10 December 2007
By: Roxana Deduleasa, Gadget News Editor
Take A Look At LG's Bizzaro PC12 Radio!
The Table Top radio is soon to be released

LG PC12 Radio Gizmodo

Don't you think the new PC12 radio from LG looks like a little dinosaur egg? Well, it might, but some say its annoyingly oval shape looks rather futuristic. So, I suppose you could kill that prehistoric idea and think of it as a device coming from the future, or given by the aliens as a Thanksgiving gift. Either ways, technically speaking, the bizarro LG's PC12 radio is nothing to die for. It is packed with a HD radio tuner, which is a better alternative to the usual AM/FM tuner, an easy to use night stand alarm clock and a "never-ending" screen display.The best part is to come. The portable device comes with an integrated USB port, which basically means this baby can stream tunes from flash drives or MP3 players. According to Gizmodo, when creating the table top device, LG "utilized the latest technologies to make the PC12 one of the most innovative and visually appealing audio systems." Moreover, " PC12 is the first system to offer HD Radio technology. HD Radio enhances AM and FM broadcasting with an increased number of channels and improved sound quality. In addition to playing AM and FM stations with HD sound, the HD radio displays broadcast information including station name, artists and song titles. "The only major issue of this nice tabletop radio is the fact that the weird design line is actually taking a lot of its functionality, as there is no extra space for wide stereo speakers or comfortable to use controls. As you can see there is a rather bloody red frame which makes the radio look like it has just been cut in two. Sadly, it's too soon for pricing and availability details! We are just a few, but there are many of you, Softpedia users, out there. That's why we thought it would be a good idea to create an email address for you to help us a little in finding gadgets we missed. Interesting links are bound to be posted with recognition going mainly to those who submit. The address is.
Page 1 Copyright (c) 2001-2011 Softpedia. All rights reserved. Softpedia and Softpedia logo are registered trademarks of SoftNews NET SRL.

Journal of Neurochemistry, 2004, 89, 134141
doi:10.1111/j.1471-4159.2003.02327.x
Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on b-amyloid-induced toxicity in PC12 cells
Teresa Iuvone, Giuseppe Esposito, Ramona Esposito, Rita Santamaria, Massimo Di Rosa and Angelo A. Izzo
Department of Experimental Pharmacology, University of Naples Federico II, Naples, Italy
Abstract Alzheimers disease is widely held to be associated with oxidative stress due, in part, to the membrane action of b-amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non-psychoactive component of the marijuana plant (Cannabis sativa) on b-amyloid peptide-induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to b-amyloid peptide (1 lg/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell-signalling cascade) appearance, DNA fragmentation and increased intracellular calcium.
Treatment of the cells with cannabidiol (10)7)10)4 M) prior to b-amyloid peptide exposure signicantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against b-amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro-caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection. Keywords: Alzheimers disease, apoptosis, b-amyloid, cannabidiol, cannabinoid, neuroprotection. J. Neurochem. (2004) 89, 134141.
Alzheimers disease (AD) is the most common age-related neurodegenerative disorder (Koo et al. 1999). Regional neuronal degeneration, synaptic loss, presence of neurobrillary tangles (NFTs) (Terry 1963) and senile plaques (Braak and Braak 1997) are specic hallmarks of this disease. While NFTs are the result of disposition of hyper-phosphorylated tau protein (Lee et al. 1991), senile plaques are complex extracellular lesions composed of a core of b-amyloid (Ab) aggregates, surrounded by activated astrocytes, and dystrophic neuritis (Itagaki et al. 1989; Cotman et al. 1996). Excessive accumulation of Ab peptide has been proposed as a pivotal event in the pathogenesis of AD, although the precise mechanism by which Ab induces neuronal death is still unknown (Hensley et al. 1994; Troy et al. 2001). Proposed mechanisms include production of oxygen free radicals (Behl et al. 1994), modication of cytosolic calcium homeostasis (Mattson 1992; Ueda et al. 1997), the Wnt pathway and activation of nuclear factor-kB (Green and Peers 2002; Caricasole et al. 2003). The cysteine proteases, known as caspases, are essential mediators of many of the pathways involved in executing the apoptotic programme
following Ab accumulation (Gervais et al. 1999). Caspasemediated apoptosis can be modulated by several agents, including antioxidants (Behl et al. 1994), calcium channel blockers (Weiss et al. 1994) and growth factors (Mattson et al. 1993). Cannabinoids are a group of C21 compounds occurring in the glandular hairs of Cannabis sativa (Indian hemp) and consequently in hashish and marijuana, the well known drug
Received July 17, 2003; revised manuscript received November 27, 2003; accepted November 28, 2003. Address correspondence and reprint requests to Giuseppe Esposito or Angelo A. Izzo, Department of Experimental Pharmacology, University of Naples Federico II, via D. Montesano 49, 80131 Naples, Italy. E-mail: giuesp@interfree.it; aaizzo@unina.it Abbreviations used: Ab, b-amyloid; AD, Alzheimers disease; [Ca2+]i, changes in intracellular free calcium levels; CBD, cannabidiol; DCF, 2,7dichlorouorescein; DMEM, Dulbeccos modied Eagles medium; DMSO, dimethylsulfoxide; H2DCF-DA, dichlorouorescin-diacetate; MDA, malonyl dialdehyde; MTT, 3(4,5-dimethylthiazol-2yl)2,5-diphenyl-2H-tetrazolium bromide; NFTs, neurobrillary tangles; O.D., optical density; PBS, phosphate-buffered saline; RFU, relative uorescence unit; ROS, reactive oxygen species; D9-THC, D9-tetrahydrocannabinol.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 134141
Cannabidiol and b-amyloid
of abuse (Samuelsson 1999; Nocerino et al. 2000). The most important representative cannabinoid is D9-tetrahydrocannabinol (D9-THC), which has psychoactive properties depending upon the interaction with the cannabinoid CB1 receptors within the brain (Pertwee 1997). Good quality hashish contains 410% and marijuana 0.12.7% D9-THC (Samuelsson 1999). Cannabidiol, the main component of the glandular hairs (up to 15%), is a non-psychoactive cannabinoid; it exerts a plethora of pharmacological effects, including anti-convulsive, sedative, hypnotic, anti-psychotic, anti-nausea and anti-inammatory actions (Mechoulam et al. 2002). Cannabidiol is a potent antioxidant compound and it has been recently proposed to have a neuroprotective role during ischemic damage (Hampson et al. 1998, 2000). The aim of the present study is to evaluate the possible neuroprotective effect of cannabidiol on b-amyloid-induced neurotoxicity. For this purpose we evaluated the effect of cannabidiol on cell viability, reactive oxygen species (ROS) formation and membrane lipoperoxidation, as well as neuronal apoptosis in cultured rat pheochromocytoma PC12 cells exposed to Ab.

cannabidiol (10)5 M) was evaluated in the presence of the CB1 receptor antagonist SR141716A (10 min before cannabidiol). The concentration of Ab was selected on the basis of our laboratory experience (i.e. a concentration which produced a submaximal effect on cell viability, lipid peroxidation and ROS formation) (data not shown). MTT cell viability assay Cell viability was determined using a 3(4,5-dimethylthiazol-2yl)2,5diphenyl-2H-tetrazolium bromide (MTT) conversion assay (Mosman 1983). Briey, the cells were plated in 96-well culture plates at the density of cells/well and allowed to adhere at 37C for 2 h. Thereafter, the medium was replaced with fresh medium and the cells were incubated with Ab peptide (1 lg/mL) in the presence or absence of cannabidiol (10)4)10)7 M). After 24 h, 25 lL MTT (5 mg/mL in DMEM) were added and the cells incubated for an additional 3 h at 37C. After this time, the cells were lysed and the dark blue crystals solubilized with 125 lL of a solution containing 50% (v/v) N,N, dimethylformamide, 20% (w/v) sodium dodecylsulfate, with an adjusted pH of 4.5. The optical density (O.D.) of each well was measured with a microplate spectrophotometer (Titertek Multiscan MCC/340; Labsystem) equipped with a 620 nm lter. The cell viability in response to the treatment with Ab peptide in the presence or absence of cannabidiol was calculated as percentage of cell viability (O.D. treated/O.D. control) 100. Measurement of reactive oxygen species (ROS) The formation of ROS was evaluated by means of the probe 2,7dichlorouorescein (DCF) according to the method described by Bass et al. (1983) and Le Bel et al. (1992). Briey, PC12 cells were seeded at a density of cells/well into 96-well plates and allowed to grow for 48 h. 2,7-Dichlorouorescein-diacetate (H2DCF-DA, Sigma) was then added directly to the growth medium at a nal concentration of 5 lM and the cells incubated for 1 h at 37C. H2DCFDA is a non-uorescent permeant molecule which diffuses passively into cells; the acetates are then cleaved by intracellular esterases to form H2DCF which is thereby trapped within the cell. In the presence of intracellular ROS, H2DCF is rapidly oxidized to the highly uorescent DCF. Therefore, cells were washed twice with phosphatebuffered saline (PBS), placed in fresh medium and treated with Ab (1 lg/mL) with or without cannabidiol (10)7)10)4 M) for 24 h. After treatment, cells were washed twice with PBS and the plates placed in a uorescent microplate reader (LS 55 Luminescence Spectrometer; Perkin Elmer, Beaconseld, Bucks, UK). Fluorescence was monitored using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Results were expressed as Relative Fluorescence Units (RFU). In some experiments, the effect of the cannabinoid receptor agonist CP55,940 (10)8)10)5 M) was also evaluated. Lipid peroxidation assay Malonyl dialdehyde (MDA), as the most abundant lipid peroxidation product from PC12 cells, was measured by the thiobarbituric acid colorimetric assay (Mihara and Uchiama 1978). Briey, 24 h after treatment with Ab (1 lg/mL) (in the presence or absence of cannabidiol 10)4)10)7 M), the cells were washed three times with 1 PBS, then scraped in 1 PBS containing 0.5 mM EDTA and 1.13 mM butyl-hydroxytoluene. Cell lysis was performed by means of six cycles of freezing and thawing. To 450 lL of cellular lysate was

Materials and methods

Materials All the materials for cell culture were purchased from Biowittaker (Caravaggio, BG, Italy). Fetal calf serum and horse serum were from Hyclone (Logan, UT, USA). Human b-amyloid peptide (fragment 142) and CP55,940 {()-cis-3-[2-hydroxy-4-(, dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol)} were from Tocris Cookson (Ballwin, UK). Antibody anti-caspase 3 was from Calbiochem (San Diego, CA, USA). Anti-mouse IgG was from Dako (Glostrup, Denmark). SR141716A [(N-piperidin-1-yl)-5(4-chlorophenyl)-12,4-dichlorophenyl)-4-methyl-1H-pyrazole3-carboxamide hydrochloride] was a gift from Dr Madaleine Mosse (SANOFI-Reserche). Cannabidiol, Vitamin E (DL-a-tocopherol) and all the other reagents used, unless otherwise stated, were from Sigma (Milan, Italy). Ab fragment peptide was dissolved in pyrogen-free water, cannabidiol, vitamin E and SR141716A in dimethylsulfoxide (DMSO). The nal DMSO concentration in the assay wells, irrespective of cannabinoid concentration, was always adjusted to 0.01%, a concentration that in preliminary experiments was found to have no effect on the response under study. Cell culture PC12 cells (American Tissue Culture Catalogue number CRL1721) were grown in Dulbeccos modied Eagles medium (DMEM) supplemented with 5% fetal calf serum, 15% horse serum, 2 mM glutamine, 100 U/mL penicillin and 100 lg/mL streptomycin at 37C in 5% CO2/95% air. Conuent cells were detached, counted and seeded into Petri dishes (10 cm diameter) at a density of cells/mL and allowed to adhere for 24 h at 37C. Thereafter, the medium was replaced with fresh medium and cells were treated with Ab (1 lg/mL) in the presence or absence of cannabidiol (10)7)10)4 M, given immediately before Ab). In some experiments, the effect of

136 T. Iuvone et al.

added 1 mL 10% (w/v) trichloroacetic acid. After centrifugation at 1000 g for 10 min, 1.3 mL 0.5% (w/v) thiobarbituric acid were added and the mixture was heated at 100C for 20 min. After cooling, MDA formation was recorded (Absorbance 530 nm and Absorbance 550 nm) in a Perkin Elmer (Massachussets, MA, USA) spectrouorimeter and the results are presented as ng MDA/cells. Preparation of cytosolic fractions Extracts of PC12 cells stimulated for 24 h with Ab (1 lg/mL) in the presence or absence of cannabidiol (10)7)10)4 M) were prepared as previously described (Esposito et al. 2002). Briey, harvested cells (1 106) were washed twice with ice-cold PBS and centrifuged at 180 g for 10 min at 4C. The cell pellet was resuspended in 100 lL of ice-cold hypotonic lysis buffer [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulfonyl uoride, 1.5 lg/mL soybean trypsin inhibitor, pepstatin A 7 lg/mL, leupeptin 5 lg/mL, 0.1 mM benzamidine, 0.5 mM dithiothreitol (DTT)] and incubated in ice for 15 min. The cells were lysed by rapid passage through a syringe needle ve or six times and the cytoplasmic fraction was then obtained by centrifugation at g for 1 min. Western blot analysis Immunoblotting analysis of caspase 3 protein was performed on a cytosolic fraction of PC12 cells treated as described (Samali et al. 1999). Cytosolic fraction proteins were mixed with gel loading buffer (50 mM Tris, 10% SDS, 10% glycerol 2-mercaptoethanol, 2 mg bromophenol/ml) in a ratio of 1 : 1, boiled for 5 min and centrifuged at g for 10 min. Protein concentration was determined and equivalent amounts (50 lg) of each sample were separated under reducing conditions in 12% SDSpolyacrylamide minigel. The proteins were transferred onto nitrocellulose membrane according to the manufacturers instructions (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked by incubation at 4C overnight in high salt buffer (50 mM Trizma base, 500 mM NaCl, 0.05% Tween20) containing 5% bovine serum albumin; they were then incubated for 1 h with anti-caspase 3 antibody (1 : 2000) (Chemicon, Temecula, CA, USA) for 1 h at room temperature, followed by incubation with horseradish peroxidase (HRP)-conjugate secondary antibody (Dako). The immune complexes were developed using enhanced chemiluminescence detection reagents (Amersham, Cologno Monzese, Italy) according to the manufacturers instructions and exposed to Kodak XOmat lm. The protein bands of caspase 3 and pro-caspase 3 on X-ray lm were scanned and densitometrically analysed with a GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, CA, USA). Results are expressed as the ratio pro-caspase/caspase 3 (a low ratio is indicative of apoptosis). DNA fragmentation analysis To prepare cellular DNA, PC12 cells (4 106, incubated for 24 h with or without Ab, alone, or in presence of cannabidiol 10)5 M and 104 M) were detached from 10 cm culture dishes and the cell suspension was centrifuged at 100 g for 10 min (Gschwind and Huber 1995). The cell pellet was then washed twice with ice-cold PBS and cellular DNA was isolated using a DNA isolation kit (Roche, Basel, Switzerland) according the manufacturers instructions. A 20 lL aliquot of each DNA sample was analysed on a 1.5% agarose gel containing ethidium bromide (1 lg/mL) in TBE buffer (100 mM Tris, 90 mM boric acid, 1 mM EDTA) and run for 90 min

at 70 V. After electrophoresis, the DNA was visualized under UV light and photographed. Measurement of intracellular calcium levels Changes in intracellular free calcium levels ([Ca2+]i) in a suspension of PC12 cells were determined by the fura-2 method (Sandirasegarane et al. 1994; Zhou et al. 1996). Upon binding calcium, fura-2 shifts its excitation wavelength from 380 to 340 nm, and the ratio of the uorescence caused by excitation at 340 nm to that at 380 nm (R340/380) reects changes in [Ca2+]i (Zhou et al. 1996). PC12 cells were incubated with 5 lM fura-2/acetoxymethyl ester (fura-2/AM) in HBH (concentration in mM: NaCl 137, KCl 5.4, MgCl2 0.49, KH2PO4 0.44, Na2HPO4 0.64, NaHCO3 3, glucose 5.5, CaCl2 1.26 and HEPES 20, pH 7.4) in a 37C incubator for 60 min, and washed twice before taking an aliquot for [Ca2+]i measurement. Each aliquot contained cells suspended in a total volume of 200 lL HBH. Basal [Ca2+]i values were obtained after 2 min of equilibration and [Ca2+]i measurements were performed 2, 5, 10 and 15 min after Ab administration. As Ab produced a maximal increase 5 min after its administration, the effect of cannabidiol (10)6)10)4 M, administered 15 min before Ab 1 lg/mL) or vitamin E (10)5 M, used as a reference compound) was assessed at this time-point. Measurements were performed with a spectrouorometer with an LS55 luminescence spectrometer (Perkin Elmer). Statistical analysis Results were expressed as the mean SEM of n experiments. Statistical analysis was determined with ANOVA and multiple comparisons were performed by Bonferronis test, with p < 0.05 considered signicant.

Results

Cell viability MTT assay was used to detect cell viability. Incubation of PC12 cells with Ab (1 lg/mL) for 24 h caused 38.8 2.2% of cell death (Fig. 1). Cannabidiol (10)7)10)4 M), added to the cells immediately before treatment with Ab, signicantly reduced cell death. The effect of cannabidiol was signicant for all the concentrations tested. The cannabinoid receptor agonist CP55,940 also reduced Ab-induced cell death (cell death: Ab 37.0 3.8; CP55,940 10)8 M + Ab 13.6 7.9; CP55,940 10)7 M + Ab 21.7 9.0; CP55,940 10)6 M + Ab 8.5 4.2, p < 0.05, CP55,940 10)5 M + Ab 6.6 3.8, p < 0.05, n 34). Measurement of reactive oxygen species (ROS) ROS accumulation, estimated using a converting reaction of the probe DCFH2 to DCF, was signicantly (p < 0.001) increased after incubation with Ab (1 lg/mL), as indicated by the increase in RFU (Fig. 2). Incubation of PC12 with cannabidiol (10)7)10)4 M) signicantly inhibited Ab-induced increase in ROS accumulation (Fig. 2). The effect of cannabidiol (10)5 M) was not signicantly modied by the cannabinod CB1 receptor antagonist SR141716A (10)5 M) (RFU: control 53 5; Ab 146 11; cannabidiol +

peroxidation. Exposure of PC12 cells to Ab (1 lg/mL) caused a signicant increase in MDA levels (indicating that lipid peroxidation occurs) (Fig. 3). Lipid peroxidation of Ab-treated cells was concentration-dependently reduced by cannabidiol (10)7)10)4 M). The effect of cannabidiol (10)5 M) was not signicantly modied by the cannabinod CB1 receptor antagonist SR141716A 10)5 M (MDA ng/106 cells: untreated cells 31 3; Ab 85 4; cannabidiol + Ab 45 4; Ab + cannabidiol + SR141716A 42 3; n 5). Given alone, SR141716A (10)5 M) did not change MDA levels either in untreated cells (MDA ng/106 cells: untreated cells 31 3; SR141716A 32 3; n 5) or cells incubated with Ab (MDA ng/106 cells: Ab 85 4; Ab + SR141716A 81 4; n 5).
Fig. 1 Effect of cannabidiol (10)7)10)4 M) on b-amyloid (Ab, 1 lg/mL)-induced cell death. Cell death, evaluated by the reduction of the tetrazolium salt MTT, was assessed 24 h after incubation with Ab. Cannabidiol was added immediately before Ab. Results, expressed as the percentage of cell death, are the means SEM of ve experiments in triplicate. Untreated cells were assumed to be vital (100% viability). ***p < 0.001 versus Ab.
Apoptosis assay We evaluated the appearance of the caspase 3 band in a cellular extract of PC12 cells by western blot analysis as a hallmark of apoptosis. In our time-course experiments, we showed that PC12 cells start to undergo to apoptosis 4 h after treatment with Ab (1 lg/mL), but a marked apoptosis was evident at 6 h after treatment (Fig. 4a). Analysis of the procaspase/total caspase 3 ratio showed that Ab signicantly induced apoptosis (as indicated by the decreased ratio) 6 h after treatment, and this increase was signicantly counteracted by cannabidiol (10)4 M) (Fig. 4b). These results suggest that apoptosis in Ab-treated PC12 cells is mediated by caspase 3, and the protective action of cannabidiol may, at least in part, be attributed to inhibition of the caspase cascade.
Fig. 2 Effect of cannabidiol (10)7)10)4 M) on b-amyloid (Ab, 1 lg/mL)-induced formation of reactive oxygen species (ROS). ROS formation, evaluated by the oxidation of 2,7-dichlorouorescein (H2DCF) to the uorescent 2,7-dichlorouorescein (DCF), was assessed 24 h after incubation with Ab. Cannabidiol was added immediately before Ab. Results are the means SEM of six experiments in triplicate. p < 0.001 versus control (untreated cells); *p < 0.05 and ***p < 0.001 versus Ab.
Ab 61 11; Ab + cannabidiol + SR141716A 66 6; n 5). Given alone, SR141716A (10)5 M) did not modify ROS accumulation either in untreated cells or in cells incubated with Ab (data not shown). Cannabidiol did not affect viability in untreated cells (data not shown). Lipid peroxidation assay The thiobarbituric acid colorimetric assay, which measures malondialdehyde (MDA) levels, was used to quantify lipid

Fig. 3 Effect of cannabidiol (10)7)10)4 M) on b-amyloid (Ab, 1 lg/mL)-induced lipid peroxidation. Lipid peroxidation, evaluated by the thiobarbituric acid colorimetric assay [which measures malondialdehyde (MDA)], was assessed 24 h after incubation with Ab. Cannabidiol was added immediately before Ab. Results, expressed as concentration of MDA, are the means SEM of six experiments in triplicate. p < 0.001 versus control (untreated cells); **p < 0.01 and ***p < 0.001 versus Ab.

138 T. Iuvone et al.

Fig. 4 Effect of cannabidiol (CBD, 10)4 M) on b-amyloid (1 lg/mL)induced caspase 3 activation. (a) Representative western blot analysis of caspase 3 activation from its inactive precursor, pro-caspase 3, in PC12 cell homogenate treated with and without Ab and in the presence of cannabidiol; arrows indicate cleaved (activated) caspase 3 at about 17 kDa and its precursor, pro-caspase, at about 43 kDa. (b) Ratio pro-caspase/total caspase 3 (a decreased ratio is indicative of apoptosis). **p < 0.01 versus control cells (at 6 h), p < 0.01 versus b-amyloid (at 6 h), n 3 separate experiments.
DNA fragmentation Experiments on DNA fragmentation are shown in Fig. 5. Untreated PC12 cells showed no detectable DNA fragmentation. By contrast, DNA extracted from PC12 cells after 24 h of exposure to Ab was clearly fragmented. Pretreatment with cannabidiol (10)5 M and 10)4 M) reduced Ab-induced DNA ladders, the effect being more evident for the 10)4 M concentration. Intracellular calcium levels Ab (1 lg/mL) produced a signicant increase in [Ca2+]i 215 min after its administration, with a maximal effect after 5 min (data not shown). Therefore, the effects of cannabidiol were assessed at this time point. Ab-induced changes in [Ca2+]i were reduced by both cannabidiol (R340/380: basal 0.90 0.02; Ab 1.20 0.06; Ab + cannabidiol 10)6 M 1.10 0.07; Ab + cannabidiol 10)5 M 1.08 0.07; Ab + cannabidiol 10)4 M 0.91 0.07; n 34, p < 0.05 for the 10)5 M and 10)4 M concentrations) and vitamin E 10)5 M (R340/380: basal 0.90 0.02; Ab 1.20 0.06; Ab + vitamin E 0.93 0.03; n 3, p < 0.05).

Discussion

Fig. 5 DNA fragmentation in PC12 untreated cells and in PC12 cells treated with b-amyloid (1 lg/mL, alone or in the presence of cannabidiol 10)4 M and 10)5 M). Cellular DNA was extracted and visualized on agarose gel as described in the Materials and methods section. Each lane was loaded with the same amount of DNA. DNA size markers used were: (left) phage lambda DNA digested with Hind III restriction enzyme; (right) pBR322 DNA digested with Hinf I restriction enzyme.

The accumulation of plaques containing Ab is an invariant feature of AD pathology and there is abundant evidence suggesting that Ab contributes to the aetiology of AD (Carlson 2003; Golde 2003). In the present paper we report that cannabidiol, a major non-psychotropic component of
marijuana, prevents Ab-induced neurotoxicity, ROS production and lipid peroxidation; it also inhibits caspase 3 appearance (from the inactive precursor pro-caspase) in PC12 cells. The MTT assay has been widely used as an index of cell survival and proliferation. Previous investigators have shown that cannabidiol produces a modest or no reduction in C6 glioma cell viability after 6 days of incubation (Jacobsson et al. 2000). Here, we have shown that exposure of PC12 cells to Ab produces a reduction in cell viability, and that cannabidiol protects, in a concentration-dependent manner, Ab-induced neurotoxicity. Consistent with these results, cannabidiol has been shown to reduce glutamate-, N-methylD-aspartate- and kainate-induced toxicity in rat cortical neurones (Hampson et al. 1998). The cellular events involving free radical oxidative stress may be one basic pathway leading to cell degeneration. Increased oxidative stress and disturbed defensive mechanisms occur in the brain of AD patients, which might result in a self-propagating cascade of neurodegenerative events (Markesbery 1997). ROS have been shown to be involved in cell damage and death induced by amyloid peptides (Mattson et al. 1993; Brera et al. 2000) and antioxidant therapy has led to an improvement in AD patients. Excessive ROS can lead to a number of detrimental effects, including lipid peroxidation. In our experiments, PC12 cells exhibited
increased ROS levels and lipid peroxidation following exposure to Ab peptide. The ability of Ab to increase both ROS and lipid peroxidation has been documented previously in a number of neuronal cell types, including PC12 cells (Xiao et al. 2002; Onoue et al. 2002; Guan and Nordberg 2003). However, we have shown for the rst time that cannabidiol reduced, in a concentration-dependent manner, Ab-induced ROS accumulation and lipid peroxidation. Since antioxidants are known to attenuate Ab-induced oxidative injury (Cash et al. 2002), it is likely that the previously reported antioxidant properties of cannabidiol could contribute to its benecial effect. Other studies have shown that cannabidiol antagonizes the oxidative stress induced by retinoid anhydroretinol in lymphoblastoid cells (Chen and Buck 2000) and prevents hydrogen peroxide-induced oxidative damage in neuronal cultures (Hampson et al. 1998). In contrast to D9-THC, cannabidiol is not a cannabinoid receptor agonist (Mechoulam et al. 2002); nevertheless, cannabidiol may enhance the endocannabinoid action at CB1 receptors by its ability to block the uptake and the enzymatic degradation of the endocannabinoid anandamide (Bisogno et al. 2001). Furthermore, Petitet et al. (1998) have shown that cannabidiol, in the micromolar range, behaves as a CB1 antagonist in the rat brain. The importance of CB1 receptors in neuroprotection has been previously documented (Grundy et al. 2001; Van der Stelt et al. 2001; Marsicano et al. 2002). Milton (2002) showed that the endogenous cannabinoids anandamide and noladin ether have a protective effect on Ab-induced toxicity through activation of CB1 receptors in human teratocarcinoma cells. However, it is very unlikely that the protective (anti-oxidative) effect of cannabidiol observed here involves CB1 receptors as the potent and selective CB1 receptor antagonist, SR141716A, at a concentration previously shown to be fully effective (Esposito et al. 2001, 2002), did not show a protective effect (when administered alone), nor did it modify the protective action of cannabidiol. In agreement with our results, Molderings et al. (2002) showed (by using binding and PCR techniques) that rat pheochromocytoma PC12 cells are not endowed with CB1 receptors. It is interesting to note that in these cells, we have shown that the cannabinoid agonist CP55,290 also has a protective effect on Ab-induced toxicity, thus conrming the existence of a non-CB1 mediated protective effect of cannabinoids. Several experimental studies suggest an association between deposition of Ab, oxidative stress and apoptosis associated with AD and ageing (Kokoszka et al. 2001; Marx 2001). The Ab peptide has been shown to induce apoptosis in neurones, including PC12 cells, which may be responsible for neuronal death in AD (Martin et al. 2001; Troy et al. 2001). Apoptosis is associated with the activation of a family of aspartic acid-specic cysteine proteases, referred to as caspases (Nicholson and Thornberry 1997). There are at least 14 caspases identied in mammalian cells that are synthesized

as inactive precursor molecules, pro-caspase, and are converted by proteolytic cleavage to the active heterodimer (Thornberry and Lazebnik 1998). The activation of procaspase 3 to caspase 3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signalling pathways (Nicholson and Thornberry 1997). In the present study, we have shown that cannabidiol increased pro-caspase 3 levels and, in parallel, reduced caspase 3 levels in Ab-treated PC12 cells, suggesting that the cannabinoid could exert a protective role at the execution phase of apoptosis. The inhibitory effect of cannabidiol on apoptosis was strengthened by the observation that this cannabinoid inhibited the fragmentation of DNA (a hallmark of apoptosis) induced by Ab. The fragmentation of DNA after Ab has been previously documented (Gschwind and Huber 1995). The pharmacological manipulation of neuronal calcium homeostasis may be a useful therapeutic approach in neurodegenerative pathologies, including AD, and a rise in [Ca2+]i has been suggested to be responsible for toxic effects of Ab (Mattson 2002). Previous investigators have shown that Ab-induced neurotoxic actions in PC12 cells are associated with an increase in [Ca2+]i and that this effect is blocked by antioxidants such as vitamin E (Zhou et al. 1996). In addition, it has been suggested that the increase in [Ca2+]i induced by Ab in PC12 cells is not the result of an inux of calcium through voltage-dependent calcium channels, but rather it is the result of a free radical-mediated process. In the present study we have shown that cannabidiol counteracted the Ab-induced increase in [Ca2+]i, which is in agreement with the antioxidant properties of this cannabinoid reported here and elsewhere (Hampson et al. 1998). In summary, the results reported herein indicate, for the rst time, that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against Ab insult. Inhibition of the activity of the apoptotic enzyme caspase 3 is likely to be involved in signalling pathways for the neurotrophic effect of cannabidiol. Given the low toxicity of cannabinol shown in humans (Cunha et al. 1980; Consroe et al. 1991), this non-psychoactive component of marijuana may play an important role in counteracting neuronal cell death occurring in Alzheimers disease.

Acknowledgements

We are grateful to Dr Madaleine Mosse from SANOFI-Recerche (Montpellier, France) for the gift of cannabinoid antagonist. This work was supported by the Enrico and Enrica Sovena Foundation, SESIRCA (Regione Campania) and Conanziamento Murst.

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