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Ferulic acid: a natural antioxidant against oxidative stress induced by oligomeric A-beta on sea urchin embryo.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition both of extracellular amyloid beta peptide (A[beta]) and intracellular neurofibrillar tangles (NFT). These hallmarks have been associated with loss of neurons in the brain and consequent learning and memory deficit. AP is the main component of the senile plaques and is believed to play a central role in the development and progress of AD. A[beta] is produced by the sequential and 13-secretase proteolytic cleavage of the large transmembrane amyloid precursor protein (APP) (Nunan and Small, 2000). Although the aggregation of [beta]-amyloid is thought to be a critical step in the pathogenesis of AD, there is a growing consensus that those smaller soluble AP oligomers rather than mature amyloid fibrils are the more pathogenic species in AD (Refolo et al., 1995; Lambert et al., 1998; Walsh et aL, 2002). However, the mechanism by which these oligomers mediate toxicity remains unclear. In several experimental or transgenic AD models, it has been demonstrated that excessive oligomeric Ap production causes pathological cellular effects, including oxidative stress that, in turn, induces neurodegeneration via apoptosis (Magrane et al., 2005; Oddo et al., 2006; Oakley et al., 2006; De Felice et al., 2009; Picone et al., 2009). Generation of reactive oxygen species (ROS) is the result of normal cellular processes involving oxygen, and in healthy conditions, a variety of mechanisms protect the cells against excess ROS with peroxide-eliminating enzymes (Drage, 2002). However, different stimuli can produce chronic or abrupt increases in ROS levels above a physiological threshold, interfering with normal cellular mechanisms and triggering cell death. A large number of studies indicate that oxidative stress could be important in AD etiology and progression due to its perturbation of mitochondrial integrity and cellular energy homeostasis (Reddy, 2006). In light of these results, suppression or reduction of oxidative stress could be a promising preventive or therapeutic intervention for AD patients. Indeed, many antioxidant compounds, such as vitamin E (Subramaniam et at., 1998), nordihydroguai-arctic acid (NDGA) (Goodman et al., 1994), and nicotine (Kihara et al., 1997), have been demonstrated to protect the brain from AP neurotoxicity. Ferulic acid (FA) (4-hydroxy-3-methoxycinnamic acid), a natural antioxidant present in plant cell walls, has high anti-inflammatory and antioxidant properties, being able to act as a scavenger of free radicals (Ozaki, 1992; Graf, 1992; Scott et at., 1993; Fernandez et at., 1998; Kikuzaki et al., 2002; Kanski et at., 2002; Ogiwara et al., 2002). Further, administration of FA induces resistance to Ap toxicity in mice and has been suggested to be useful as a chemo-preventive agent against AD (Yan et al., 2001). However, little information is available about its molecular mechanism of action.

ROS generation plays a role in the activation of extracellular signal-regulated kinase (ERK) pathways, as demonstrated by studies using ROS inhibitors (Zhang et aL, 2000, 2003; Ramachandiran et al., 2002; Lee et at., 2003; Nabeyrat et al., 2003; Kohda et aL, 2003; Sinha et at., 2004; Lee et at., 2005; Matsunaga et at., 2005). Depending on the cell type and the nature of the injury, activation of the Ras/Raf/ERK pathway is associated with either the extrinsic or intrinsic apoptotic pathway (Wang et at., 2000; Zhang et al., 2003, 2004; Kim et al., 2003; Nesterov et al., 2004; Schweyer et al., 2004; Basu and Tu, 2005; Li et at., 2005; Jo et at., 2005; Wu et at., 2005; Martin et al., 2006; Glotin et al., 2006; Liu et al., 2008; Yang et al., 2008). Moreover, several studies indicate that upregulation of the tumor suppressor p53 may be an important mechanism of Ras/Rat/ERK-induced apoptosis (Persons et al., 2000; She et at., 2000; Woessmann et al., 2002; Shih et al., 2002; Brown and Benchimol, 2006; Liu et at., 2008). All these results suggest that use of antioxidants could be a strategy to inhibit oxidative stress and activation of the signaling cascade.

Model organisms are widely used to explore potential causes and treatments for human diseases when human experimentation would be unfeasible or unethical. Although classically, researchers have modeled human diseases in cell lines or mouse, simple model systems such as the nematode Caenorhabditis elegans, the zebrafish Danio rerio, the fruit fly Drosophila melanogaster, the ascidian Ciona intestina-Its, and sea urchin, transgenic or not, have also been used to study human neurodegenerative diseases (Virata and Zeller, 2010; Di Carlo, 2011). These model systems can offer many advantages in obtaining information about the toxic mechanisms underlying the diseases and can easily be used to test the efficacy of putative neuroprotective compounds.

The animal model used in this study was the sea urchin, an organism that has significantly contributed to our understanding of many biological processes including gene regulation, molecular embryology, fertilization, cell and evolutionary biology, population genetics, and toxicology (Giudice, 1973; Di Carlo et al., 1996; Romancino et at., 2001). Part of its value as a model organism is its close phylogenetic relationship to humans. Sequencing of the Strongylocentrotus purpuratus genome has revealed the presence of about 7000 genes shared with humans, including genes associated with neurodegenerative pathologies (Sodergren et at., 2006). This finding agrees both with the presence of neurons and neuritis in the ciliary band, esophagus, and intestine, and with the existence of genes encoding known neurogenic transcription factors (Sodergren et al., 2006). In addition, Paracentrotus lividus embryos have been succes4u1ly utilized to investigate toxicity induced by different A1.3 aggregation forms (Carrotta et al., 2006; Pel-licana et al., 2009). Moreover, an antigen related to the human APP, called PAPP, has been identified and a relation between AP aggregation forms and activation of distinct apoptotic pathways demonstrated (Pelbeano et al., 2009). In sea urchins, some signaling molecules and regulatory pathways present in higher organisms are well conserved. Some evidence, for example, indicates the presence of the mitogen-activated protein kinase (MAPK) gene family, which is the ERK-mediating signaling required for primary mesenchyme cell ingression, spiculogenesis, and mesoderm differentiation--all mechanisms relevant for sea urchin embryonic development (Rottinger et at., 2004; Fernandez-Serra et al., 2004). Further, exposure of sea urchin embryos to ultraviolet radiation causes ROS production and leads to apoptosis via p53 gene upregulation (Lesser et al., 2003; Lin et al., 2008). Transcription factors of the Fork-head (Fox) gene family have been identified in many metazoans, including sea urchin, where they play an important role in such biological processes as apoptotic gene activation (Calnan and Brunet, 2008). Here we use the sea urchin Paracentrotus lividus as a model system to try to understand how FA protects from A[beta]-induced oxidative stress.

Materials and Methods

Recombinant A[beta]42 protein purification and oligomer production

Recombinant AP42 (rA[beta]42) peptide was obtained after cloning in pQE30 vector and expression as a fusion protein linked to a polyhistidine peptide (Carrotta et al., 2006). The protein expression was induced with 1 mmol [1.sup.-1] isopropyl-[beta]-D-thiogalactopyranoside (IPTG); after growing for 5 h at 37 [degrees]C, the cells were chilled in ice and harvested by centrifugation at 4000 X g for 15 min at 4 [degrees]C. Preparative SDS-PAGE of protein extracted from induced pQE30-Ap42 was carried out. A band corresponding to the induced protein was excised from the gel and electroeluted in 50 mmol [1.sup.-1] [NH.sub.4]C[O.sub.3] at 60 mA at 4 [degrees]C overnight, utilizing a Bio-Rad apparatus (Hercules, CA). The recovered samples were dried in a speed vacuum. Under physiological pH conditions, rA[beta]42 forms, in vitro, small oligomers, whereas at acidic pH large fibrillar aggregates are produced. To obtain exclusively small oligomers, the powder of rA[beta]42 was dissolved in 0.01 mol Tris-HCI buffer, pH 7.2, and the solution was readily characterized by dynamic light scattering at T = 15 [degrees]C (Carrotta et at., 2006).

Eggs and embryos treatment

Paracentrotus lividu.Y sea urchin eggs were demembraned by fertilization in 2 mmol [1.sup.-1] PABA. Approximately 1000 embryos were added to artificial seawater (ASW) in 24-well plates, and when the two-cell stage was reached, purified rA[beta]42 oligomers (1.5 [micro]mol[1.sup.-1] and 3 [micro]mol [1.sup.-1]), alone or combined with FA at 25 or 40 [micro]]mol [1.sup.-1], were added. Untreated embryos or embryos treated with FA were utilized as control. After 48 11, the effect on morphogenesis was observed by microscopic inspection, and representative pictures of the samples were recorded using a Zeiss Axioscope 2 microscope. All the assays were repeated three times with different batches. The successive assays were performed using rA[beta]42 at 1.5 [micro]mol [1.sup.-1] and FA at 40 [micro]mol [1.sup.-1].

Analysis of reactive oxygen species (ROS) generation

To assess ROS generation, the embryos were untreated (control) or treated with rA[beta]42 oligomers alone, or combined with FA, or with FA alone, or with H202 as control, for 48 h. Afterward, the embryos were incubated with 1 [micro]mol [1.sup.-1] dichlorofluorescein diacetate (DCFH-DA) in PBS for 10 min at room temperature in the dark. The use of DCFH-DA is founded on the inability of any fluorescent fluorescein derivative to emit fluorescence after being oxidized by hydrogen peroxide. In healthy cells, DCFH-DA crosses cell membranes and is enzymatically hydrolyzed by intracellular esterases to non-fluorescent DCFH. In the presence of ROS, DCFH is oxidized to highly fluorescent di-chlorofluorescein (DCF). The intensity of fluorescence is directly proportional to the hydrogen peroxide concentration. After the treatment with DCFH-DA, the embryos were washed in PBS (137 mmol [1.sup.-1] NaC1, 2.7 mmol [1.sup.-1] KC1, 8 mmol [Na.sub.3][PO.sub.4], pH 7.4) and fluorescence images were obtained with an Axioscope 2 microscope (Zeiss) and captured with an Axiocam digital camera (Zeiss) interfaced with a computer. Fluorescence emission green (529 nm) was evaluated by fluorimeter (Microplate reader Wallac Victor 2 1420 Multilabel Counter, Perkin Elmer) with a 488-nm excitation laser.

Mitochondrial membrane potential assay

Mitochondrial membrane potential was measured directly by using the MitoProbe JC-1 assay kit (Molecular Probes, Eugene, OR). The kit employs a unique cationic dye (5,5',6,61-tetrachloro-1,1',3,3 ' -tetraethy lbenzimidazolylcar-bocyanine iodide) to signal the loss of the mitochondrial membrane potential. At physiological membrane potential, JC-1 forms red fluorescent aggregates in the mitochondrion. In stressed cells, instead, the mitochondrial membrane potential collapses, and JC-1 cannot accumulate in the mitochondria but remains in the cytoplasm in the green fluorescent monomeric form. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Sea urchin embryos were untreated (control) or treated with rA[beta]42 oligomers alone or combined with FA, or with FA alone for 48 h. Afterward, the embryos were incubated with 2 mmol JC-I fluorescent dye in PBS for 30 min; CCCP (carbonyl cyanide 3-chlorophenylhydrazone) (50 mmol [1.sup.-1]), a proton ionophore that destroys the membrane potential across the mitochondrial membrane, was used as control. The fluorescence emission shift of JC-1 from red (590 nm) to green (529 nm) was evaluated by fluorimeter (Microplate reader Wallac Victor 2 1420 Multilabel Counter, Perkin Elmer) with a 488-nm excitation laser. The red and green fluorescence was visualized with an Axioscope 2 microscope (Zeiss).

Immunofluorescence assay

P. lividus embryos were untreated (control) or treated with rA)942 oligomers alone, or combined with FA, or with FA alone for 48 h. After washing in ASW, the embryos were fixed in freshly prepared 4% paraformaldehyde in PBS for 30 min, and kept at 4 [degrees]C. After three washes in PBS, the embryos were incubated for 1 h with 3% bovine serum albumin (BSA)/PBS. Then the samples were incubated with anti-p-ERK (1:100; Santa Cruz), or anti-p53 (1:100; Santa Cruz), or anti-Foxo3a (1:100 Cell Signaling), at 4 [degrees]C overnight. After three washes in PBST, the samples were treated with anti-rabbit TRITC-conjugate secondary antibody (1: 300 SIGMA), and anti-mouse FITC conjugate secondary antibodies (1:300 SIGMA). Fluorescent images were observed with an Axioscope 2 microscope (Zeiss) and captured with an Axiocam digital camera (Zeiss) interfaced with a computer.

Apoptosis and Caspase3 activity assays

A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Promega) was performed according to the manufacturer's instructions. The embryos were treated as described above and fixed in 4% paraformaldehyde in PBS for 30 min. After three washes in PBS, the embryos were permeabilized with 0.2% Triton X-100 in PBS for 5 min, rinsed with PBS, and incubated with a TUNEL reaction mixture (enzyme and nucleotides) in a humidified atmosphere at 37 [degrees]C for 1 h. Staining was obtained by using a peroxidase substrate, hydrogen peroxide, and the stable chromogen diaminobenzidine (DAB). After these incubations, the samples were rinsed three times in PBS and analyzed under a Zeiss Axioscope microscope. Caspase-3 activity was measured using commercially available luminescent assays (caspase-Glo 3/7 assay systems, Promega). Caspase reagent was added directly to the embryos growing in white 96-well plates (100 embryos in 100 [micro]l of seawater), and the embryos were incubated for 15-30 min before recording luminescence with a Wallac Victor 2 1420 Multilabel Counter (Perkin Elmer).

Statistical analysis

All experiments were repeated three times (n = 3), and in each experiment the samples were taken in triplicate. The results are presented as mean [+ or -] SD. Statistical evaluation was conducted by ANOVA, followed by a Bonferroni test for analysis of significance. Results with a P value <0.05 were considered statistically significant, *P < 0.05, **P < 0.01.

Results

Ferulic acid protects Paracentrotus lividus embryos against rA[beta]42 oligomers toxicity

To examine whether FA is able to reverse cytotoxicity induced by AP oligomers on P. lividus embryo, a morphological assay was performed. Under stress, sea urchin responds by altering morphology, and a correlation between abnormal embryos and toxicity can be detected.

Two-cell-stage P. lividus embryos were incubated with two rA[beta]42 oligomer concentrations (1.5 [micro]mol [1.sup-1] and 3 [micro],mol [1.sup-1]) and left to develop for 48 h until the control reached the pluteus stage (Fig. 1). Depending on the oligomer concentration, different morphological defects were found in the embryos. At the lower rA[beta]42 oligomer concentration (1.5 [micro]mol [1.sup-1]), P. lividus failed to complete morphogenesis, displaying a range of phenotypes including prismoid form; whereas at the higher oligomer concentration (3 [micro]mol [1.sup-1]), the embryos were completely degenerated (Fig. 1C). When different FA concentrations (25 or 40 [micro]mol [1.sup-1]) were added, the embryo morphology was recovered proportionally depending on the damage intensity. FA addition thus allowed for obtaining either embryos with a decreased cellular disorder or embiyos resembling prisms and plutei. The result of this experiment gives significant evidence of the protective effect of FA on oligomeric rA[beta]42-induced toxicity on sea urchin embryo.

Ferulic acid scavenges ROS produced by amyloid beta peptide

Some evidence suggests that AP is able to generate free radicals and oxidative damage. To investigate whether the protective effect produced by FA is due to its ability to act as a free radical scavenger, we performed an experiment to measure intracellular ROS formation. We utilized a fluorometric assay in which the emitted fluorescence is proportionally correlated to the hydrogen peroxide concentration inside the cell. P. lividus embryos after addition of rA[beta]42 were incubated without or with FA, and the emitted fluorescence was analyzed by microscopic observation. The intensity of fluorescence, corresponding to the intracellular ROS presence, increased when the embryos were incubated with rA[beta]42 or with [H.sub.2][0.sub.2] used as control. In contrast, FA, in agreement with its antioxidant properties, lowered the intensities of fluorescence elicited by rA[beta]42 or [H.sub.2][0.sub.2]. No fluorescent signal was detected in untreated or FA-treated embryos (Fig. 2A). We quantified the green fluorescence by fluorimetric analysis (Fig. 2B).

Ferulic acid restores the nzitochondrial membrane potential

Generation of ROS leads to mitochondrial oxidative damage and induces collapse of the electrochemical gradient across the mitochondrial membrane. To confirm that FA reduces ROS generation, we measured mitochondrial membrane potential (Alin) using the JC-1 assay, in which mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio (Fig. 3). P. lividus embryos were untreated or treated with rA[beta]42 alone or with FA and with CCCP as controls. Both rA[beta]42 and CCCP treatments significantly depolarized the [DELTA][[PSI].sub.m], and the embryos mainly showed an intense green fluorescence. In contrast, when FA was added after rA[beta]42 treatment, the [DELTA][[PSI].sub.m], was similar to control conditions, and the embryos mainly showed an intense red fluorescence, indicating that FA had counteracted amyloid beta toxicity. We quantified the red and green fluorescence by fluorimeter, and the mitochondrial membrane depolarization is indicated by a decrease in the red/green fluorescence intensity ratio (Fig. 3B).

Foxo3a is inhibited by ferulic acid administration

Foxo3a is a member of the forkhead transcription factor gene family that is able to activate apoptotic genes, especially as a consequence of oxidative stress generation. To explore whether the expression of this protein may be regulated by the treatments here utilized, we performed an immunohistochemistry experiment using an antibody against Foxo3a. The images of P. lividus embryos treated with rA[beta]42 oligomers without or with FA are shown in Figure 4A. Intense fluorescence is detected when sea urchin embryos were treated with rA[beta]42 alone, whereas a faint signal is detectable when embryos were treated with rA[beta]42 and FA together. We quantified the red fluorescence (Fig. 4B). FA, inhibiting ROS production, downregulated Foxo3a expression and perhaps the apoptotic genes activated by it.

Ferulic acid reduces apoptosis induced by rAP42 oligomers

A consequence of oxidative stress can be apoptosis activation. To investigate whether FA action affects apoptosis, we fixed survived or rescued P. lividus embryos and tested them with the TUNEL assay. Only a few stained nuclei were detected in the control embryos, probably as a result of physiological events, whereas embryos treated with oligomers showed a large number of well-stained nuclei, indicating that nuclear fragmentation was occurring. Upon combined treatment with rA1342 and FA, a drastic reduction in the number of stained nuclei was visible, demonstrating that FA had counteracted amyloid-induced cell death (Fig. 5A).

A[beta]-induced apoptosis occurs mainly through a caspase-dependent pathway even if others have been described (Nakagawa et al., 2000). To investigate whether FA is able to reverse caspase activation, the same samples indicated above were submitted to a caspase-activation assay (Fig. 5B). As expected, rA[beta]42 oligomers activated caspase 3 enzyme, whereas administration of FA combined with rA[beta]42 oligomers inhibited its activation, demonstrating that FA had neutralized the degenerative amyloid effect.

Ferulic acid reduces extracellular signal-regulated kinase activation and p53 upregulation induced by rA[beta]42

Among the MAPK family, a role for the ERK1/2 pathway in AD neuronal death has been proposed. (Chong et al., 2006). Moreover, ERK activation and p53 upregulation are associated with cell death induced by ROS (Cagnol and Chambard, 2009). Since in sea urchin, as demonstrated above, ROS generation and cell death occur after treatment with rA[beta]42 oligomers, we explored whether these molecules are also activated under this stimulus and whether their expression can be downregulated after FA treatment. By immunofluorescence experiments, we observed that rA[beta]42 treatment induced a considerable activation of ERK and a significantly higher p53 protein level with respect to the control. Moreover, merged images showed colocalization of these proteins around the esophagus, suggesting that p53 upregulation was dependent on ERK activation. In contrast, when the embryos were simultaneously treated with both rA[beta]42 and FA, levels of ERK activation and p53 were reduced (Fig. 6).

Discussion

Increasing evidence suggests that many of the biochemical changes in neurodegeneration may be secondary to oxidative stress and mitochondrial abnormalities including deficits in key enzymes of mitochondrial oxidative metabolism. The use of antioxidants has been recognized as an important countermeasure against conditions in which oxidative stress is implicated. Ferulic acid exhibits a wide range of therapeutic properties such as anti-diabetic, anticancer, anti-ageing, and neuroprotective effects (Srinivasan et al., 2007). Many of FA therapeutic potentials can be attributed to its anti-inflammatory activity and, in particular, to its antioxidant capacity. The latter is due to the presence in its structure of a phenolic hydroxyl group and extended side chain conjugation that are able to donate electrons to quench the free radicals. Moreover, FA is involved in the chelation of metals that play a crucial role in oxidative damage to biological materials (Srinivasan et al., 2007). Thus, the use of FA could be a preventive method to scavenge or capture free radicals before they attack important cellular targets. Further, as already suggested (Kanski et al., 2002), even in the absence of experimental evidence, we speculate on the basis of its structural similarity to salicylic acid, which can cross the blood-brain barrier, that FA should be able to enter the central nervous system.

All this evidence suggests that FA could be of interest for Alzheimer's disease therapy. To address this hypothesis, we investigated the protective effect of FA against A/342-induced toxicity using sea urchin as an in vivo model system. This system represents a good model for testing the effect of physical and chemical stresses and could be employed for a first screening of several new drugs, which could be used in future clinical trials. We had already demonstrated that the embryo of Paracentrorus lividus is very sensitive to treatment with rA[beta]42 oligomers (Carrotta et at., 2006; PellicanO et al., 2009), suggesting that some altered biochemical mechanisms, occurring in AD, could be mimed in this animal model. To validate this model system as a possible candidate for AD drug screening, we tested the ability of FA to offer protection from the oxidative stress and subsequent apoptosis induced by rA[beta]42 oligomers.

As a first step we demonstrated, by morphological assay, that FA reduced embryo perturbation induced by oligomeric rA[beta]42, indicating that P. lividus is able to respond to FA treatment. ROS generation and mitochondrial dysfunction are two effects of beta amyloid presence in both in vitro and in vivo systems (Reddy, 2006). After rA[beta]42 oligomers were administered, P. lividus embryos responded with ROS generation and disruption of mitochondrial membrane potential as in higher model systems, and FA counteracted these dysfunctions. Similarly, after FA treatment, apoptosis and caspase3 activation were affected, indicating that its antioxidant effect had interfered with the degenerative process. Although A[beta] toxicity was classically considered to be due to its extracellular deposits, cellular and biochemical studies have recently provided evidence that this peptide accumulates inside neurons, targeting mitochondria, where it contributes to disease progression (Lustbader et al., 2004). It has, indeed, been demonstrated that the mitochondria] protein A[beta]-binding alcohol dehydrogenase (ABAD) is a direct molecular link between Ap and mitochondrial toxicity (Lustbader et al., 2004). These data were confirmed by using a peptide that specifically inhibits ABAD-A[beta] interaction and suppresses A[beta] free radical generation and apoptosis induction in neurons (Lustbader et al., 2004). Then, by using in vivo and in vitro approaches, it has been demonstrated that Af3 is transported into mitochondria via the translocase of the outer membrane, localized within the mitochondria] cristae (Hansson et al., 2008). Moreover, biochemical studies suggest that formation of the mitochondria] permeability transition pore is involved in A[beta]-mediated mitochondrial dysfunction (Moreira etal., 2001). Thus, the comprehension that the mitochondrion is at the intersection of cell life and death has made it a promising target for drug discovery and therapeutic interventions, and FA could be a good candidate for these processes.

Furthermore, in agreement with the evolutionarily conserved mechanisms activated by rA[beta]42 toxicity and inhibited by FA in sea urchin, some molecules involved in these processes are present. Modulation of the level of Foxo3a expression after treatment with rA[beta]42 without or with FA was observed. Foxo3a is a transcription factor that, depending on its requirement, is localized in the cytoplasm bound to 14.3.3.3 protein or in the nucleus, where it activates different classes of genes among apoptotic genes (Calnan and Brunet, 2008). When a cell is exposed to molecules such as H202 or A[beta] that induce oxidative stress through a path including phosphorylation by JNK. Foxo3a is translocated into the nucleus, where it activates apoptotic genes. In the Strongylocentrotus purpuratus genome, Spfoxo genes have been characterized and a role in specification of the skeletogenic lineage and biomineralization has been assigned (Tu et al., 2006). Here, we demonstrate that, even in sea urchin, Foxo is involved in the signaling activated by oxidative stress as it occurs in higher organisms. Treatment with rA[beta]42 increases Foxo levels, which are reduced by administration of FA. Moreover, it is well known that ROS presence can initiate and sustain ERK activation; here we show that, in response to oxidative stress induced by rA[beta]42 oligomers, embryos of P. lividus activate ERK and upregu-late p53, but that addition of FA reduces the levels of ERK and p53, thus counteracting cell degeneration.

Conclusions

Ferulic acid could represent an exciting challenge for treatment of pathologies in which oxidative stress is involved and particularly in Alzheimer's disease. Furthermore, Paracentrotus lividus represents a good model system for the study of mitochondria] dysfunction in the neurodegenerative process. This species can also be employed for testing new drugs. Thus, the use of the sea urchin model may open a window for new therapeutic strategies aimed at preserving or improving mitochondria] function.

Acknowledgments

We thank Mr. Luca Caruana for his useful technical assistance. This work was partially supported by the Italian Ministry of University and Research with the PRIN project ("Sviluppo di una strategia molecolare per la prevenzione dell'aggregazione proteica e della fibrillogenesi: un ap-proccio biofisico") and by the Italian Ministry of Economy and Finance with the "PNR-CNR Aging Program 20122014" project.

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PASQUALE PICONE, DOMENICO NUZZO, AND MARTA DI CARLO *

Istituto di Biomedicina ed Immunologia Molecolare (IBIM)-CNR, via Ugo La Malfa 153, 90146, Palermo, Italy

Received 28 June 2012: accepted 27 December 2012.

* To whom correspondence should be addressed. E-mail: dicarlo@ibim.cnr.it

Abbreviations: AD, Alzheimer's disease: APP, amyloid precursor protein: A[beta]. amyloid beta peptide: CCCP, carbonyl cyanide 3-chlorophenyl-hydrazone; DCFS-DA, dichlorofluorescein diacetate; ERK, extracellular signal-regulated kinase: FA, ferulic acid (4-hydroxy-3-methoxycinnamic acid): ROS, reactive oxygen species; TUNEL. terminal deoxynucleotidyl transferase dUTP nick end labeling [assay].
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Author:Picone, Pasquale; Nuzzo, Domenico; Di Carlo, Marta
Publication:The Biological Bulletin
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Date:Feb 1, 2013
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