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NEUROPHARMACOLOGY
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, University of Piemonte Orientale "Amedeo Avogadro", Novara, Italy
Received August 25, 2006; accepted October 25, 2006.
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Abstract |
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). NAEs are produced from the corresponding N-acylphosphatidylethanolamines through the action of Ca2+-activated D-type phosphodiesterases. Under physiological conditions, NAE occur at low levels in virtually all mammalian cells, tissues, and certain body fluids (Bisogno et al., 1999), and their production is increased by stress, especially under conditions of cell and tissue degeneration (Hansen et al., 2001). Under these conditions, the greatest increase regards saturated and monounsaturated members of this class of endolipids, all cannabinoid receptor-inactive compounds (Schmid and Berdyshev, 2002).
N-Palmitoylethanolamine (PEA) is a fully saturated, bioactive, and endogenous NAE, first identified half a century ago in lipid extracts of various tissues (Bachur et al., 1965). PEA is endowed with antiallergic, anti-inflammatory, and antinociceptive properties (Calignano et al., 1998) and may elicit a wide range of in vivo effects that includes analgesia, inhibition of food intake, reduction of gastrointestinal motility, inhibition of cancer cell proliferation, and cytoprotection (Lambert and Di Marzo, 1999). Under physiological conditions, PEA is present at high concentrations in the central nervous system (Cadas et al., 1997), where its concentrations significantly increase under pathological conditions, like brain ischemia (Franklin et al., 2003; Berger et al., 2004), excitotoxicity (Di Marzo et al., 1994; Hansen et al., 1995), and neuroinflammation (Darmani et al., 2005). In vitro and in vivo experiments suggest that PEA has neuroprotective activity; it protects cerebellar granule neurons against excitotoxicity (Skaper et al., 1996), inhibits electroshock- and chemically induced seizures in mice (Sheerin et al., 2004), and enhances microglial cell motility (Franklin et al., 2003). PEA exhibits poor affinity for cannabinoid CB1 or CB2 receptors (Sheskin et al., 1997), but, surprisingly, its antinociceptive effects are inhibited by SR144528, a selective CB2 receptor antagonist (Calignano et al., 1998). PEA produces a 2-fold decrease in the Ki value for the AEA binding at the transient receptor potential-type vanilloid 1 receptor (De Petrocellis et al., 2001) and acts as an endogenous ligand for the peroxisome proliferator-activated receptor-
(Lo Verme et al., 2005). PEA is hydrolyzed by at least two enzymes: the fatty acid amide hydrolase (FAAH) (Schmid and Berdyshev, 2002) and the N-acylethanolamine-hydrolyzing acid amidase (Ueda et al., 2001). FAAH and N-acylethanolamine-hydrolyzing acid amidase are widely expressed in mammalian tissues (Ueda et al., 2001; Cravatt and Lichtman, 2002), and mice lacking the faah gene show reduced PEA hydrolysis and corresponding increased PEA levels in brain and liver tissues (Patel et al., 2005).
Taken together, these observations suggest that PEA is endowed with pharmacological properties potentially useful for inducing neuroprotection; however, the in vivo efficacy of PEA is low due to its rapid hydrolytic inactivation, and PEA does not qualify as suitable drug candidate. To overcome this problem and as part of a series of investigations on new neuroprotective agents, we have synthesized a series of homologs of PEA, characterizing their activities on two in vitro models of neurodegeneration (oxidative stress and excitotoxicity). The affinities and/or activities of these compounds for a series of molecular endpoints highly sensitive to variation in the polar head of endolipids [CB1,CB2, transient receptor potential-type vanilloid 1 receptor, FAAH, and anandamide membrane transporter (AMT)] have been published recently (Appendino et al., 2006). We present evidence that oxyhomologation of the amide bond of PEA, although leading to an increased stability toward amido-hydrolyzing enzymes, also potentiates the neuroprotective properties of the parent compound and propose a mechanistic rationale for this effect.
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Materials and Methods |
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; Appendino et al., 2006). (+)-MK 801 was obtained from Tocris Bioscience (Bristol, UK). AM251 and AM630 were from Alexis Bioscience (Vinci, Italy). Dulbecco's modified Eagle's medium (DMEM), DMEM-nutrient mixture Ham's F12, neurobasal medium, B27 supplement, and fetal calf serum were from Invitrogen (Milan, Italy). L-Glutamine, penicillin, streptomycin, all-trans-retinoic acid, tert-butylhydroperoxide (t-BOOH), vitamin E (-tocopherol), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Milan, Italy). All other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany).
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PEA, PEA oxyhomologs, and fatty acid hydroxamates were dissolved in dimethyl sulfoxide; all other drugs used were dissolved in the experimental media. Final drug concentrations were obtained by dilution of stock solutions into the experimental media. Final concentration of organic solvents was always less than 0.1%, and they have no effects on cell viability. Drugs were added to the experimental buffer 1 h before the insults and were maintained for the entire experiment. Cells exposed to solvent alone were considered as controls.
Cell Cultures. Human neuroblastoma cell lines, SH-SY5Y and SK-N-BE, were cultured in DMEM-nutrient mixture Ham's F12 and DMEM, respectively, supplemented with 10% (v/v) fetal calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). The cell culture medium was replaced every 2 days, and the cultures were maintained at 37°C, 95% air-5% CO2 in a humidified incubator. The cells were differentiated into neuron-like type by treatment with all-trans-retinoic acid (10 µM) that was added to the cell culture medium every day for either 7 (SH-SY5Y) or 14 days (SK-N-BE). The day before the experiments, differentiated cells were plated in a six-well culture plate (0.5–1 x 106 cells/well).
In Vitro Models of Neurodegeneration
Oxidative Stress. The in vitro model of oxidative stress was performed as described previously (Lombardi et al., 2002). In brief, differentiated SH-SY5Y cells were washed twice with phosphate-buffered saline (PBS) and exposed to t-BOOH (100 µM) in the experimental buffer (138 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM PBS, and 10 mM glucose, pH 7.4) for 3 h at 37°C.
Excitotoxicity. For the in vitro model of excitotoxicity, differentiated SK-N-BE cells were washed twice with PBS and incubated in neurobasal medium supplemented with B27 (16 h at 37°C) to increase their sensitivity to the excitotoxic insult. Afterward, the medium was changed, and cells were exposed to 1 mM L-glutamate for 24 h at 37°C.
Evaluation of Cell Viability and Cell Death
Cell Viability. Cell viability was evaluated with the MTT assay. Absorbance was measured at 570 to 630 nm by using an Ultramark microplate reader (Bio-Rad Laboratories, Milan, Italy). The percentage of neuroprotection was calculated as follows: percentage of neuroprotection = 100 – [(x – z) x 100/(x – y)], where x is the absorbance read in control samples, y is the absorbance read in t-BOOH- or L-glutamate-treated samples, and z is the absorbance read in drug- and either t-BOOH- or L-glutamate-treated samples.
Cell Death. Cell death was evaluated by measuring the lactic acid dehydrogenase (LDH) activity in the experimental medium at the end of the experiment by using a commercial kit (Roche Diagnostic, Penzberg, Germany) according to the manufacturer's instructions.
Studies on the Mechanisms of Action
Determination of Free Radical Production. Free radical production was measured by incubating the cells with the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate as described previously (Lombardi et al., 2002). Accumulation of 2',7'-dichlorofluorescein in the cells was measured as an increase in fluorescence at 525 nm, when the sample was excited at 488 nm using a Jasco (FP-777) spectrofluorometer (Jasco International, Tokyo, Japan).
Determination of Lipid Peroxidation. Lipid peroxidation was evaluated by measuring the levels of thiobarbituric acid-reacting substances (TBARS) in the cells at the end of the experiments as described previously (Miglio et al., 2004). In brief, the cells were washed and harvested with ice-cold 50 mM phosphate buffer, pH 7.4, then 500 µl of 1% thiobarbituric acid and 500 µl of 8 N HCl were added to each sample. The samples were boiled for 20 min and subsequently cooled with tap water. 1-Butanol (1.5 ml) was then added to the samples, and the mixture was shaken for 2 min. After centrifugation at 2000g for 10 min, the fluorescence intensity at 550 (excitation) and 532 nm (emission) in the butanol phase was measured by a Jasco (FP-777) spectrofluorometer.
Measurement of Intracellular Calcium Concentration. Intracellular calcium concentration was measured at single cell level by using a digital calcium image system. Fura-2-loaded cells (5 x 105 cells/ml) were firmly attached (15 min at 37°C) to poly-L-lysine (0.1 mg/ml)-coated thin (0.2-mm) round glass coverslips (4 cm), washed, transferred to a perfusion chamber (Bioptechs, Butler, PA), and mounted on an inverted microscope (Eclipse TE 300; Nikon, Tokyo, Japan). Experiments were performed at a chamber temperature of 37°C. All measurements were taken at 40-fold magnification. Excitation wavelengths were alternately selected at 340 and 380 nm by a monochromator system (Polychrome IV; TILL Photonics, Gräfelfing, Germany), and fluorescence, filtered at 505 nm, was taken with a gray-scale charge-coupled device camera (SensiCam; PCO, Kelheim, Germany). Images were acquired and analyzed with Axon Imaging Workbench 4.0 software (Axon Instruments, Union City, CA). Data were expressed as the fluorescence ratio (Fr) mean of n monitored cells when they were excited at 340 and 380 nm, respectively.
Reverse Transcriptase-Polymerase Chain Reaction Analysis. Total RNA was extracted from cells with the GeneElute Mammalian Total RNA Kit (Sigma-Aldrich) and treated (1 h, 25°C) with 5 U of DNase I, RNase-free (Roche Diagnostics). DNase was then inactivated by heating for 5 min at 95°C. Human thalamic RNA was purchased from Clontech Laboratories (Milan, Italy) and used as the positive internal control. Resulting RNA was reverse-transcribed with oligo(dT) primers using 10 µg of total RNA and the Thermo-Script reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis system (Invitrogen). PCR was performed in a 25-µl reaction mixture containing 2 µg of cDNA, 2.5 µl of x10 buffer, 1.5 µl of 50 mM MgCl2, 0.5 µl of a 10 mM dNTPs mix (Invitrogen), 2.5 U of TaqDNA polymerase (Invitrogen), and 2.5 µl of each primer (Table 1). RT-PCR amplicons were resolved in a 2% agarose gel by electrophoresis and visualized with ethidium bromide.
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Data Analysis. Results are expressed as means ± S.E.M. of at least six experiments. Statistical significance was evaluated by Student's t test for paired varieties. Differences were considered statistically significant when p < 0.05. Origin version 6.0 (Microcal Software, Northampton, MA) was used as a nonlinear regression model for analysis of the concentration-response data to obtain EC50.
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Results |
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), we studied possible neuroprotective effects of PEA (1a), PEA oxyhomologs (1b– d), and fatty acid hydroxamates (2a– c) against t-BOOH-induced (100 µM, 3h) oxidative stress. t-BOOH significantly reduced the percentage of cell survival (49.1 ± 4.0%; p < 0.01; n = 6) in comparison with controls (t-BOOH-untreated cells). During t-BOOH exposure, cell morphology was continuously observed by phase-contrast microscopy, and progressive necrosis of cell body and neurites was evident (data not shown). When the cells were treated with increasing concentrations (0.1–30 µM) of PEA, the percentage of cell death was significantly reduced (p < 0.05; n = 6). The maximal neuroprotective effect was 26.3 ± 7.5% at 30 µM PEA (Fig. 2a). Experimental problems, due to both low water solubility and toxic effects of PEA, did not allow us to use higher concentrations of this compound.
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Finally, to clarify the role of the hydroxamic group, the neuroprotective effects of three fatty acid hydroxamates (2a– c) were tested with the same experimental protocol. The N-hydroxypalmitamide (2a; 10 µM) abolished the effects of t-BOOH (the EC50 calculated value was 1.0 µM), whereas the N-hydroxynonamide (2b) was both less effective (the maximal neuroprotection observed was 36.4 ± 4.7% at 30 µM) and less potent (the EC50 calculated value was 6.2 µM) than 2a. No neuroprotective effects were observed for the N-hydroxyicosamide (2c) at all concentrations tested (Fig. 2c).
Effects of Cannabinoid Receptor Antagonists. Previous results from our laboratories show that PEA oxyhomologs have poor affinity for CB1/CB2 receptors and do not inhibit either the FAAH-mediated AEA hydrolysis or the AMT-mediated AEA cellular uptake (Appendino et al., 2006). To determine whether the neuroprotective effects evoked by these compounds are mediated by cannabinoid receptors, we repeated the above experiments in the presence of AM251, a selective cannabinoid CB1 receptor antagonist (Gatley et al., 1996), and AM630, a selective cannabinoid CB2 receptor antagonist (Ross et al., 1999). Neither AM251 (10 µM) nor AM630 (10 µM) antagonized the neuroprotective effects induced by increasing concentration (0.1–30 µM) of either the compound 1b (Fig. 3a) or 2a (Fig. 3b). The EC50 calculated values in the absence or presence of AM251 or AM630, respectively, were 1.7, 1.8, and 2.1 µM for 1b and 0.9, 1.2, and 1.2 µM for 2a.
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; Annunziato et al., 2003). To study the mechanisms underlying the neuroprotective effects of the 1b and 2a compounds, we measured TBARS and free radical levels in t-BOOH (100 µM; 3 h)-treated cells in the absence or presence of increasing concentrations of PEA, 1b, or 2a. t-BOOH treatment (100 µM; 3 h) induced a significant increase in both the TBARS (47.8 ± 8.6%; p < 0.01; n = 6) and the cellular free radical levels (11-fold increase of the measured fluorescence; p < 0.01; n = 6) if compared with t-BOOH-untreated cells. The compounds 1b and 2a significantly prevented these increases in a concentration-dependent manner. Both 1b and 2a abolished TBARS formation at 10 µM, and the EC50 calculated values were 0.12 and 0.34 µM, respectively. On the contrary, the fold of free radical increase was only partially reduced by either 1b or 2a (81.3 ± 2.8% and 57.1 ± 5.2%, respectively; p < 0.01; n = 6) at 1 µM. PEA did not prevent either TBARS formation or free radical increase at all concentrations tested (Fig. 4, a and b).
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Moreover, to determine whether these compounds are capable of preventing the [Ca2+]i increase elicited by free radicals (Annunziato et al., 2003), we measured the [Ca2+]i increase evoked by t-BOOH treatment (100 µM; 3 h) in fura-2-loaded cells treated or not with compounds 1b or 2a. Both compounds, added to the experimental medium 60 min before t-BOOH, prevented the [Ca2+]i increase elicited by oxidative stress (Fig. 4c).
Excitotoxicity. Since controversial results have been reported on the neuroprotective effects of PEA against excitotoxicity (Skaper et al., 1996; Andersson et al., 2000), we studied the effects of PEA and the new PEA homologs (1b and 2a) in an experimental model of excitotoxicity. Differentiated SK-N-BE neuroblastoma cells express NR1, NR2A, and NR2B subunits of the NMDA receptor, as assessed by RT-PCR analysis (Fig. 5, line 2). When these cells were exposed to L-glutamate (1 mM; 24 h), a significant increase (60.5 ± 2.8%; p < 0.01; n = 6 in comparison with L-glutamate-untreated cells) in the level of LDH activity was measured in the experimental medium at the end of experiments. (+)-MK 801 (1 µM), a noncompetitive NMDA receptor antagonist, was able to antagonize L-glutamate-induced LDH increase (Fig. 6a). When cells were exposed to L-glutamate (1 mM; 24 h) in the presence of increasing concentration (0.01–30 µM) of either 1b or 2a, the levels of LDH activity were significantly decreased. The maximal protection (68.4 ± 0.1% and 67.2 ± 3.3%, respectively; p < 0.01; n = 6) was measured at 10 µM 1b or 2a; the EC50 calculated values were 0.35 and 0.69 µM, respectively. On the contrary, PEA resulted ineffective at all concentrations tested (0.01–30 µM) (Fig. 6b).
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). Therefore, we studied whether cell exposure (60 min before L-glutamate) to either 1b or 2a may prevent the delayed L-glutamate-induced [Ca2+]i increase in fura-2-loaded cells. The Fr mean measured from control cells was 0.71 ± 0.04 (n = 50). Cell exposure to L-glutamate (1 mM, 24 h) produced a significant increase in the [Ca2+]i (Fr mean = 1.22 ± 0.06; n = 54; p < 0.01) that was prevented by (+)-MK 801 (1 µM; 60 min before L-glutamate), a noncompetitive NMDA receptor antagonist (Fr mean = 0.81 ± 0.05; n = 55). Both compounds 1b (10 µM) and 2a (10 µM) significantly reduced (–50 ± 10% in comparison with L-glutamate-treated cells) the L-glutamate-induced [Ca2+]i increase (Fr means were 0.91 ± 0.07 and 0.92 ± 0.08, respectively; n = 52 for each compound; p < 0.05), whereas PEA (up to 10 µM) had no effect (Fig. 7). The significant (p < 0.05) [Ca2+]i increase still present, compared with control (L-glutamate-untreated cells), in cells exposed to 1b and 2a supports the residual excitotoxicity (
30%) measured (Fig. 6b).
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Discussion |
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; Barnham et al., 2004). Exogenous or endogenous compounds capable to prevent or block the progression of these harmful events may be relevant for neuroprotection (Levi and Brimble, 2004). NAE are endogenous compounds that are part of a protective physiological system providing on-demand defense in case of abnormal neuronal activity (Marsicano et al., 2003). Neurodegeneration is associated with a massive accumulation of PEA (Hansen et al., 2001), whose neuroprotective effects are, however, still a matter of debate. The beneficial effects, reported in some studies (Skaper et al., 1996), have been questioned and could not be reproduced by others (Andersson et al., 2000; Franklin et al., 2003). Recently, Zolese et al. (2005) demonstrated a combination of both antioxidative and slightly prooxidative effects of PEA on Cu2+-induced low-density lipoprotein oxidation. Antioxidative effects were observed at low PEA concentrations (0.01–0.1 µM), whereas pro-oxidative effects dominated the profile of this compound at higher concentrations (1 µM). From our findings, 30 µM PEA partially (26.3 ± 7.5% in comparison with t-BOOH-untreated cells) protected neurons against oxidative stress, but toxicity was observed at higher (>30 µM) concentrations. In addition, PEA was incapable of preventing L-glutamate-induced excitotoxicity in our experimental model. Discrepancies in the results from different laboratories might be related to differences in experimental models (i.e., cell culture type, potency of the insult, time exposure to the insult, experimental buffers, chemical stability of solutions), but it seems also clear that the problems associated to the metabolic stability of PEA are compounded by a substantially biphasic (anti- or proneurotoxic) profile of activity. PEA-metabolizing enzymes are widely expressed in mammalian tissues, and the cells used for these experiments can, in principle, metabolize PEA and PEA derivatives because they express FAAH (RT-PCR analysis; data not shown). Therefore, the stabilization of PEA molecule seems to be essential to enhance its bioactivity.
To increase the metabolic stability of PEA and shift its biphasic biological profile toward neuroprotection, we modified the polar head group of the molecule. We reasoned that oxyhomologation of the amide bond, although having a stabilizing effect toward enzymatic hydrolysis because of topologic and electronic changes in the amide bond, should also induce metal-chelating properties potentially capable to interfere with oxidative stress (Gaeta and Hider, 2005). Indeed, compared with the parent compound, the oxyhomologs of PEA (1b– d) showed greater efficacy and potency in protecting neurons against oxidative stress and excitotoxicity, with N-homologation being more efficient in this respect compared with carbonyl or alkyl oxyhomologation (compare 1b with 1c and d). Spurred by the activity of the N-hydroxyamide 1b, we also investigated the activity of its parent compound, palmitoyl hydroxamic acid (2a). This compound showed potent neuroprotective activity, even larger than that of the oxyhomologs of PEA. Shortening or lengthening of the acyl chain, as in capryl (C-9; 2b) and arachyl (C-20; 2c) hydroxamates, led to a decrease of activity, showing that the activity associated to the hydroxamate group is modulated in a specific fashion by the lipid moiety. Although hydroxamic acids have been well investigated for their bioactivity (Yoo and Jones, 2006), remarkably little is known on their N-alkyl derivatives (N-hydroxyamides). The observation that N-palmitoyl-N-(2-hydroxyethyl)hydroxylamine retains most of the neuroprotective activity of palmityl hydroxamate despite its decreased chelating properties is a significant observation, worth pursuing by in a systematic way with a larger sets of substrates.
Remarkably, oxyhomologation had little, if any, effect on the interaction of PEA with cannabinoid receptors and the enzymes involved in the degradation of endocannabinoid (Appendino et al., 2006). For these reasons, amide oxyhomologs could achieve substantially increased concentrations and a longer half-life at their sites of activity.
Rather than depending on the interaction with cannabinoid receptors and their associated proteins, the neuroprotective effects of the oxyhomologs (1b– d) or the hydroxamates (2a and b) are seemingly related to their metal-chelating and antioxidant activity (Gaeta and Hider, 2005). Moreover, neither AM251, a selective CB1 receptor antagonist, nor AM630, a selective CB2 receptor antagonist, interfered with the neuroprotective effects of 1b or 2a; compounds 1b to d showed negligible affinity for cannabinoid CB1 and CB2 receptors and did not prevent AEA hydrolysis catalyzed by FAAH or its cellular uptake mediated by the putative neuronal AMT (Appendino et al., 2006); compounds 1b and 2a prevented t-BOOH-induced lipid peroxidation/free radical accumulation; and compounds 1b and 2a prevented the [Ca2+]i increase elicited by both free radical actions (Annunziato et al., 2003) and L-glutamate activation of NMDA receptors (Choi 1988). It is therefore possible that these compounds protect neurons against oxidative insults by inhibiting chain reactions leading to free radical accumulation, seemingly by a metal-chelating mechanism, a well known property of hydroxamates (Gaeta and Hider, 2005). Direct inhibition of NMDA receptor channels is unlikely since most of the NMDA ligands are, indeed, amino acids, phosphono amino acids or amines that can be protonated under physiological conditions (structure-activity relationship studies) (Brauner-Osborne et al., 2000). Two classes of amphiphilic compounds, lysophospholipids and arachidonic acid, have been demonstrated to modulate NMDA receptors through insertion into plasma membranes (Casado and Ascher, 1998). Our compounds might elicit similar effects, but the molecular basis for the neuroprotective activity of PEA is still substantially unclear. However, it does not seem unreasonable to assume that the hydroxamate moiety potentiates the intrinsic neuroprotective properties of this compound. Taken together, our results suggest that implanting a metal-chelating motif on endolipid structures leads not only to an increase of metabolic stability but also to a potentiation of the neuroprotective properties, without substantially changing the profile of interaction with the endocannabinoid/endovanilloid system and their associated proteins.
Ion metals, particularly iron and copper, play a critical role in both oxidative stress and abnormal metal-protein interaction associated with several neurodegenerative disorders (Barnham et al., 2004; Zecca et al., 2004). Nontoxic lipophilic brain-permeable iron chelators might offer potential therapeutic benefits for these progressive diseases. An ideal chelating agent should have the capacity to first scavenging the free redox active metal, present in excess in the brain, into nontoxic and excretable metal complexes, and, secondly, to cap the metal at its labile binding site (i.e., 
-amyloid, -synuclein, prion protein, and neuromelanine), preventing any mediated toxic action (Fenton activity and/or aggregation) (Gaeta and Hider, 2005). Lipids endowed with the hydroxamate group represent an interesting addition to the growing inventory of compounds potentially capable to achieve this goal and deserve further investigation in in vivo models of neurodegeneration.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NAE, N-acylethanolamines; AEA, N-arachidonoylethanolamine; PEA, N-palmitoylethanolamine; SR144528, N-((1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide); FAAH, fatty acid amide hydrolase; AMT, anandamide membrane transporter; (+)-MK 801, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, (6-iodo-2-methyl-1-[2-(4-morpholinyl-)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)methanone; DMEM, Dulbecco's modified Eagle's medium; t-BOOH, tert-butylhydroperoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; LDH, lactic acid dehydrogenase; TBARS, thiobarbituric acid-reacting substance(s); Fr, fluorescence ratio; RT, reverse transcriptase; PCR, polymerase chain reaction; NMDA, N-methyl-D-aspartate.
Address correspondence to: Grazia Lombardi, Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche, e Farmacologiche, University of Piemonte Orientale "Amedeo Avogadro", Via Bovio, 6, 28100 Novara, Italy. E-mail: lombardi{at}pharm.unipmn.it
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0.01 versus cells treated with vehicle alone; *, p 
