ALTERATION OF THE ENDOCANNABINOID SYSTEM IN MOUSE BRAIN DURING PRION DISEASE

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S. PETROSINO,a B. MÉNARD,b N. ZSÜRGER,b V. DI MARZOa1* AND J. CHABRYb1* a Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, CNR Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli (NA), Italy b Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-Université de Nice Sophia Antipolis 660, Route des Lucioles, 06560 Valbonne, France

Abstract—Prion diseases are neurodegenerative disorders characterized by deposition of the pathological prion protein (PrPsc) within the brain of affected humans and animals. Microglial cell activation is a common feature of prion diseases; alterations of various neurotransmitter systems and neurotransmission have been also reported. Owing to its ability to modulate both neuroimmune responses and neurotransmission, it was of interest to study the brain endocannabinoid system in a prion-infected mouse model. The production of the endocannabinoid, 2-arachidonoyglycerol (2-AG), was enhanced 10 weeks post-infection, without alteration of the other endocannabinoid, anandamide. The CB2 receptor expression was up-regulated in brains of prion-infected mice as early as 10 weeks and up to 32 weeks postinfection whereas the mRNAs of other cannabinoid receptors (CBRs) remain unchanged. The observed alterations of the endocannabinoid system were specific for prion infection since no significant changes were observed in the brain of prion-resistant mice, that is, mice devoid of the Prnp gene. Our study highlights important alterations of the endocannabinoid system during early stages of the disease long before the clinical signs of the disease. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: prion, endocannabinoid, cannabinoid receptor, neurodegenerative disease, brain, mouse.

Transmissible spongiform encephalopathies (TSE), or prion diseases, are fatal neurodegenerative disorders occurring in sheep (scrapie), cattle (bovine spongiform encephalopathy) and humans (Creutzfeldt–Jakob disease). The key event in TSE is the conversion of a normal cellular prion protein (PrPc) into an abnormal isoform (PrPsc) which accumulates in tissues of infected individuals. Beside PrPsc deposition, common pathological features of TSEs are intra-neuronal vacuolization, astrogliosis, activation of microglial cells and neuronal cell death. Although PrPsc was proposed to be responsible for both transmission and pathogenicity of TSEs (Prusiner, 1998), growing evidence argues against the possibility of them being the only cause of neurodegeneration. Moreover, PrP gene knockout (Prnp/) mice fail to propagate infectivity or develop pathology (Büeler et al., 1993) supporting the idea that PrPc expression is required for both prion infectivity and neurodegeneration. Several neurotransmitter systems are damaged in prion-infected mice, mainly the serotonin, dopamine and noradrenaline systems at terminal stages (Vidal et al., 2009). Indeed, decreased brain levels not only of bioamines, but also of acetylcholine and GABA were reported at clinical stages in prion-infected rodents (Bassant et al., 1986; Bareggi et al., 2003). Recently, the endocannabinoid system, a signaling system consisting of cannabinoid CB1 and CB2 receptors and of their endogenous agonists, the endocannabinoids, was suggested to be involved in various human neurodegenerative/neuroinflammatory disorders such as Alzheimer’s disease (Benito et al., 2007; Bisogno and Di Marzo, 2008; Campillo and Paez, 2009). In this context, we previously demonstrated that several cannabinoids prevent PrPsc accumulation in cells chronically infected with sheep scrapie and murine prion strains (Dirikoc et al., 2007). Moreover, the non-psychoactive cannabinoid, cannabidiol (CBD), protects neurons against PrPsc toxicity and slightly increases the survival time of prion-infected mice (Dirikoc et al., 2007). This background prompted us to assess, for the first time, whether components of the endocannabinoid system, that is, the cannabinoid receptors (CBRs) CB1 and CB2 and their most studied endogenous agonists, arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoyglycerol (2-AG), as well as two AEA-related molecules, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) (two endogenous agonists of PPAR receptors), are altered in prion-infected mice from early to late stages of the disease. To gain evidence for a possible tight relationship between PrPc and alterations of the endocannabinoid system, prion protein-deficient mice (Prnp/) were also employed for the study.

EXPERIMENTAL PROCEDURES

Animals, microglial cell cultures and prion infection procedure

All animals were handled in accordance with good animal practice as defined by the relevant national and local animal welfare bodies. The disease progression and related molecular events occurring during 139 A strain infection are well-characterized, allowing to minimize the number of animals for each condition. To reduce pain, mice were killed at 32 wpi i.e. at the onset of terminal stages of the disease. We used Prnp/ (Büeler et al., 1993) and wt mice under the same genetic background, that is, C57BL6/J 129/Sv. For prion infection experiments, 8 weekold male mice were i.p. inoculated with 100 l of 1% brain homogenates (w/v) prepared from healthy (noted as “noninfected”) or terminally sick prion-infected mice (139 A murine strain; noted as “infected”). To rule out any possible effects caused by the inoculum and/or animal manipulation and because of their resistance to prion infection, 8 week-old male Prnp/ mice were used as negative controls and injected using the same protocol. Mouse microglia in primary culture and cell line (N11) were prepared and grown in DMEM, 10% FCS, penicillin/streptomycin as previously described (Marella and Chabry, 2004). Cells were incubated with 0.01% healthy or prion-infected brain homogenates at 37 °C for the indicated time, washed twice in PBS, then proteins were solubilized in cold lysis buffer containing Tris 5 mM pH 7.4, 1% Triton X-100, 140 mM NaCl and 5 mM EDTA and prepared for Western blotting.

Endocannabinoid extraction, purification and measurement

Animals (n3/condition) were decapitated and whole brain immediately frozen in carbon ice. Brains were dounce-homogenized and extracted with CHCl3:MeOH:Tris–HCl 50 mM pH 7.4 (1:1:1 by volume) containing 100 pmol of d8-AEA, d5-2-AG, d4-PEA and d4-OEA as internal standards. Lipid-containing organic phases were dried down, weighed and purified by open-bed chromatography on silica gel and analyzed by isotope dilution liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) (LC-APCI-MS) using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS2010) quadrupole MS via a Shimadzu APCI interface (Marsicano et al., 2002). MS analyses were carried out in the selected ionmonitoring mode. AEA, 2-AG, PEA and OEA levels were quantified on the basis of their area ratio with deuterated internal standards signal area. Amounts in pmols were normalized per mg of lipid extract. As it is often the case for endocannabinoid measurements, data were statistically evaluated by one-way ANOVA followed by the Bonferroni’s test.

mRNAs expression in mouse brain analyzed by RT-qPCR

Total RNA was isolated from mouse brain (using the Trizol® RNA extraction kit (Invitrogen)) according to the manufacturer recommendations followed by a DNase I-RNAse free (Promega) treatment. First-strand cDNA were synthesized from 2 g of total RNA with 200 U of SuperScript II reverse transcriptase (SuperScript II, Invitrogen) in the appropriate buffer in the presence of 25 g/ml oligo(dT)15, 0.5 mM desoxyribonucleotide triphosphate mix, 10 mM dithiothreitol. The reaction was incubated 50 min at 42 °C then inactivate 15 min at 70 °C. The target transcripts were amplified by means of a LightCycler 480 sequence detector system (Roche Diagnostics) using the following primers: mouse CB1R forward (5=-GGGCAAATTTCCTTGTAGCA-3=) and mouse CB1R reverse (5=-CAGGCTCAACGTGACTGAGA-3=); mouse CB2R forward (5=-GACAAGGCTCCACAAGACC-3=) and mouse CB2R reverse (5=-CTCCTTCATGGGGTTGAACT-3=); mouse PPAR forward (5=-CAAGGCCTCAGGGTACCACTAC-3=) and mouse PPAR reverse (5=-GCCGAATAGTTCGCCGAAA-3=); mouse PPAR forward (5=-CCAATGGTTGCTGATTACAAATATG-3=) and mouse PPAR reverse (5=-AATAAGGTGGAGATGCAGGTTCTACT-3=); GAPDH forward (5=-GAACATCATCCCTGCATCCA-3=) and GAPDH reverse (5=-CCAGTGAGCTTCCCGTTCA-3=). One l of the first strand cDNA product was used for amplification (in triplicate) in a 25-l reaction solution containing 12.5 l of qPCR Master Mix Plus for SYBR® Green I (Eurogentec) and 250 nM of each primer. Negative controls without added reverse transcriptase were performed. The following conditions were used for PCR: 94 °C for 10 min; 40 amplification cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The abundance of each mRNA target was calculated relative to the expression of GAPDH mRNA, used as a reference gene. A relative abundance of 1 (arbitrary unit 1) was assigned to the expression level of the mRNA of interest from 10 week-old control mouse brains. The “comparative Ct” method and standard deviations were calculated according to User Bulletin #2 from Applied Biosystems (Section 7). (http://www3.appliedbiosystems. com/cms/groups/mcb_support/documents/generaldocuments/cms_ 040980.pdf). The non-parametric Mann–Whitney test was used to study the significance of differences between mRNA levels in the brains of non-infected and infected Prnp/ (n4/condition) or wt (n5/condition) mice. Values are expressed as meanSD. All statistical calculations were performed with Prism Software.

Western blot analysis on brain homogenates

Brains were rinsed twice in NaCl 0.9%, then homogenized in Tris 50 mM pH 7.4 containing 5 mM EDTA, and a cocktail of protease inhibitors (Roche). Homogenates were centrifuged at 1,4000 rpm for 30 min at 4 °C and the pellets were then solubilized in cold lysis buffer. For detection of PrPsc, lysates were digested with 100 g of proteinase K (PK) per mg of proteins for 45 min at 37 °C. Proteins were resuspended in 50 l of denaturing loading buffer (65 mM Tris–HCl pH 6.8 containing 5% SDS, 3 M urea, 5% -mercaptoethanol, 10% glycerol, 0.05% Bromophenol Blue) boiled and loaded onto a 12% polyacrylamide gel. Proteins were separated by sodium dode cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose membrane and revealed with the appropriate primary antibody (i.e. anti-PrP SAF83, antiCB2R and anti-actin from Santa Cruz) by chemoluminescence with a LAS3000 detector (Fuji). Data were standardized using the labeling obtained on the same blot with the anti-CB2R antibody and the anti-actin antibody. Densitometry analyses were performed with the “NIH” IMAGE software, computing five independent experiments and the results are expressed as fold of increase (SD) by comparing the corrected labeling of CB2R immuno-positive bands in infected versus healthy brains. Statistical analyses were performed using the non-parametric Mann–Whitney test ** P0.001.

RESULTS

Brain concentrations of endocannabinoids, PEA and OEA in prion-infected mouse brains

We measured the concentrations of 2-AG, AEA, PEA and OEA in whole brains of healthy and prion-infected mice by isotope-dilution LC-MS. The results can be summarized as follows: (1) brain 2-AG levels did not change with age in non-infected animals, but were significantly higher 10 wpi (week post-infection) with the 139 A prion strain as compared to the corresponding non-infected mice and infected mice 2- and 24 wpi (Fig. 1); (2) brain 2-AG levels did not vary significantly in either healthy- or prion-injected Prnp/ mice (Fig. 1); (3) AEA brain levels in prion-infected mice were never significantly altered with respect to the corresponding non-infected mice, nor did they significantly change with aging (in pmol/mg extracted lipids: non-infected, 2 wpi, 0.310.18; 10 wpi, 0.240.17; 24 wpi, 0.280.09; infected, 2 wpi, 0.260.08; 10 wpi, 0.340.06; 24 wpi, 0.460.05; meansSD, n3); likewise, no statistically significant changes were found in Prnp/ mice (in pmol/mg extracted lipids: non-infected, 2 wpi, 0.270.21; 10 wpi, 0.190.08; 24 wpi, 0.290.03; infected, 2 wpi, 3.12.1; 10 wpi, 0.310.17; 24 wpi, 0.420.11; means SD, n3); (4) PEA brain levels were never significantly altered with age or prion infection, in either wt (in pmol/mg extracted lipids: non-infected, 2 wpi, 7.66.3; 10 wpi, 3.50.5; 24 wpi, 3.30.08; infected, 2 wpi, 3.40.6; 10 wpi, 4.80.5; 24 wpi, 4.10.3; meansSD, n3) or Prnp/ (in pmol/mg extracted lipids: non-infected, 2 wpi, 14.711.4; 10 wpi, 2.70.1; 24 wpi, 9.65.5; infected, 2 wpi, 16.19.7; 10 wpi, 9.55.5; 24 wpi, 6.20.3; meansSD, n3) mice; (5) OEA brain levels were never significantly altered with age or prion infection, in either wt (in pmol/mg extracted lipids: noninfected, 2 wpi, 11.77.8; 10 weeks, 7.61.6; 24 wpi, 7.10.5; infected, 2 wpi, 7.21.4; 10 wpi, 9.61.0; 24 wpi, 7.60.6; meansSD, n3) or Prnp/ (in pmol/mg extracted lipids: non-infected, 2 wpi, 19.710.1; 10 wpi, 4.40.6; 24 wpi, 13.95.4; infected, 2 wpi, 23.513.6; 10 wpi, 14.86.9; 24 wpi, 6.60.1; meansSD, n3) mice. Because of the absence of PrPc expression, PrPsc formation is hampered in Prnp/ mice, which as a consequence, are resistant to prion diseases (Büeler et al., 1993). In summary, among the analyzed compounds, 2-AG was the only one the cerebral concentrations of which increased specifically during prion disease development.

Cannabinoid receptors in prion-infected mouse brains

Using quantitative PCR, we studied the mRNA expression of well-characterized cannabinoid receptors, that is, CB1R and CB2R in brains of healthy and prion-infected mice. Total mRNAs were extracted from brains of healthy and prion-infected wt and Prnp/ mice at 2, 10, 24 and 32 weeks after i.p. inoculation (Fig. 2A, B). As early as 10 wpi, the levels of CB2R mRNA significantly increased during the development of the disease, up to 16-fold at 32 wpi (Fig. 2B), whereas no significant variation of the levels of CB1R (Fig. 2B), PPAR and PPAR receptors was observed (Fig. 2C). The elevation of CB2R mRNA level was specific of the development of prion pathogenesis since no variation was observed either in brains of mice inoculated with healthy brain homogenate (“non-infected,” Fig. 2A) or in Prnp/ brains taken as controls (Fig. 2B). To check for the expression of the CB2R, proteins extracted from brains of prion-infected or non-infected mice were submitted to electrophoresis and blotted with an anti-CB2R antibody (Fig. 2D). The level of expression of the CB2R was increased by 3- and 8-fold, factors 24 and 32 weeks postinfection respectively, as compared to non-infected controls (Fig. 2D, E). In preliminary experiments, we showed that neither the levels of CB2R nor that of PrPc varied in brain mice inoculated with healthy brain homogenate (ctrl). Based on these data, protein lysates from brains of noninfected mice 24 wpi were taken as control (Fig. 2D, E). The level of PrPc was virtually unchanged in the brains of non-infected and infected mice all along the disease (Fig. 2D, middle). As expected, PrPsc was absent in brain of lifespan, indicating that the absence of PrPc expression has no influence on the level of expression of endocannabinoid receptors.

DISCUSSION

The aim of the present study was to investigate the pattern of expression of various components of the endocannabinoid system during the time course of a murine model of prion disease. Among the tested endocannabinoids in brain extracts, 2-AG was the only one to be up-regulated 10 wpi, that is, during the asymptomatic stage. This increase of 2-AG levels was strictly dependent on prion disease progression. In contrast, no significant variation of the cerebral concentrations of AEA and its anti-inflammatory congeners and PPAR ligands, PEA and OEA, was observed in direct relation with prion disease development. Focusing on the 10 wpi time point, it is interesting to note that CB2R expression (mRNA and protein) was specifically and selectively increased in the brain of prioninfected mouse. The up-regulation of CB2R expression persisted and even increased until the later stages of the disease (i.e. 24 and 32 wpi), whereas the expression level of the other receptors investigated (i.e. CB1R and PPAR or ) remained unchanged. The CB2R belongs to the seven trans-membrane G-protein-coupled receptor family and is abundantly expressed in immune cells, such as macrophages, T- and B-cells and microglial cells. Indeed, we show here that the level of CB2R expression is upregulated in microglial cells exposed to prions. The presence of activated microglia in the vicinity of PrPsc deposits is a common feature in affected humans and animals (Williams et al., 1997). Activated microglia migrate toward PrPsc deposits, where they may initiate and/or exacerbate neuronal cell damage. However, the signaling molecules that trigger microglial cell recruitment are still only partly identified in the context of prion diseases (Marella and Chabry, 2004). Some pathological situations accompanied by microglial activation result in the selective increase production of 2-AG, which triggers microglial cell migration in a CB2R-dependent manner and can be antagonized by the non-psychoactive cannabinoid, cannabidiol (Walter et al., 2003). Interestingly, we have shown previously that cannabidiol prevents PrPsc-induced microglia cell migration (Dirikoc et al., 2007). Thus, 2-AG and CB2R could be involved in microglial cell recruitment in TSEs. We provided unprecedented evidence that the concentrations of one of the two major endocannabinoids in the brain, 2-AG, and the expression levels of its major molecular target, the CB2R, are elevated following prion infection long before the onset of clinical signs. Interestingly, both 2-AG and CB2R are strongly up-regulated in the brain of animal models or patients with other neurodegenerative/ neuroinflammatory diseases (Benito et al., 2007; Bisogno and Di Marzo, 2008; Campillo and Paez, 2009). Since, the increase of both 2-AG and CB2R is already fully developed in mice 10 wpi, such alteration follows closely, at least time-wise, the neuroinvasion of prions or/and is concomitant to the onset cerebral accumulation of PrPsc. In view of the previously reported pro-homeostatic, neuroprotective and anti-inflammatory functions of the endocannabinoid system, and, particularly, of CB2R (Benito et al., 2008; Bisogno and Di Marzo, 2008; Palazuelos et al., 2009; Sagredo et al., 2009), this alteration might represent an adaptive response aimed at counteracting the consequences of prion diseases. Indeed, it is broadly accepted that the endocannabinoid system might represent an early onset self-defense mechanism against brain injury (Bisogno and Di Marzo, 2007). Thus, further studies are required to assess whether local changes of these mediators closely follow, in space and time, the cerebral accumulation of PrPsc irrespective of its effects. Through the use of pharmacological tools manipulating either levels or actions of endocannabinoids, future studies will establish if the observed alterations represent an endogenous response aimed at counteracting the consequences of prion infection, or if they participate in the neuroinflammatory response. In both cases, the “2-AG/ CB2R” pair might constitute a new target for the development of therapies against TSEs.

Acknowledgments—The authors are grateful to Dr. C. Weissmann for providing Prnp/ mice, Dr. G. Lambeau for the gift of PPAR and PPAR primers and Dr. J. Grassi for -PrP antibodies. This work was supported in part by the “Fondation Alliance BioSecure” Paris, France.

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