WIN 55,212-2, Agonist of Cannabinoid Receptors, Prevents Amyloid β1-42 Effects on Astrocytes in Primary Culture

Alzheimer´s disease (AD), a neurodegenerative illness involving synaptic dysfunction with extracellular accumulation of Aβ1-42 toxic peptide, glial activation, inflammatory response and oxidative stress, can lead to neuronal death. Endogenous cannabinoid system is implicated in physiological and physiopathological events in central nervous system (CNS), and changes in this system are related to many human diseases, including AD. However, studies on the effects of cannabinoids on astrocytes functions are scarce. In primary cultured astrocytes we studied cellular viability using MTT assay. Inflammatory and oxidative stress mediators were determined by ELISA and Western-blot techniques both in the presence and absence of Aβ1-42 peptide. Effects of WIN 55,212-2 (a synthetic cannabinoid) on cell viability, inflammatory mediators and oxidative stress were also determined. Aβ1-42 diminished astrocytes viability, increased TNF-α and IL-1β levels and p-65, COX-2 and iNOS protein expression while decreased PPAR-γ and antioxidant enzyme Cu/Zn SOD. WIN 55,212-2 pretreatment prevents all effects elicited by Aβ1-42. Furthermore, cannabinoid WIN 55,212-2 also increased cell viability and PPAR-γ expression in control astrocytes. In conclusion cannabinoid WIN 55,212-2 increases cell viability and anti-inflammatory response in cultured astrocytes. Moreover, WIN 55,212-2 increases expression of anti-oxidant Cu/Zn SOD and is able to prevent inflammation induced by Aβ1-42 in cultured astrocytes. Further studies would be needed to assess the possible beneficial effects of cannabinoids in Alzheimer's disease patients.


Introduction
Alzheimer's disease (AD) is a common neurodegenerative disease implicated in the aging process, affecting nearly 50% of people over 75 [1,2]. It involves neurofibrillary degeneration, extracellular accumulation of beta-amyloid peptide (Aβ) and synaptic dysfunction, resulting in neural cell death in the hippocampus and cerebral cortex, and in activation of glial cells [3,4].

Primary culture of cortical astrocytes
All animals were handled according to the recommendations of the Bioethics Committee of the School of Medicine of the University of Valencia, Spain. Ethics committee specifically approved this study. Cortical astrocytes were isolated from rat fetuses of 21 days gestation. Fetuses were obtained by cesarean section and decapitated. Cerebral cortices were removed and triturated 10-15 times through a Pasteur pipette with 10 ml DMEM. The cell suspension was filtered through nylon mesh with a pore size of 90 μm and re-suspended in DMEM containing 20% fetal bovine serum (FBS), supplemented with L-glutamine (1%), HEPES (10 mM), fungizone (1%), and antibiotics (1%). Cells were plated on T75 culture flask and maintained in a humidified atmosphere of 5% CO2/95% air at 37°C during 15 days. After 4 days of culture, the FBS was maintained at 20% and after 1 week of culture, the FBS content was reduced to 10%, and the medium was changed twice a week. The purity of astrocytes was assessed by immunofluorescence using anti-glial fibrillary acidic protein (astrocyte marker; Sigma-Aldrich), anti-CD-68 (microglial marker; Serotec), anti-myelin basic protein (olygodendroglial marker; Sigma-Aldrich) and anti-microtubule-associated protein 2 (neuronal marker; Sigma-Aldrich). The astrocyte cultures were found to be at least 99% glial fibrillary acidic protein positive. No cells were found to express CD-68, myelin basic protein, or microtubule-associated protein-2.

Cell treatments
Ten days after seeding, WIN (10 μM) was added to culture flasks. Twenty-four hours later, 10 μM Aβ 1-42 (toxic peptide) or Aβ 40-1 (control peptide) (Sigma-Aldrich) were added to the flasks. Aβ 1-42 concentration used in our study is in the range of toxic concentrations of the peptide [21,22]. Before incubation, the peptides were diluted in 100 μM of phosphate-buffered saline (PBS) and incubated for 24 h at 37º C. Assays were performed 24 h after peptide addition.

MTT assay
Cell viability was determined by MTT assay. The MTT assay is a well-established, widely used and easily reproducible method for the assessment of cell viability and cytotoxicity [23,24]. Astrocytes were plated in 96-well culture plate and incubated with WIN during 24h. Subsequently, Aβ 40-1 (control) and Aβ 1-42 peptides were added to wells for another 24h. After cell treatments, the medium was removed and cells were incubated with red free medium and MTT solution [0.5 mg/ml, prepared in phosphate buffer saline (PBS) solution] for 4 h at 37ºC. Finally, the medium was removed and formazan particles were dissolved in dimethyl sulfoxide. Cell viability, defined as the relative amount of MTT reduction, was determined by spectrophotometry at 570 nm.

Cytokine determination, IL-1 and TNFα
Astrocytes were seeded as previously published [12]. At the time of assay, the red phenol medium was removed and replaced by PBS containing 1 mg/ml bovine serum albumin, either in the presence or absence of Aβ 1-42 (10 μM). IL-1β and TNF-α concentration (pg/ml) were ascertained using ELISA kits (Pierce Biotechnology, Inc.).

Western blot analysis
Cultured cells were treated with lysis buffer and mechanically degraded to release the proteins. Protein concentration was determined using modified Lowry method [25]. Loading buffer (0.125 M Tris-HCl, pH 6.8, 2% SDS, 0.5% (v/v) 2-mercaptoethanol, 1% bromophenolblue and 19% glycerol) was added to protein sample and heated for 5 min at 95ºC. Proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes in a humid environment using a transfer buffer (25mM Tris, 190mM glycine, 20% methanol). Membranes were blocked with 5% milk in TBS-T (0.05% Tween-20) and incubated with primary antibodies overnight at 4ºC. Membranes were washed 3 times with wash buffer TBS-T and incubated with a secondary anti-rabbit IgG or anti-mouse IgG (Cell Signalling Technologies Danvers, MA) antibody conjugated to the enzyme horseradish peroxidase (HRP) for 1 h. Membranes were washed three times and proteins were detected using the ECL method as specified by the manufacturer. Autoradiography signals were assessed using digital image system ImageQuant LAS 4000 (GE Healthcare).

Statistical analyses
Values are expressed as mean±S.D. Differences between groups were assessed by one-way analysis of variance (ANOVA). Statistical significance was accepted at P 0.05. Data sets in which F was significant were examined by a modified t-test.

Protective role of WIN on cell viability
The role of WIN on cell viability was studied using MTT conversion assay. Fig 1A shows that incubation with WIN at different concentrations induced a significant increase in cell viability at 10 μM. Consequently, that concentration was used in future experiments. Astrocytes previously incubated with 10 μM Aβ 1-42 for 24 h significantly decreased cell viability compared to control cells ( Fig 1B). Furthermore, pretreating astrocytes during 48 h with WIN (10 μM) prevented the decrease in cell viability induced by Aβ 1-42 (WIN + Aβ), conversely WIN (1, 2, 5μM) did not have any effect ( Fig 1B).

Effect of Aβ 1-42 and WIN on PPAR-γ protein expression
Pro-inflammatory gene expression is downregulated by PPARs family [26]. We found that pretreatment with WIN (10 μM) increased PPAR-γ expression compared to control cells (Fig 5). Incubation with Aβ 1-42 significantly decreased PPAR-γ expression that was prevented by WIN pretreatment. Effect of Aβ 1-42 and WIN on Cu/Zn SOD and Mn SOD protein expression Superoxide dismutase is a key antioxidant enzyme. In our study, incubation with Aβ 1-42 decreased Cu/Zn SOD expression in astrocytes in primary culture which was prevented by WIN pretreatment, evidencing that WIN could play a neuro-protective role against oxidative stress induced by Aβ 1-42 peptide (Fig 6A). On the other hand, our results indicated that Mn SOD protein expression is increased in presence of Aβ 1-42 . Pretreatment with WIN did not prevent Mn SOD increase induced by Aβ 1-42 (Fig 6B).

Discussion
Oxidative stress and inflammation are the main mechanisms in the progression of various neurodegenerative diseases, including AD [27][28][29][30]. In our study, we determined different markers involved in inflammation and oxidative stress induced by the Aβ 1-42 peptide in primary cultures of astrocytes, with the aim to assess the antioxidant and anti-inflammatory effects of cannabinoid WIN. We found that WIN significantly increased astrocytes viability compared to control cells. Furthermore, WIN prevented the decrease in astrocytes viability induced by Aβ  .
It has been shown that cannabinoids preserve neurons from Aβ exposure by activating MAP kinase cascade [31] and by anti-oxidative and anti-apoptotic effects [32]. Moreover, some studies demonstrated that cannabinoids protect glial cells from death [33,34]. Nevertheless, in cancer, where cells are highly proliferative and undifferentiated, treatment with cannabinoids can block cell proliferation in a dose dependent manner [35][36][37][38], demonstrating that the effects of cannabinoids on cell viability are probably dependent on cell type [39] and developmental stage [40].
Expression of CB1 [41] and CB2 [42] receptors in rat culture astrocytes have been published and also dual activation of both cannabinoid receptors by WIN (the mixed non-selective CB1/ CB2 agonist) in rat cortical astrocytes have been detected [41] On the other hand, WIN confers its protective and anti-inflammatory effects against Aβ injury through both CB1 and CB2 receptors [43]. Given that our results there is expression of both types of cannabinoid receptors, CB1 and CB2, it is likely that the effect of WIN observed in our study is due to the interaction with both types of receptors, consistent with published results by Fakhfouri and cols [44].
We found that WIN prevented the increase of inflammatory mediators IL-1β, TNF-α, NF-κB, iNOS and COX-2, as well as the decrease of the anti-inflammatory mediator PPAR-γ induced by Aβ 1-42 in astrocytes in primary culture. The inflammatory process is a characteristic mechanism in the development of AD, and pro-inflammatory agents are involved in the progression of cell damage [45,46,47,12]. Moreover, it is known that astrocytes participate in the inflammatory process induced by Aβ 1-42 [27,28,48]. Initially, inflammation is beneficial since it produces pro-inflammatory substances involved in tissue protection, limiting the survival and proliferation of cells exposed to toxic agents, such as Aβ 1-42 [49,50]. However, sustained inflammatory response could lead to neurotoxic damage or cell death [12,51,8]. NF-κB proteins are up-regulated in inflammation conditions such as astroglial activation induced by Aβ 1-42 oligomers [52]. In this regard, we found an increase in NF-kB/p-65 expression in astrocytes after addition of Aβ 1-42 that was prevented by WIN pretreatment. Valles and collaborators  [53] found that the cytokine-receptor complex is able to bind to cytokines and other proteins of the extracellular matrix, producing inflammatory signals which could be important in pathologies such as Alzheimer's disease [53,54]. In agreement with our results, different authors have reported that cannabinoids mitigate neural cell activation in the neuroinflammatory response induced by Aβ 1-42 , reducing the levels of pro-inflammatory molecules such as IL-1β, TNF-α, COX-2 and iNOS [55,56,57]. Likewise, the activation of cannabinoid receptors diminishes the release of IL-1β, IL-6 and TNF-α in microglial cells [58,59,60] as well as COX-2 and iNOS [61]. Studies conducted in rats pretreated with the Aβ peptide found that WIN prevented cognitive impairment, glial activation and neuronal loss [19,62,63], and also reduced COX-2, iNOS and TNF-α levels [63,64].
Kainu et al. [65] demonstrated for the first time the presence of mRNA and protein PPAR-γ in CNS cells. Subsequent studies have detected PPAR-γ expression in microglial and astrocytic cells [66,12]. PPAR-γ agonists protect against Aβ-induced inflammatory and neuronal damage [67,68], thus making neurons and astrocytes potential therapeutic targets for PPAR-γ ligands [69,12]. Astrocytes also express the largest levels of PPAR-γ in the neural tissues [70,71]. As other authors [72,73,74], we found a decreased expression of PPAR-γ in astrocytes treated with Aβ 1-42 . Esposito et al. [56] showed in neurons that cannabinoids may act as neuro-protective agents by PPAR-γ activation. In this study, we demonstrate an increase in this protein expression in astrocytic cells previously incubated with WIN. Furthermore, we found for the first time that WIN prevents PPAR-γ expression decrease induced by Aβ 1-42 peptide in astrocytes in primary culture. There is strong evidence to suggest that some cannabinoids can act on  PPARs through either direct or indirect pathways. In order to directly act on nuclear transcriptional factors PPARs, exogenous cannabinoids need to pass through plasma membrane and be transported into nucleus which may involve certain membrane and intracellular transporters. However, we still cannot rule out that cannabinoids effects could be indirect through the binding of other cellular targets which in turn induces PPARs activation [75]. In fact, WIN attenuates amyloid-beta-induced neuroinflammation in rats through activation of cannabinoid receptors and PPAR-γ pathway [44].
Different authors have demonstrated the role of oxidative stress in AD [76][77][78][79]. The cumulative damage caused by free radicals induces alterations in the activity or expression of antioxidant enzymes like catalase or SOD. These enzymes were found to be decreased in both CNS and peripheral tissues of AD patients [80,81]. In this sense, we demonstrate that Cu/Zn SOD is decreased in astrocytes treated with Aβ 1-42 . Our results are consistent with those reported by other authors, highlighting the role of oxidative stress in the development of AD [82]. New substances are under research to reduce damage caused by oxidative stress in this disease. Widely distributed in the body, cannabinoids receptors were discovered few decades ago and are still under research [57]. Few studies address the effect of cannabinoids on oxidative stress. For instance, cannabinoids were found to prevent or antagonize oxidative stress toxicity in cortical neurons in cultures [83,84], and in lymphoblastic cells [85]. Studies with PC12 cells exposed to Aβ 1-42 peptide demonstrated that cannabinoids reduced reactive oxygen species production and membrane lipid oxidation [86,32]. Our results provide evidence that Aβ 1-42 decreases Cu/Zn SOD expression in astrocytes in primary culture, and pretreatment with WIN increases Cu/Zn SOD expression, preventing the decrease caused by Aβ 1-42 . These findings indicate that cannabinoids could act as a protective agent against oxidative stress caused by Aβ  . In Fig 7 the set of results are summarized. However our results indicate that Aβ 1-42 elevated Mn SOD protein expression, increasing mitochondrial biogenesis mechanism, such as we previously published [74]. Pretreatment with WIN did not prevent Mn SOD overexpression induced by Aβ 1-42. Mn SOD plays a role in the adaptive response which protects brain cells from damage, as in the case of AD. In fact, Mn SOD preserves neurons against oxidative stress [87] and protects developing neurons from β-amyloid toxicity [88]. This enzyme catalyzes the conversion of superoxide radicals to molecular oxygen and H 2 O 2 , whereas glutathione peroxidase, peroxiredoxin reductase and catalase neutralize H 2 O 2 . Overexpression of Mn SOD induces cognitive recovery and reduces Aβ levels in AD animal models [89]. Furthermore, Mn SOD deficiency increases β-amyloid levels and amyloid plaque burden, promoting the development of behavioural disturbances [90].
Preclinical data suggest a beneficial role of some cannabinoids for treatment of different diseases. Dronabidol, an oil-based solution of Δ9-THC, is used as anti-emetic and appetite stimulant [91]. Δ9-THC also decreases agitation present in the advanced stage of AD [92]. In 2003, the FDA granted the patent for cannabinoids as antioxidants and neuro-protectants (U.S. Department of Health and Human Services). Despite these promising preliminary results, the clinical utility of cannabinoids in AD is still to be determined [93].

Conclusions
Taken together, our findings show that cannabinoid WIN increases cell viability and anti-inflammatory response in astrocytes in primary culture and prevents cell death induced by Aβ  . Furthermore, WIN increases expression of anti-oxidant Cu/Zn SOD and is able to prevent inflammation induced by Aβ 1-42 in astrocytes. In this sense, clinical studies are needed to evaluate the neuro-protective effects of cannabinoids in Alzheimer´s disease.