsaturated
synaptosomes by Ab1–42.                                                fatty acids (PUFA) leads to the formation of reactive alde-
                                                                       hydes, among which one of the most neurotoxic is HNE
                                                                       (Esterbauer et al., 1991; Lauderback et al., 2001). This
FAEE In Vivo Protects Against Ab1–42-Induced                           alkenal reacts with proteins forming stable covalent
Protein Oxidation and Lipid Peroxidation                               adducts to histidine, lysine, and cysteine residues via Mi-
      Protein carbonyls and 3-NT levels were measured as               chael addition (Berlett and Stadtman, 1997; Butterfield,
markers of protein oxidation (Berlett and Stadtman, 1997;              2002; Butterfield et al., 2002b; Butterfield and Lauder-
Lauderback et al., 2001; Sultana et al., 2005c). Protein car-          back, 2002). The extent of this reaction can be measured
bonyl groups are incorporated into proteins by direct oxi-             immunochemically by quantifying the levels of HNE-
dation of certain amino acid side chains, by peptide back-             bound proteins. Figure 4 shows the HNE-bound protein
bone scission, or by Michael addition reactions with prod-             levels in synaptosomes isolated from gerbils previously
ucts of lipid peroxidation or glycoxidation (Berlett and               injected with FAEE or with DMSO and incubated
Stadtman, 1997; Butterfield and Lauderback, 2002). Oxida-               in vitro with 10 lM Ab1–42 for 6 hr. Consistently with
tive stress could also stimulate additional damage via the             the protein oxidation results shown above, we observed
overexpression of inducibile nitric oxide synthase (iNOS)              in vivo protection by FAEE against 10 lM Ab-induced
and the action of constitutive neuronal NOS (nNOS) that                lipid peroxidation. HNE levels were found to be higher
leads to increased levels of 3-NT. Figure 2 shows the car-             in Ab1–42-treated synaptosomes isolated from DMSO-
bonyl levels in synaptosomes isolated from DMSO- and                   injected gerbils (P < 0.01), whereas Ab1–42-treated synap-
from FAEE-injected gerbils that were subsequently treated              tosomes isolated from FAEE-injected gerbils showed
with 10 lM Ab1–42. The level of carbonyls was found to                 reduced levels of HNE-bound proteins (P < 0.005).
be significantly higher (P < 0.01) in Ab1–42-treated synap-             These results are consistent with recent in vitro data
tosomes previously isolated from DMSO-injected gerbils.                obtained on primary neuronal cultures (Sultana et al.,
FAEE in vivo treatment protects subsequently isolated syn-             2005c), indicating that FAEE acts as a potent antioxidant,
aptosomes against Ab1–42-induced oxidative protein dam-                thus preventing protein oxidation and lipid peroxidation.
age (P < 0.005). The antioxidant properties of FAEE were
further confirmed by measuring 3-NT levels, formed by
reaction of reactive nitrogen species (RNS) with proteins              FAEE Leads to Elevated HO-1 and HSP72
(Castegna et al., 2003; Sultana et al., 2006). Figure 3 shows          Protein Levels
the protective effects of FAEE on Ab1–42-induced forma-                      It has been well documented that oxidative stress
tion of 3-NT. Synaptosomes isolated from DMSO-injected                 conditions induce expression of the so-called stress
gerbils showed increased levels of 3-NT (P < 0.05) when                response proteins, such as the HSP family and HO sys-
treated in vitro with 10 lM Ab1–42, whereas synaptosomes               tem (Polla et al., 1996; Fauconneau et al., 2002; Calabr-
isolated from FAEE-injected gerbils and treated with                   ese et al., 2004b; Poon et al., 2004). Figure 5a,b shows
Ab1–42 were completely protected (P < 0.01).                           the increased levels of both HO-1 and HSP72 protein

Journal of Neuroscience Research DOI 10.1002/jnr
422      Perluigi et al.

Fig. 4. Protective effect of FAEE on Ab-induced lipid peroxidation
(HNE levels). HNE levels were determined as described in Materials
and Methods. Ctr, synaptosomes isolated from DMSO/FAEE-
injected gerbils; FAEE, synaptosomes isolated from FAEE-injected
gerbils with no further treatment; Ab1–42, synaptosomes isolated from
saline-injected gerbils and treated with 10 lM Ab1–42 for 6 hr; Ab1–42
þ FAEE, synaptosomes isolated from FAEE-injected gerbils and
treated with 10 lM Ab1–42 for 6 hr. Data are mean 6 SEM expressed
as percentage of control values (n ¼ 6). *P < 0.01, Ab1–42 vs. control;
**P < 0.005, Ab1–42 vs. Ab1–42 þ FAEE.

levels in synaptosomes isolated from DMSO-injected
gerbils and then treated with Ab1–42 for 6 hr (P <
0.05). This effect becomes more pronounced in synapto-
somes isolated from FAEE-injected gerbils that were
subsequently treated with Ab1–42 (P < 0.01), suggesting
that both oxidative stress and FAEE facilitate increased
HO-1 and HSP72 levels. Consistently with this sugges-
tion, an increased level of HSP72 in synaptosomes iso-
lated rom FAEE-injected gerbils not treated with Ab1–42
was observed (Fig. 6a). We confirmed, based on this
finding and on our previous data (Scapagnini et al.,
2004; Sultana et al., 2005c; Joshi et al., 2006), the ability
of FAEE independently to lead to elevated levels of the
stress response proteins HO-1 and HSP72, a process that
might represent an efficient antioxidant system against
Ab-induced neurotoxicity.

FAEE Leads to Lower Levels of iNOS
      Considerable evidence demonstrates the involve-                     Fig. 5. Western immunoblot analysis of synaptosomes for HO-1 (a),
ment of neuroinflammatory processes in AD brain                            HSP72 (b), and iNOS (c) protein levels. Samples containing 50 lg of
(McGeer et al., 2000; Togo et al., 2004; Tuppo and                        protein were loaded onto 10% SDS-PAGE gels, and the blots were
Arias, 2005). Neurotoxic amounts of RNS are formed                        probed with the polyclonal anti-HO-1 (1:2,000), anti-HSP70 (1:500), and
                                                                          anti-iNOS (1:1,000) antibodies, respectively, for 2 hr. Immunoblots were
by the activity of iNOS (Heneka and Feinstein, 2001;                      scanned by densitometry, and all values were normalized to b-actin. Den-
Haas et al., 2002). In the current study, we show that                    sitometric values represent mean 6 SEM obtained from three independent
iNOS protein levels are sharply increased in synapto-                     experiments.The figures show a representative experiment (one of three),
somes isolated from DMSO-injected gerbils treated with                    with each lane in duplicate. Significant differences were assessed by
Ab1–42 compared with control synaptosomes (DMSO-                          ANOVA. *P < 0.05, control vs. Ab1–42; **P < 0.01, Ab1–42 þ FAEE.
injected gerbils; P < 0.05). In vivo FAEE treatment
decreased iNOS protein levels in synaptosomes isolated
from FAEE-injected gerbils treated with Ab1–42 (Fig.                                          DISCUSSION
6a). Interestingly, we also observed that treatment of                        Oxidative stress induced by Ab in vivo plays a
FAEE alone is able to decrease the protein level of                       prominent role in the neurodegeneration associated with
iNOS (Fig. 6b).                                                           AD (Friedlich and Butcher, 1994; Smith et al., 1998;

                                                                                                   Journal of Neuroscience Research DOI 10.1002/jnr
                                                                       In Vivo Neuroprotection Against Ab1–42 by FAEE          423

                                                                      tive damage and apoptosis in cultured neurons (Kanski
                                                                      et al., 2002; Zhang et al., 2003) and inhibits lipopolysac-
                                                                      charide (LPS)-induced production of tumor necrosis fac-
                                                                      tor-a and macrophage inflammatory protein-2 in a mu-
                                                                      rine macrophage cell line (Sakai et al., 1997).
                                                                             In addition to the radical-scavenging activity of an
                                                                      antioxidant, both its polarity and its three-dimensional
                                                                      interaction with lipid bilayers may contribute to its anti-
                                                                      oxidant activity. Synaptic membranes are particularly
                                                                      vulnerable to oxidative stress, so the ability of an antioxi-
                                                                      dant to act at membrane sites because of its high lipophi-
                                                                      licity results in a higher antioxidant potential. The syn-
                                                                      apse is one of the primary targets of Ab-mediated neu-
                                                                      rotoxicity, so the affinity of FAEE with lipid substrates
                                                                      might be an important factor in modulating Ab-induced
                                                                      oxidative damage.
                                                                             Many studies have shown that the oxidative damage
                                                                      associated with AD is represented by lipid peroxidation
                                                                      (Sayre et al., 1997; Markesbery and Lovell, 1998; Lauder-
                                                                      back et al., 2001), nitration (Smith et al., 1997; Castegna
Fig. 6. Representative Western blots showing in vivo effects of       et al., 2003; Sultana et al., 2006), reactive carbonyls pro-
FAEE alone in synaptosomes isolated from FAEE-injected gerbils        duction (Aksenov et al., 2001; Butterfield, 2002; Butter-
(150 mg/kg body weight). Samples containing 50 lg protein were        field et al., 2002b; Butterfield and Lauderback, 2002; Sul-
analyzed by SDS-gel electrophoresis and immunoblotting as described   tana et al., 2005a,b), and nucleic acid oxidation (Mecocci
in Materials and Methods. a: HSP72. b: iNOS.
                                                                      et al., 1993; Wang et al., 2005), which are all increased in
                                                                      vulnerable neurons of diseased brain. In the current study,
Butterfield, 2002; Butterfield et al., 2002b; Butterfield                carbonyl levels and 3-NT levels were found to be
and Lauderback, 2002; Drake et al., 2003; Boyd-Kimball                decreased in Ab1–42-treated synaptosomes isolated from
et al., 2005b). Therefore, a safe and effective, brain-ac-            FAEE-injected gerbils compared with control synapto-
cessible drug, one that both possesses antioxidant proper-            somes. In addition, in vivo FAEE treatment resulted in
ties and has the property of leading to elevated levels of            protective effects against Ab1–42-induced lipid peroxida-
neuroprotective proteins while leading to decreased lev-              tion. HNE is one of the most reactive and toxic end
els of a potentially harmful protein, might prove to be               products of lipid peroxidation and is thought to interfere
beneficial in treating the symptoms of AD or slowing its               with normal cellular functions in AD brain tissues (Ester-
onset. Based on the notion that ethyl ferulate showed                 bauer et al., 1991; Sayre et al., 1997; Markesbery and
increased antioxidant properties compared with ferulic                Lovell, 1998; Butterfield and Lauderback, 2002).
acid and that its higher lipophilicity might improve its                     There are a variety of genes encoding proteins that
brain accessibility (Kikuzaki et al., 2002; Scapagnini                possess antioxidant properties. Of particular interest in
et al., 2004), the current study provides evidence that in            the CNS is HO-1, which has been reported to operate
vivo FAEE treatment exerts protective effects against                 as a fundamental defensive mechanism for neurons
Ab1–42-induced oxidative stress in our experimental                   exposed to an oxidant challenge (Maines, 2000; Calabr-
model, while leading to elevated levels of HO-1 and                   ese et al., 2004b; Mancuso, 2004; Poon et al., 2004). A
HSP-72 and decreased levels of i-NOS.                                 growing body of evidence reveals that HO-1 and one of
       Synapse loss is believed to be an early pathological           the reaction products catalyzed by HO-1, biliverdin,
event in AD (Mattson et al., 1998), and synaptosomes                  which is rapidly converted into bilirubin in mammalian
have been shown to be oxidized by treatment with                      cells, are potent antioxidants at lower levels (Dore et al.,
Ab1–42 (Lauderback et al., 2001, 2002; Butterfield, 2002;              1999; Takata et al., 2002; Calabrese et al., 2003; Man-
Butterfield et al., 2002b; Butterfield and Lauderback,                  cuso, 2004). HO-1, also known as HSP32, is a member
2002). In the present study, we have shown that the lev-              of the HSP family of chaperone proteins that are crucial
els of ROS decreased in synaptosomes isolated from                    for recovery from stress-induced protein damage (Cal-
FAEE-injected gerbils and treated ex vivo with Ab1–42                 abrese et al., 2002; Mancuso, 2004; Poon et al., 2004).
compared with CTR synaptosomes (DMSO-injected                         In AD cortex and hippocampus, HO-1 has been shown
gerbils). Our data demonstrate the ability of FAEE to act             to be overexpressed and colocalizes to senile plaques and
in vivo as a potent free radical scavenger. Because of its            neurofibrillary tangles (Schipper, 2000).
phenolic nucleus and an extended side chain conjugation                      The 72-kDa HSP (HSP72) is a stress-inducible pro-
(Kanski et al., 2002), FAEE readily traps free radical spe-           tein that belongs to the HSP70 chaperone family. HSP72
cies such as hydroxyl and peroxyl radicals by forming a               shows very low expression levels in brain under physio-
resonance-stabilized phenoxy radical (Kanski et al., 2002;            logical conditions, but it is induced after certain oxidative
Sultana et al., 2005c). FA attenuates iron-induced oxida-             stresses (Bergeron et al., 1996; Poon et al., 2004). Evi-

Journal of Neuroscience Research DOI 10.1002/jnr
424     Perluigi et al.

dence indicates that HSP72 may contribute to cellular             age neurons (Mander and Brown, 2005). Therefore, de-
protection against a variety of stresses by preventing pro-       velopment of new compounds that can modulate these
tein aggregation, by assisting in the refolding of damaged        disease-linked biological processes might provide insight
proteins, and by serving as a chaperone for nascent poly-         into alternative therapeutic approaches and future identi-
peptides along ribosomes (Mayer and Bukau, 2005).                 fication of new drug targets (Ishii et al., 2000; Yan
      HSP induction not only is a signal for detection of         et al., 2003). Consistent with this notion, we found
physiological stress but is utilized by the cells in the repair   increased levels of iNOS and nitrated proteins (3-NT) in
process following a wide range of injuries, to prevent            synaptosomes isolated from DMSO-injected gerbils and
damage resulting from the accumulation of nonnative               then treated with Ab. We observed in the current study
proteins (Kelly and Yenari, 2002). In the present study,          a significant reduction of iNOS levels and 3-NT levels
we have shown that the levels of both HO-1 and HSP72              in synaptosomes isolated from FAEE-injected gerbils and
were elevated by Ab treatment as a cellular response to           treated ex vivo with Ab1–42. We have also shown that
the oxidative injury cascade activated by the peptide.            i.p. injection of FAEE alone is able to decrease levels of
      Our findings of even more increased levels of these          iNOS in synaptosomes. We suggest that, besides the
stress response proteins in Ab1–42-treated synaptosomes           effect of FAEE alone on iNOS, the protective effect of
isolated from FAEE-injected gerbils suggest that the acti-        FAEE relies on its ability to block the activation of
vation of HSPs (HSP72 and HO-1) is a protective                   iNOS induced by Ab1–42. Thus, FAEE not only is able
mechanism exerted by FAEE against Ab-induced oxida-               to decrease the basal level of iNOS but also has a
tive stress. Several studies have indicated that synapto-         marked ability to prevent the Ab1–42-dependent increase
somes have the capacity for protein synthesis (Steward            of iNOS, thus modulating the inflammatory process and
et al., 1991; Jimenez et al., 2002; Witzmann et al.,              the oxidative burden cascade activated by nitric oxide.
2005), and recent findings have shown, by using two-                      In conclusion, the present study demonstrates the
dimensional gel electrophoresis, that synaptosomes dis-           ability of FAEE to act as a potent antioxidant in vivo,
play differential expression in protein levels under vari-        thus providing neuroprotection against Ab-induced oxi-
ous conditions (Boyd-Kimball et al., 2005a). We have              dative stress. Our data suggest that the ester derivative of
previously demonstrated that HO-1 expression is                   ferulic acid, FAEE, shows higher lipophilicity with
increased in FAEE-treated astrocytes (Scapagnini et al.,          increased ability to penetrate the blood–brain barrier.
2004) and neurons (Sultana et al., 2005c) as a protective         We hypothesize a multifaceted mechanism of in vivo
mechanism against oxidative stress, with relevance to             neuroprotection by this compound: 1) FAEE is a potent
preconditioning. Consistent with these in vitro results,          free radical scavenger by significantly attenuating ROS
we have demonstrated in the current study that FAEE               production, protein oxidation, and lipid peroxidation; 2)
alone is able to lead to elevated HSP72 levels in synap-          FAEE is also neuroprotective by leading to elevated lev-
tosomes, thus confirming the likely ability of FAEE to             els of stress response proteins, such as HO-1 and HSP72;
provide neuroprotection in part by stimulating the stress         and 3) FAEE modulates neuroinflammatory processes
response. The heat shock response contributes to estab-           mediated by iNOS. Further studies are required to gain
lishing a cytoprotective state in a variety of metabolic          insight into the potential use of FAEE in the treatment
disturbances and injuries, including hypoxia, stroke, epi-        of AD and other oxidative stress-related disorders. Inves-
lepsy, cell and tissue trauma, neurodegenerative disease,         tigations on the use of FAEE on animal models of AD
and aging (Calabrese et al., 2004b; Latchman, 2004;               are in progress in our laboratory.
Mancuso, 2004). This notion has opened new perspec-
tives in medicine and pharmacology, in that molecules                         ACKNOWLEDGMENTS
activating this defense mechanism appear to be candi-
dates for novel cytoprotective strategies. In agreement               This work was supported in part by NIH grants
with this observation, we suggest that FAEE could pro-            AG-10836 and AG-05119 to D.A.B.
vide neuroprotection against Ab toxicity by modulating
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                                                                                                          Journal of Neuroscience Research DOI 10.1002/jnr

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