Contrasting protective effects of cannabinoids against oxidative stress and amyloid-β evoked neurotoxicity in vitro

NeuroToxicology 33 (2012) 138–146

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NeuroToxicology

Contrasting protective effects of cannabinoids against oxidative stress and amyloid-b evoked neurotoxicity in vitro Benjamin S. Harvey a, Katharina S. Ohlsson b, Jesper L.V. Ma˚a˚g c, Ian F. Musgrave a, Scott D. Smid a,* a

Discipline of Pharmacology, School of Medical Sciences, Faculty of Health Sciences, The University of Adelaide, SA, Australia Institute of Neuroscience and Physiology, The Sahlgrenska Academy, Go¨teborg University, Sweden c Department of Pharmaceutical Biosciences, Uppsala University, Sweden b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 July 2011 Accepted 23 December 2011 Available online 3 January 2012

Cannabinoids have been widely reported to have neuroprotective properties in vitro and in vivo. In this study we compared the effects of CB1 and CB2 receptor-selective ligands, the endocannabinoid anandamide and the phytocannabinoid cannabidiol, against oxidative stress and the toxic hallmark Alzheimer’s protein, b-amyloid (Ab) in neuronal cell lines. PC12 or SH-SY5Y cells were selectively exposed to either hydrogen peroxide, tert-butyl hydroperoxide or Ab, alone or in the presence of the CB1 specific agonist arachidonyl-20 -chloroethylamide (ACEA), CB2 specific agonist JWH-015, anandamide or cannabidiol. Cannabidiol improved cell viability in response to tert-butyl hydroperoxide in PC12 and SHSY5Y cells, while hydrogen peroxide-mediated toxicity was unaffected by cannabidiol pretreatment. Ab exposure evoked a loss of cell viability in PC12 cells. Of the cannabinoids tested, only anandamide was able to inhibit Ab-evoked neurotoxicity. ACEA had no effect on Ab-evoked neurotoxicity, suggesting a CB1 receptor-independent effect of anandamide. JWH-015 pretreatment was also without protective influence on PC12 cells from either pro-oxidant or Ab exposure. None of the cannabinoids directly inhibited or disrupted preformed Ab fibrils and aggregates. In conclusion, the endocannabinoid anandamide protects neuronal cells from Ab exposure via a pathway unrelated to CB1 or CB2 receptor activation. The protective effect of cannabidiol against oxidative stress does not confer protection against Ab exposure, suggesting divergent pathways for neuroprotection of these two cannabinoids. ß 2011 Elsevier Inc. All rights reserved.

Keywords: b-Amyloid Cannabinoid Neuroprotection Oxidative stress

1. Introduction Neuronal death that occurs in neurodegenerative disorders such as Alzheimer’s Disease (AD) is believed to be caused in part by increased levels of the neurotoxic b-amyloid (Ab) peptide in the brain (Yankner and Lu, 2009). The neurotoxicity of Ab is attributable to the generation of oligomers, fibrils and reactive oxygen species (Behl et al., 1994; Bernstein et al., 2009; Carrano et al., 2011). Oxidative stress causes neuronal damage via lipid peroxidation, DNA damage, protein oxidation and inflammation (Casetta et al., 2005). Recognition of the role of oxidative stress in AD pathogenesis has led researchers to investigate the potential for antioxidants to ameliorate or offset the underlying neurodegeneration (Craggs and Kalaria, 2011). As a therapeutic strategy there is some promise in

* Corresponding author at: Discipline of Pharmacology, School of Medical Sciences, Faculty of Health Sciences, The University of Adelaide, Adelaide, SA 5005, Australia. Tel.: +61 8 83035287; fax: +61 7 82240685. E-mail address: [email protected] (S.D. Smid). 0161-813X/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2011.12.015

the development of AD treatments that encompass both pharmacological and antioxidant properties (Sebestik et al., 2010). Antioxidant therapy is usually considered within the realm of natural products derived from dietary sources, as distinct from the limited pharmacotherapeutic interventions for AD treatment (Wollen, 2010). However, there is the recognition of overlap in that some dietary polyphenols may have direct anti-amyloid effects in addition to antioxidant properties (Bieschke et al., 2010; Richard et al., 2011; Wollen, 2010). Seemingly included in this neuroprotective pleitropy are the cannabinoids, which have been shown to mediate neuroprotection both through their actions at distinct CB1 and CB2 receptors and, at least for some cannabinoids, through an antioxidant capacity (Bisogno and Di Marzo, 2008). Cannabinoids have been shown to protect neuronal cells from Ab exposure (Iuvone et al., 2004; Milton, 2002) and prevent experimental Ab-evoked brain pathology in vivo (Ramirez et al., 2005). Part of the direct cellular neuroprotective action of cannabinoids may also be as a result of activating anti-apoptotic pathways in response to oxidative stress (Milton, 2002; Mnich et al., 2010) and inhibiting Ab-mediated lysosomal destabilisation (Noonan et al., 2010). The capacity of

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cannabinoids to directly alter Ab fibrillisation and aggregate formation has yet to be explored. The non-psychoactive phytocannabinoid, cannabidiol has also demonstrated neuroprotective effects in vitro and in vivo sufficient to be regarded as a potential therapeutic agent in the treatment of neurodegenerative disease (Iuvone et al., 2009). Neuronal cells pretreated with cannabidiol maintain higher levels of viability associated with reduced nitrite and iNOS expression following Ab exposure (Esposito et al., 2006b). Levels of ROS, lipid peroxidation products and caspase-3 are also reduced (Iuvone et al., 2004), in addition to reduced hyperphosphorylation of tau protein in Abexposed PC12 cells (Esposito et al., 2006a). Mice administered cannabidiol before Ab treatment also show a reduction in inflammatory markers (Esposito et al., 2007). The mechanism behind which cannabidiol provides for neuronal cell protection cannot be fully attributed to its antioxidant capacity and is confounded by a lack of clear elucidation of its pharmacology (Scuderi et al., 2009). The demonstration of an action of cannabidiol at CB2 receptors also merits further investigation (Castillo et al., 2010; Thomas et al., 2007). In light of the questions that remain related to the neuroprotective activity of cannabidiol and the endocannabinoids, the following study compared the neuroprotective profile of cannabidiol and select cannabinoid ligands in two neuronal cell lines. One, a rat phaeochromocytoma (PC12) peripheral neuronal cell line with a well established neurotoxic profile to Ab exposure and two, a human neuroblastoma (SH-SY5Y) cell line which exemplifies a population of central neuronal cells typically susceptible to oxidative stress (Halliwell, 2006). Effects of cannabidiol on oxidative stress and Ab fibril-evoked neurotoxicity were then compared with known antioxidants, in addition to cannabinoid receptor-selective ligands and an endocannabinoid in an effort to better understand the antioxidant and direct anti-amyloid role of this phytocannabinoid. Direct measurements of extended Ab fibrillisation and aggregate formation were also compared in addition to characterising CB1 and CB2 receptor expression on the neuronal cell lines used in this study. 2. Materials and methods

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rabbit (ab6717) were obtained from Abcam (Cambridge, UK). Primary rabbit antibody to CB1 receptor (product number 209550) was obtained from Calbiochem (Alexandria, NSW, Australia). 2.2. PC12 and SH-SY5Y cell culture Rat phaeochromocytoma cells (Ordway PC12) cells were kindly donated by Professor John Piletz (Department of Psychiatry and Behavioural Neuroscience, Loyola University Medical Center, IL, USA). PC12 cells were maintained in 75 cm3 culture flasks containing 20 ml of RPMI-1640 media supplemented with 5% FCS, 1% L-glutamine, 1% NEAA and 1% penicillin/ streptomycin. Flasks also contained 5 ml of conditioned media from the previous flask containing growth factors. Cells were maintained in an incubator at 37 8C with 5% CO2. Experiments and cell maintenance were performed in a laminar flow hood under sterile conditions. Cells were passaged every 4–5 days and detached from flasks using 1 trypsin EDTA. Cell counts were performed using trypan blue to stain non viable cells, which were excluded from the count. Ninety-six well plates were used for cell viability experiments and cells were seeded in RPMI1640 with 10% FCS at a density of 2  104 cells per well. Undifferentiated cells were then allowed to equilibrate for 24 h before the experiment. SH-SY5Y human neuroblastoma cell lines were obtained from ATCC (Manassas, VA, USA). SH-SY5Y cells were maintained in 75 cm2 culture flasks with 20 ml of 1:1 DMEM/Ham’s F-12 medium supplemented with 10% FCS, 1% NEAA, 1% penicillin/ streptomycin. Cells were split twice weekly using PBS to detach adherent cells. Cell counts were performed using trypan blue to stain non viable cells, which were excluded from the count. All experiments and cell maintenance was performed in a laminar flow hood under sterile conditions. Cells were grown in an incubator with 5% CO2 at 37 8C. 96-well plates were used for experiments and undifferentiated SH-SY5Y cells were seeded in the medium described above at a density of 2  104 cells per well. Plates were then allowed to equilibrate for 24 h prior to experimentation, with cells maintained in an undifferentiated state.

2.1. Drugs, chemicals and antibodies 2.3. Ab preparation and treatment in PC12 cells Ab protein fragment (1–40), thiazolyl blue tetrazolium bromide (MTT), sodium deoxycholate, Trolox, butylated hydroxytoluene (BHT), trypan blue, thioflavin T, bromophenol blue, b-mercaptoethanol, anandamide (AEA), arachidonyl-20 -chloroethylamide (ACEA), b-catalase, Roswell Park Memorial Institute 1640 (RPMI) medium, Dulbecco’s Modified Eagles Medium (DMEM), tert-butyl hydroperoxide (t-BHP), poly-L-lysine solution, Triton X-100 and foetal calf serum (FCS) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Ham’s F-12 nutrient mixture was obtained from Invitrogen (Mulgrave, VIC, Australia). JWH-015 was obtained from Tocris Bioscience (Bristol, UK). H2O2 was obtained from Merck (Kilsyth, VIC, Australia). Cannabidiol was obtained from Cayman Chemical (Ann Arbour, MI, USA). Scrambled sequence Ab(1–40) protein (sAb) was used as a control for non-fibrillar peptide and was obtained from AnaSpec (Fremont, CA, USA). Non essential amino acids (NEAA), penicillin/streptomycin, 10 trypsin EDTA, L-glutamine, methanol and phosphate buffered saline (PBS) at pH 7.4 were obtained from Thermo Fisher Scientific (Scoresby, VIC, Australia). Dimethyl sulphoxide (DMSO), Tris base and Tris–HCl were obtained from Amresco (OH, USA). Bovine serum albumin (BSA) was obtained from Bovogen Biologicals (East Keilor, VIC, Australia). SDS and glycine were obtained from Biorad (Gladesville, NSW, Australia). Rabbit polyclonal primary antibody to CB2 receptor (ab3561) and goat polyclonal secondary FITC-conjugated antibody to

Both Ab(1–40) and scrambled sequence Ab(1–40) (sAb) were prepared as per manufacturer’s recommendations. One percent ammonium hydroxide was added directly to the vial containing lyophilised amyloid to yield a protein concentration of 3.8 mM and the vial gently shaken. Sterile PBS was added to prepare a final concentration of 100 mM. Amyloid was then dispensed into aliquots and incubated at 37 8C with 5% CO2 for 72 h to allow for fibril formation. Ab or sAb was then frozen at 70 8C until required. Fibrillar Ab or sAb diluted in PBS (at final concentrations of 0–1.0 mM) was added to the plates following 15 min pretreatment with either vehicle or test cannabinoids. As SH-SY5Y cells displayed a weak and variable toxicity to Ab(1–40), only PC12 cells were subsequently used for Ab-evoked neurotoxicity experiments. PC12 cells were incubated at increasing concentrations of Ab (0.1, 0.2. 0.3, 0.5 and 1.0 mM), alone or in the presence of the following cannabinoids: cannabidiol, anandamide (endocannabinoid), ACEA (CB1 receptor-selective agonist) or JWH-015 (CB2 receptor-selective agonist) at single test concentrations previously shown to be active in neuronal tissue or cell culture studies (Esposito et al., 2006a; Hampson et al., 1998; Iuvone et al., 2004; Milton, 2002; Tolon et al., 2009; Velez-Pardo et al., 2010). Plates were then incubated for 48 h prior to determining cell viability via the MTT assay.

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2.4. Effects of antioxidants and cannabinoids on oxidative stress in PC12 and SH-SY5Y cells Hydrogen peroxide (H2O2) is a well established pro-oxidant in neuronal cells. tert-Butyl hydroperoxide (t-BHP) elicits greater lipid peroxidation than H2O2 (Hix et al., 2000; Liu et al., 2005). Plates were incubated over a concentration range of either H2O2 (0–200 mM; PC12 cells only) or tert-butyl hydroperoxide (0– 150 mM in PC12 cells, 0–50 mM in SH-SY5Y cells) based on preliminary studies which demonstrated a differential sensitivity to pro-oxidants between the neuronal cell lines. Cells were incubated for 24 h, alone or in the presence of a single test compound (either one cannabinoid ligand or one antioxidant) and the MTT assay subsequently performed on each plate to determine cell viability. The 96 well plate layout typically consisted of a single pro-oxidant compound used at increasing concentrations (PC12 cells: 50 mM increments for H2O2, 25 mM for t-BHP; SH-SY5Y cells: 5–10 mM increments for t-BHP only) in the presence of single test concentrations of either cannabinoid or antioxidant compounds. Cells were pretreated with test compounds 15 min prior to the addition of pro-oxidants. Neuronal cell lines were pretreated with established synthetic antioxidants Trolox (100 mM) or butylated hydroxytoluene (BHT; 50 mM) in addition to b-catalase at effective in vitro concentrations (Demerle-Pallardy et al., 2000; Kim et al., 2005; Saito et al., 2007). In separate experiments, SH-SY5Y cells were pretreated with either of the following cannabinoids: cannabidiol (0.01– 10 mM) or JWH-015 (0.01–1 mM). PC12 cells were treated with cannabidiol (1 or 10 mM). 2.5. Cell viability measurements Cell viability was determined using the thiazolyl blue tetrazolium bromide (MTT) cytotoxicity assay. MTT is taken up by viable cells and converted in the mitochondria to a soluble blue formazan product. The intensity of developed colour is proportional to the level of active mitochondria in living cells. After incubation, 96-well plates had all media removed and replaced with serum-free media containing 0.25 mg/ml of MTT. The plate was further incubated for 2 h at 37 8C with 5% CO2, then MTT solution removed and cells lysed with DMSO. The plate was then placed on an orbital shaker for 6 min before determining absorbance at 570 nm using a PolarStar Galaxy microplate reader (BMG Labtech, Durham, NC, USA). 2.6. Immunohistochemical expression of CB1 and CB2 receptors Glass coverslips coated for 24 h with poly-L-lysine were placed into a 6-well plate and PC12 or SH-SY5Y cells were added at a density of 2  105 cells/ml in 1 ml of RPMI-1640 medium or 1:1 DMEM/Ham’s F-12 respectively. The plate was then incubated for 24 h at 37 8C with 5% CO2. Media was removed by aspiration from the coverslips which were then washed 3 times in cold sterile PBS (pH 7.4). Cells were fixed in 3.7% formaldehyde for 10 min at room temperature and then washed twice with cold PBS. The cells were permeabilised in 0.1% Triton X-100 for 10 min at room temperature then washed 3 times in cold PBS. Non-specific antibody binding was blocked for 1 h in blocking solution of 5% BSA in cold PBS. Media was aspirated from the wells and coverslips washed with PBS. Primary CB1 (Calbiochem; 209550) and CB2 (Abcam; ab3561) receptor antibody dilutions were prepared (1:200, 1:500, 1:1000) in 0.5% BSA in PBS and then incubated with cells for 20 h at 4 8C. After incubation the media was aspirated and coverslips washed 3 times with PBS. Cells were treated with secondary FITC-conjugated goat polyclonal antirabbit antibody (Abcam; ab6717) for 1 h in the dark. All media was

then removed and coverslips washed in PBS and mounted on glass slides using 90% glycerol and sealed with clear nail varnish. Slides were viewed using an Olympus BHB fluorescence microscope (Olympus Corporation, Tokyo, Japan) at a magnification of 20 with a FITC filter in place using excitation and emission wavelengths of 495 nm and 521 nm respectively. Images were taken with a Nikon digital sight DS-5MC camera and processed using Nikon NIS-Elements BR software (Nikon Corporation, Tokyo, Japan). 2.7. Western blot expression of CB1 and CB2 receptors Western blotting was performed to detect the expression of cannabinoid receptors in the PC12 and SH-SY5Y neuronal cell lines. Cells were washed in cold PBS and lysed with ice-cold RIPA buffer containing protease inhibitors (aprotinin 2 mg/ml, pepstatin A 1 mg/ml, PMSF 1 mM and EDTA 5 mM). The cells were scraped into a microfuge tube and agitated on ice for 30 min prior to centrifugation at 12,000 rpm at 4 8C. Following the determination of protein concentrations via BCA assay (Thermo Fisher Scientific, Scoresby, VIC, Australia), lysate supernatant was stored at 70 8C until needed. Samples were prepared by mixing lysate supernatant with Laemmli’s sample buffer 1:1, boiled in a water bath for 3 min and cooled on ice. Protein samples (20 mg) were then loaded onto precast Mini-Protean TGX minigels together with protein standards (all from Bio-Rad, Gladesville, NSW, Australia) and run at 200 V in SDS-PAGE running buffer for 30 min. The proteins were then transferred to a nitrocellulose membrane in Towbin blotting buffer at 100 V for 70 min. Filtered skim milk powder (5%) in Trisbuffered saline with Tween-20 (TBST) was used as blocking buffer for 1 h under agitation at 4 8C. The primary antibodies used were the anti-cannabinoid CB1 receptor or anti-cannabinoid CB2 receptor as used in the immunohistochemical studies (Section 2.6) at a dilution of 1:1000 in 0.25% skim milk powder. The membrane was incubated with the primary antibody overnight at 4 8C under agitation and then washed with TBST. Goat anti-rabbit IgG DyLight 800-conjugated secondary antibody (Thermo Fisher Scientific, Scoresby, VIC, Australia) was incubated with the membrane at a dilution of 1:10,000 in TBST under constant agitation for 1 h at room temperature. Thereafter, the membrane was washed with TBST before visualisation using a near-infrared imaging system (Odyssey Li-COR Biosciences, Lincoln, NE, USA). Positive control tissues for CB1 receptor (human colonic muscularis propria) and CB2 receptor (human colonic mucosa) subject to concomitant Western blot analysis yielded strong immunoreactivity in regions of known molecular weights of the CB1 (60 kDa) and CB2 (40 kDa) receptors, confirming the specificity of the primary antibodies used. 2.8. ThT assay and electron microscopy of Ab fibril and aggregate formation The fluorescent dye Thioflavin T (ThT) binds selectively to fibrils over time with fluorescence increasing proportionally to the amount of fibrils in solution. The ThT assay was used to determine if 72 h was sufficient time to allow for fibril formation (Hudson et al., 2009b) and if fibrils were directly affected by cannabinoid ligands. The cannabinoids tested were cannabidiol (10 mM), AEA (10 mM), ACEA (1 mM) and JHW-015 (1 mM) at the highest concentrations used in viability studies. Cannabinoid ligands were tested in the absence of Ab to control for any direct quenching of ThT fluorescence and scrambled Ab(1–40) was tested as a nonfibrillar protein control. ThT (10 mM in PBS) was added to wells on a black fluorescent microplate, followed by addition of either fibrillar Ab (10 mM) or scrambled amyloid Ab(1–40) (10 mM) and

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the test cannabinoid. The plate was placed in a PolarStar Galaxy microplate reader at 37 8C and fluorescence readings were taken every 30 min for a period of 48 h, the equivalent exposure period for cell viability experiments. Excitation and emission wavelengths were 440 nm and 490 nm respectively. Transmission Electron Microscopy (TEM) was used to visualise Ab aggregates and fibrils and to confirm fibril formation had occurred. A 5 ml sample containing preformed Ab (10 mM) which had been incubated in PBS at 37 8C for 72 h was then immediately transferred onto a 400 mesh formvar carbon coated nickel electron microscopy grid (Proscitech, Kirwan, QLD, Australia). After 1 min this sample was blotted dry and 10 ml of contrast dye containing 2% uranyl acetate (Proscitech, Kirwan, QLD, Australia) was placed onto the grid, left for 1 min and blotted dry. Grids were then loaded onto a specimen holder and then into a Philips CM100 80 kV transmission Electron Microscope (Philips Research, The Netherlands). Sample grids were then viewed using a magnification of 34,000–92,000. 2.9. Statistical analysis Data obtained from the MTT assay was analysed via a two-way analysis of variance (ANOVA) to assess the effects of cannabinoid ligands against H2O2, t-BHP or Ab-evoked toxicity vs. vehicle control. Bonferroni’s post hoc test was used to determine the p value at each concentration vs. appropriate controls. Area under the curve (AUC) was calculated for each Thioflavin T (ThT) trace. AUC values were compared using a one-way ANOVA with a Dunnett’s post hoc test to compare cannabinoid ligand treatments against Ab. A significance value of p < 0.05 was used for all experiments. Analysis and production of graphs was performed in GraphPad Prism 5 for Windows (GraphPad Software, San Diego, USA).

Fig. 1. Comparison of effects of (a) antioxidants: butylated hydroxytoluene (BHT 50 mM) or trolox (100 mM) and (b) b-catalase (460 U/ml) on cell viability following 24 h exposure to hydrogen peroxide in PC12 cells. The conventional synthetic antioxidants BHT and trolox did not attenuate neurotoxicity induced by hydrogen peroxide; however b-catalase completely prevented the loss of cell viability (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05 vs. Vehicle.

3. Results 3.1. Effects of antioxidants and cannabinoids on cell viability following oxidative stress There was an overall reduction in cell viability with increasing H2O2 exposure in PC12 cells (Fig. 1). However, SH-SY5Y cells were extremely labile in the presence of H2O2 and we were unable to reliably test this cell line further when using this pro-oxidant. There was no significant inhibition in the loss of PC12 cell viability evoked by H2O2 (Fig. 1a) in the presence of the potent antioxidants butylated hydroxytoluene (BHT; 50 mM) and Trolox (100 mM). Other natural antioxidants such as ascorbic acid also had no influence when tested (data not shown). However, b-catalase (Fig. 1b) significantly prevented toxicity across a range of hydrogen peroxide concentrations (***p < 0.001, **p < 0.01, * p < 0.05 vs. Vehicle at 200, 100 and 50 mM H2O2 respectively). Cannabidiol (1–10 mM) provided no significant protection against hydrogen peroxide-evoked cell loss in PC12 cells (Fig. 2a). Cannabidiol at a high concentration (10 mM) however was protective against oxidative stress when PC12 cells were exposed to 50–100 mM t-BHP (Fig. 2b; ***p < 0.001 vs. Vehicle at 50, 75 and 100 mM t-BHP). SH-SY5Y cells appeared more sensitive to the neurotoxicity elicited by t-BHP compared to PC12 cells, as equivalent concentrations of t-BHP evoked greater overall loss of SH-SY5Y cell viability when compared to PC12 cells (e.g. 80% viability in PC12 cells vs. 20% viability in SH-SY5Y cells at 50 mM t-BHP, Fig. 2b vs. Fig. 3). This required both a lower concentration range of t-BHP to be used comparatively with SH-SY5Y cells and toxicity occurred over a more narrow concentration range of t-BHP (0–50 mM in SH-SY5Y cells vs. 0–150 mM for PC12 cells). While the CB2 receptor-selective agonist

Fig. 2. Contrasting effects of cannabidiol (CBD; 1–10 mM) pretreatment on PC12 cell viability following (a) hydrogen peroxide and (b) tert-butyl hydroperoxide (t-BHP) exposure. Cannabidiol (10 mM) significantly increased PC12 cell viability over the range 50–100 mM of t-BHP exposure (n = 3) but had no effect on hydrogen peroxide evoked neurotoxicity. ***p < 0.001 vs. Vehicle.

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Fig. 3. Effects of (a) the CB2 receptor agonist JWH-015 and (b) cannabidiol (CBD) on exposure to tert-butyl hydroperoxide (t-BHP) in SH-SY5Y cells. JWH-015 exerted no protective effect on t-BHP-evoked loss of cell viability (a); however cannabidiol high concentration (CBD; 10 mM) significantly attenuated neurotoxicity to oxidative stress induced by t-BHP (b) (n = 3). **p < 0.01, *p < 0.05 vs. Vehicle.

JWH-015 (0.01–1 mM) failed to elicit any significant neuroprotection in response to t-BHP in SH-SY5Y cells (Fig. 3a), SH-SY5Y cells were significantly protected against oxidative stress from t-BHP exposure following cannabidiol (10 mM) pretreatment (Fig. 3b: *p < 0.05 vs. Vehicle at 25, 35 and 40 mM t-BHP, **p < 0.01 vs. Vehicle at 30 mM t-BHP).

Fig. 4. Effects of (a) the CB2 receptor agonist JWH-015 and (b) cannabidiol (CBD) on exposure to Ab(1–40) in PC12 cells. Neither the CB2 specific agonist nor the phytocannabinoid cannabidiol inhibited neurotoxicity in response to Ab(1–40) (n = 3).

fluorescence up to 48 h. These results confirmed that fibrils or aggregates were continuing to form under conditions similar to those used in the cell viability experiments with Ab (48 h incubation following 72 h fibrillisation). None of the cannabinoid ligands tested in the cell viability experiments significantly modified ThT fluorescence (Fig. 6b and c), indicating no direct

3.2. Effects of antioxidants and cannabinoids on cell viability following b-amyloid exposure SH-SY5Y cells also displayed variable and inconsistent toxicity to Ab(1–40) exposure and so only PC12 cells were subsequently used in Ab-treated cell viability experiments. Incubation with Ab(1–40) resulted in a reduction in PC12 cell viability over the concentration range 0.02–1 mM (Figs. 4 and 5). However, neither the CB2-receptor selective agonist JWH-015 (0.001–0.1 mM; Fig. 4a) nor cannabidiol (0.01–10 mM; Fig. 4b) significantly prevented the Ab-evoked loss of PC12 cell viability. Incubation of PC12 cells with scrambled sequence b-amyloid (1–40) had no effect on cell viability (data not shown). In PC12 cells, the endocannabinoid anandamide (10 mM) significantly inhibited Ab(1–40)-evoked neuronal cell loss over the 0.3–0.5 mM Ab range (Fig. 5a; *p < 0.05 vs. Vehicle). This effect was not observed when using the CB1 receptor-selective agonist ACEA (1 mM), which provided no significant protection against Ab mediated toxicity at any of Ab concentrations (Fig. 5b). 3.3. Results of ThT assay and microscopy of Ab fibril and aggregate formation Transmission electron microscopy of cell-free suspensions of Ab revealed fibril formation over 72 h of incubation at 37 8C (Fig. 6a). The results of the ThT assay confirmed the formation of fibrillar (b sheet) structures of Ab(1–40) protein (Fig. 6b). Increased ThT fluorescence developed rapidly within the incubation period up to 20 h and thereafter maintained increments in

Fig. 5. Effects of (a) the endocannabinoid anandamide (AEA 10 mM; n = 7) and (b) the CB1-selective agonist arachidonyl-20 -chloroethylamide (ACEA, 1 mM; n = 5) on exposure to b-amyloid (Ab1–40) toxicity in PC12 cells. There was a significant attenuation in neurotoxicity in response to Ab1–40 in the presence of anandamide (a) but not ACEA (b). *p < 0.05 vs. Vehicle.

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profile indicative of cannabinoid receptor antibody specificity (not shown). CB2 receptor immunoreactivity by contrast was not observed in either PC12 or SH-SY5Y cells via immunohistochemistry (not shown); however, Western blot analysis revealed a faint band of CB2 receptor immunoreactivity in both PC12 and SH-SY5Y neuronal cell lines corresponding to the expected molecular weight of the CB2 receptor at 40 kDa (Fig. 7d). 4. Discussion

Fig. 6. (a) Transmission Electron Microscopy (TEM) image of cell free suspension of Ab(1–40) (10 mM) incubated at 37 8C for 72 h in PBS, demonstrating the formation of amyloid fibrils. (b) Representative trace of ThT fluorescence in cell free suspension with prefibrillised Ab(1–40) (10 mM), alone or in the presence of cannabinoid ligands over 48 h at 37 8C. ThT fluorescence increases over this period are indicative of further amyloid-b fibril/aggregate formation. Cannabidiol (CBD; 10 mM), arachidonyl-20 -chloroethylamide (ACEA; 1 mM), anandamide (AEA; 10 mM) and the CB2 receptor-selective agonist JWH-015 (JWH; 1 mM) did not significantly influence ThT binding, indicating a lack of direct effect on Ab fibril/ aggregate formation. No ThT fluorescence was observed when cannabinoid ligands were incubated in the absence of Ab. Output normalised against baseline ThT (without Ab) at equivalent time points. (c) Area under the curve (AUC) measurements of ThT fluorescence over 48 h of Ab(1–40) exposure, alone or with cannabinoid ligand pretreatment. Despite a trend for reduced overall ThT fluorescence in the presence of cannabidiol, there was no significant effect of the cannabinoid ligands on ThT fluorescence. n = 4.

disruption by cannabinoids on preformed Ab fibrils and aggregates. Scrambled sequence amyloid Ab(1–40) did not produce any ThT fluorescence (data not shown). Despite a trend towards a reduction in ThT fluorescence in the presence of cannabidiol, overall area under the curve measurements for total ThT fluorescence indicated no significant effect of any of the cannabinoid ligands tested on Ab fibril formation (Fig. 6c). 3.4. CB1 and CB2 receptor expression in PC12 and SH-SY5Y cell lines Immunohistochemical studies revealed expression of the CB1 receptor in both neuronal cell lines (Fig. 7a and b). Western blot analysis demonstrated a dense band of immunoreactivity corresponding to the expected size of the CB1 receptor (60 kDa) in both cell lines (Fig. 7c), with strong expression in PC12 cells. Omission controls were negative for staining and positive controls for CB1 receptor (human colonic muscularis propria) and CB2 receptor (human colonic mucosa) expressed an immunoreactivity

The results of this study demonstrate a protective effect of anandamide but not cannabidiol against neurotoxicity associated with Ab exposure. Cannabidiol’s protection against oxidative stress and lipid peroxidation in neuronal cells was either dissociated from or lacking sufficient efficacy against Ab fibril and aggregate-evoked neurotoxicity. This is in contrast to studies that have demonstrated neuroprotection against amyloid b from cannabidiol pretreatment in PC12 cells under similar incubation conditions (Esposito et al., 2006a; Iuvone et al., 2004). The main difference between those studies and the present findings is likely in the structural form of the Ab used; where previous studies have incubated cells with non-fibrillar Ab, we have used Ab in its preformed (fibrillar) state at the onset of incubation in PC12 cells. Our results demonstrate that cannabidiol is ineffective in protecting PC12 cells against exposure to preformed Ab fibrils. This suggests that the neuroprotective efficacy of cannabidiol is dependent on Ab fibril formation occurring during cell exposure, which implies either a direct influence on fibril formation or interference with Ab fibril uptake or processing by cannabidiol. Recent findings indicate that neuronal cells concentrate Ab via endosomes or lysosomes into high molecular weight aggregates, which can then further seed extracellular fibril formation upon extrusion (Hu et al., 2009). Endocannabinoids such as anandamide have been shown to stabilise lysosomes as part of their capacity to protect against Ab-mediated neurotoxicity (Noonan et al., 2010); hence it would be interesting to compare this property with cannabidiol. Other relevant differences may relate to the extent of differentiation of neuronal cells prior to Ab exposure, which alters the sensitivity of PC12 cells to cannabinoids (Tahir et al., 1992). Anandamide, in contrast to cannabidiol, provided protection to PC12 cells against Ab-mediated neurotoxicity. These results support a previous study which demonstrated inhibition of neuronal cell loss arising from Ab exposure with prior anandamide treatment, in which it was likely the Ab was also in a fibrillar state following a 24 h Ab pre-incubation (Milton, 2002). Anandamide has previously been shown to attenuate neurotoxicity following oxidative stress (Kim et al., 2005; Mnich et al., 2010), however it can in itself exhibit neurotoxicity at concentrations typically greater than 10 mM (Fowler et al., 2010; Sarker et al., 2000). We did not test anandamide’s effects on either hydrogen peroxide or tertbutyl hydroperoxide (t-BHP)-evoked cell death directly, because the higher anandamide concentrations required for antioxidant capacity were neurotoxic, as has been observed previously (Noonan et al., 2010; Sarker and Maruyama, 2003). Previous studies have shown no beneficial effect of anandamide on hydrogen peroxide-mediated PC12 cell death (Mnich et al., 2010). Although activity of anandamide on neuronal cells seemingly occurs independently of CB1 and CB2 receptor activation (Maccarrone et al., 2000; Sarker and Maruyama, 2003), both CB1 and CB2 receptor-dependent neuroprotective actions of anandamide have been demonstrated in extraneuronal cells such as microglia (Eljaschewitsch et al., 2006). Indeed, there is strong supporting evidence that selective cannabinoid receptor activation in glia and microglia provide neuroprotection through both CB1 and CB2 receptors (Iuvone et al., 2007; Klegeris et al., 2003;

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Fig. 7. Photomicrographs of (a) PC12 cells and (b) SH-SY5Y cells demonstrating immunoreactivity for CB1 receptor (antibody from Calbiochem (209550)). Scale = 50 mm. Primary antibody for CB1 receptor diluted 1:200. No staining to CB2 receptors was visible when using anti-CB2 receptor antibody (Abcam; ab3561, data not shown). (c) Western blot demonstrating CB1 receptor immunoreactivity in SH-SY5Y and PC12 cell lysates, referenced to the expected 60 kDa region corresponding to the CB1 receptor. PC12 cells in particular exhibited a strong band of immunoreactivity. (d) Western blot of SH-SY5Y and PC12 cell lysates for CB2 receptor expression (Abcam; ab3561), demonstrating a faint band of immunoreactivity in the region corresponding to the expected 40 kDa molecular weight of the receptor in both neuronal cell lines.

Ramirez et al., 2005). However, our results support a lack of direct cannabinoid receptor activity associated with anandamide’s protective actions, as there was no similar neuroprotection demonstrated when cells were pretreated with either CB1 or CB2 receptor-selective agonists, ACEA and JWH-015 respectively. Other CB1/CB2 receptor-independent effects of anandamide include actions at orphan G protein-coupled receptors and transient receptor potential (TRP) receptors (De Petrocellis and Di Marzo, 2010). Metabolic conversion of anandamide via COX-2 into biologically active prostamides and lipoxygenase mediators (Fowler, 2007) and neuroprotective activity at PPARa receptors (Sun et al., 2007) underscore the pleitropic role of endocannabinoids that may account for some of their non-cannabinoid receptor mediated actions in the present study. Cannabidiol was found to prevent neurotoxicity arising from oxidative stress in response to t-BHP in a previous study in the micromolar range (Hampson et al., 1998). The difference in cannabidiol’s efficacy between t-BHP and hydrogen peroxide exposure is perhaps not surprising when considering the effects of hydrogen peroxide were resistant to a number of potent antioxidants (as observed in Fig. 1). Indeed only b-catalase, which actively breaks down hydrogen peroxide, was found to exert protective effects in neuronal cells in our hands. However one would expect such antioxidants to exert at least some influence at lower concentrations of hydrogen peroxide used in this study. Another difference to consider however may lie in the capacity of

t-BHP to elicit preferentially more lipid peroxidation than hydrogen peroxide (Palomba et al., 2001) and may also infer that cannabidiol exerts an antioxidant capacity by operating primarily within the lipid membrane. Cannabidiol has previously been shown to inhibit reactive oxygen species and lipid peroxidation generated via Ab exposure (Iuvone et al., 2004), but can also alter microglial endocannabinoid release via an action at lipid rafts (Rimmerman et al., 2011). A lack of effect of JWH-015 on t-BHP neurotoxicity precluded a CB2 receptor-mediated protective effect of cannabidiol as the basis of protection against t-BHP. Why cannabidiol is neuroprotective against oxidative stress generated from t-BHP but not protective against Ab exposure is not clear, as part of Ab’s neurotoxicity has been attributed to the generation of oxidative stress (Iuvone et al., 2004). However there are differences in the profile of neurotoxicity between the two stressors, where hydrogen peroxide and Ab exposure produce contrasting effects on tau phosphorylation and neurotrophin receptor expression for example (Olivieri et al., 2001, 2003). It is likely therefore that Ab activates other pathways in evoking cell death that are not surmountable via antioxidant capacity alone. The results of the ThT assay preclude a direct effect of the cannabinoid ligands and anandamide on disrupting preformed Ab fibrils and/or aggregates. In our studies ThT will continue to bind to preformed Ab fibrils and aggregates well beyond 48 h, which was the time course of the cannabinoid incubations with Ab. Hence, Ab fibrils were found to be stable over the full period of cell exposure.

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While the ThT assay is prone to interference from polyphenolic compounds (Hudson et al., 2009a), we found no interaction from any of the cannabinoid ligands we tested on ThT fluorescence alone or any ligand autofluorescence. While there are no studies that have linked direct effects of cannabinoids on amyloid fibril formation or disruption, D9-THC has been found to inhibit Ab aggregation indirectly by binding an allosteric site on acetylcholinesterase, a known molecular chaperone for promoting Ab fibrillisation (Eubanks et al., 2006). The results of the immunohistochemical and Western blot studies confirm the presence of CB1 receptors on both SH-SY5Y and PC12 cells as has been previously described (Marini et al., 2009; Sarker and Maruyama, 2003; Zhang et al., 2007). Our findings of a lack of CB2 receptor immunoreactivity on either PC12 or SH-SY5Y cells are in keeping with a general lack of expression of CB2 receptors in these neuronal cell lines (Klegeris et al., 2003; Sarker and Maruyama, 2003). However, we demonstrated a faint band of CB2 receptor expression in both neuronal cell lines via Western blot, indicative of a low level of receptor expression in comparison to CB1 receptors. The low expression level may account for the lack of functional role in neuroprotection observed to cannabidiol or anandamide, as the pharmacological studies did not support a CB2 receptor-based neuroprotective action. While the neuroprotective action of CB2 receptor activation is mainly related to microglial expression, neuronal expression of CB2 receptors has been documented (Palazuelos et al., 2006) and neuroprotection ascribed to CB2 receptor activation in various models of neurotoxicity or neurodegeneration (Ferna´ndez-Ruiz et al., 2007). Expression of CB2 receptors is upregulated in Alzheimer’s disease, but occurs predominantly within neuritic plaques (Benito et al., 2003). In conclusion, the endocannabinoid anandamide protects neuronal cells from Ab exposure via a pathway unrelated to CB1 or CB2 receptor activity. The protective effect of cannabidiol demonstrated against oxidative stress in neuronal cells via tertbutyl hydroperoxide exposure does not extend to providing protection against Ab, suggesting at least some divergent pathways for neuroprotection of these two cannabinoids. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was in part funded by a grant from The Sunshine Foundation of Australia. The authors wish to acknowledge Yanqin Liu and Antonio Calabrese (Dept. Chemistry, The University of Adelaide) for assistance with the ThT assay and electron microscopy. References Behl C, Davis JB, Lesley R, Schubert D. Hydrogen peroxide mediates amyloid b protein toxicity. Cell 1994;77:817–27. Benito C, Nunez E, Tolon RM, Carrier EJ, Rabano A, Hillard CJ, et al. Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer’s disease brains J Neurosci 2003;23: 11136–41. Bernstein SL, Dupuis NF, Lazo ND, Wyttenbach T, Condron MM, Bitan G, et al. Amyloidbeta protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nat Chem 2009;1:326–31. Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Neugebauer K, et al. EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity. Proc Natl Acad Sci USA 2010;107:7710–5. Bisogno T, Di Marzo V. The role of the endocannabinoid system in Alzheimer’s disease: facts and hypotheses. Curr Pharm Des 2008;14:2299–3305. Carrano A, Hoozemans JJ, van der Vies SM, Rozemuller AJ, van Horssen J, de Vries HE. Amyloid beta induces oxidative stress-mediated blood–brain barrier changes in

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