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Accepted Manuscript Stimulation of brain glucose uptake by cannabinoid CB 2 receptors and its therapeutic potential in Alzheimer’s disease Attila Köfalvi, Cristina Lemos, Ana M. Martín-Moreno, Bárbara S. Pinheiro, Luis García-García, Miguel A. Pozo, Ângela Valério-Fernandes, Rui O. Beleza, Paula Agostinho, Ricardo J. Rodrigues, Susana J. Pasquaré, Rodrigo A. Cunha, María L. de Ceballos, Dr. PII: S0028-3908(16)30087-9 DOI: 10.1016/j.neuropharm.2016.03.015 Reference: NP 6212 To appear in: Neuropharmacology Received Date: 20 June 2015 Revised Date: 18 February 2016 Accepted Date: 7 March 2016 Please cite this article as: Köfalvi, A., Lemos, C., Martín-Moreno, A.M., Pinheiro, B.S., García-García, L., Pozo, M.A., Valério-Fernandes, Â., Beleza, R.O., Agostinho, P., Rodrigues, R.J., Pasquaré, S.J., Cunha, R.A., de Ceballos, M.L., Stimulation of brain glucose uptake by cannabinoid CB 2 receptors and its therapeutic potential in Alzheimer’s disease, Neuropharmacology (2016), doi: 10.1016/ j.neuropharm.2016.03.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Accepted Manuscript

Stimulation of brain glucose uptake by cannabinoid CB2 receptors and its therapeuticpotential in Alzheimer’s disease

Attila Köfalvi, Cristina Lemos, Ana M. Martín-Moreno, Bárbara S. Pinheiro, LuisGarcía-García, Miguel A. Pozo, Ângela Valério-Fernandes, Rui O. Beleza, PaulaAgostinho, Ricardo J. Rodrigues, Susana J. Pasquaré, Rodrigo A. Cunha, María L. deCeballos, Dr.

PII: S0028-3908(16)30087-9

DOI: 10.1016/j.neuropharm.2016.03.015

Reference: NP 6212

To appear in: Neuropharmacology

Received Date: 20 June 2015

Revised Date: 18 February 2016

Accepted Date: 7 March 2016

Please cite this article as: Köfalvi, A., Lemos, C., Martín-Moreno, A.M., Pinheiro, B.S., García-García,L., Pozo, M.A., Valério-Fernandes, Â., Beleza, R.O., Agostinho, P., Rodrigues, R.J., Pasquaré, S.J.,Cunha, R.A., de Ceballos, M.L., Stimulation of brain glucose uptake by cannabinoid CB2 receptorsand its therapeutic potential in Alzheimer’s disease, Neuropharmacology (2016), doi: 10.1016/j.neuropharm.2016.03.015.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.

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Stimulation of brain glucose uptake by cannabinoid CB2 receptors and its therapeutic potential in Alzheimer’s disease

Running title: CB2Rs stimulate cerebral glucose uptake

Attila Köfalvi1,2, Cristina Lemos1$, Ana M. Martín-Moreno3,4,# , Bárbara S. Pinheiro1$, Luis García-García5, Miguel A. Pozo5,6, Ângela Valério-Fernandes1, Rui O. Beleza1, Paula Agostinho1,7, Ricardo J. Rodrigues1,2, Susana J. Pasquaré3,7, Rodrigo A. Cunha1,8, María L. de Ceballos3,4

1CNC - Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504 Coimbra, Portugal, 2Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal, 3Neurodegeneration Group, Department of Cellular, Molecular and Developmental Neurobiology, Instituto Cajal, CSIC, Doctor Arce, 37, 28002 Madrid, Spain, 4CIBERNED; Centre for Biomedical Research on Neurodegenerative Diseases, Spain, 5CAI de Cartografía Cerebral, Instituto Pluridisciplinar, UCM, Madrid, Spain, 6PET Technology Institute, Madrid, Spain, 7Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), CONICET-Bahía Blanca and Universidad Nacional del Sur, Edificio E1, Camino La Carrindanga km 7, 8000 Bahía Blanca, Argentina, 8FMUC, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

#Present address: MD Anderson Cancer Center, Arturo Soria 270, 28033 Madrid, Spain $Present address: Experimental Psychiatry Unit, Center for Psychiatry and Psychotherapy, Innsbruck Medical University, Austria Correspondence Dr. María L. de Ceballos Neurodegeneration Group Dept. of Cellular, Molecular and Developmental Neurobiology Cajal Institute, CSIC Doctor Arce, 37 28002 Madrid, Spain Telephone:+34-91 5854716 Fax:+34-915854754 E-mail: [email protected]

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Abstract

Cannabinoid CB2 receptors (CB2Rs) are emerging as important therapeutic targets in brain disorders that typically involve neurometabolic alterations. We here addressed the possible role of CB2Rs in the regulation of glucose uptake in the mouse brain. To that aim, we have undertaken 1) measurement of 3H-deoxyglucose uptake in cultured cortical astrocytes and neurons and in acute hippocampal slices; 2) real-time visualization of fluorescently labeled deoxyglucose uptake in superfused hippocampal slices; and 3) in vivo PET imaging of cerebral 18F-fluorodeoxyglucose uptake. We now show that both selective (JWH133 and GP1a) as well as non-selective (WIN55212-2) CB2R agonists, but not the CB1R-selective agonist, ACEA, stimulate glucose uptake, in a manner that is sensitive to the CB2R-selective antagonist, AM630. Glucose uptake is stimulated in astrocytes and neurons in culture, in acute hippocampal slices, in different brain areas of young adult male C57Bl/6j and CD-1 mice, as well as in middle-aged C57Bl/6j mice. Among the endocannabinoid metabolizing enzymes, the selective inhibition of COX-2, rather than that of FAAH, MAGL or α,βDH6/12, also stimulates the uptake of glucose in hippocampal slices of middle-aged mice, an effect that was again prevented by AM630. However, we found the levels of the endocannabinoid, anandamide reduced in the hippocampus of TgAPP-2576 mice (a model of β-amyloidosis), and likely as a consequence, COX-2 inhibition failed to stimulate glucose uptake in these mice. Together, these results reveal a novel general glucoregulatory role for CB2Rs in the brain, raising therapeutic interest in CB2R agonists as nootropic agents.

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Keywords

Anandamide (AEA), β-amyloid, cerebral glucose uptake, cannabinoid CB2 receptor, cyclooxygenase-2 (COX-2), positron emission tomography (PET).

Abbreviations

2-AG, 2-arachidonoylglycerol; 3Rs, replacement, refinement and reduction of animals in research; ABDH6/12, α/β-hydrolase domain 6/12; ACEA, arachidonyl-2'-chloroethylamide; AEA, anandamide (N-arachidonoylethanolamine); CB1Rs and CB2Rs, cannabinoid CB1 and CB2 receptors; COX-2, cycloxygenase-2; DMEM, Dulbecco's modified eagle medium; 3HDG, 3H-2-deoxyglucose; DMSO, dimethyl-sulfoxide; FAAH, fatty acid amide hydrolase; 18FDG, 18F-fluoro-deoxyglucose; HEPES, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); MAGL, monoacylglycerol lipase; 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; TgAPP, transgenic amyloid precursor protein; WT, wild-type.

Chemical compounds studied in this article

AM630 (PubChem CID: 4302963); anandamide (PubChem CID: 5281969); arachidonyl-2-chloroethylamide (PubChem CID: 5311006); 2-arachidonoylglycerol (PubChem CID: 5282280); 2-deoxy-D-glucose (PubChem CID: 108223); DuP697 (PubChem CID: 3177); GP1a (PubChem CID: 10252734); JWH133 (PubChem CID: 6918505); JZL184 (PubChem CID: 25021165); LY2183240 (PubChem CID: 11507802); ouabain (PubChem CID: 439501); WIN55212-2 (PubChem CID: 5311501); WWL70 (PubChem CID:17759121).

Introduction

Brain disorders, including Alzheimer's disease (AD), often involve specific early alterations in the metabolism of glucose in the brain (Mosconi, 2005; Teune et al., 2010). The idea of alleviating symptoms of dementia by boosting cerebral energy metabolism has been toyed with for decades (Branconnier, 1983), yet safe pharmacological agents with well characterized mechanism of action are still lacking. In this sense, we have investigated here the local cerebral glucoregulatory potential of the endocannabinoid system in rodents.

The endocannabinoid system mainly consists of two established (CB1 and CB2) receptors that are activated by endocannabinoid signalling molecules, such as N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) (Di Marzo and De Petrocellis, 2012; Pertwee 2012). At the cellular level, endocannabinoid signalling via CB1 receptors (CB1Rs) regulates peripheral energy metabolism and glucose uptake (Matias et al., 2008). In the brain, acute activation of the psychoactive CB1R inhibits mitochondrial respiration and glucose metabolism in neurons and astrocytes, without robust effect on the uptake of glucose (Bénard et al., 2012; Duarte et al., 2012; Lemos et al., 2012). Similarly, the peripheral glucoregulatory role of the non-psychoactive CB2 receptors (CB2Rs) has also been recognized (Agudo et al., 2010; Romero-Zerbo et al., 2012), although this remains to be documented in the brain.

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CB2Rs were first described in peripheral organs and cells (Galiègue et al. 1995), and subsequently in activated microglial cells (Carlisle et al., 2002) - the resident immune cell of the brain. However, during the last decade, the presence and function of CB2Rs have been described in other cell types in the brain. An early evidence was provided by van Sickle et al. (2005) who identified and functionally characterized CB2Rs in brainstem neurons. Other studies have reported CB2R involvement in neurogenesis, axon guidance, and neuronal excitability (den Boon et al., 2012; Duff et al., 2013; Sierra et al., 2014; Zhang et al. 2014). Furthermore, astrocytes (in particular, activated astrocytes) are also endowed with CB2R (Sheng et al,. 2005; Benito et al., 2008; Bari et al., 2011; Rodriguez-Cueto et al., 2013). However, a recent report added that the vast majority of specific CB2R immunoreactivity in the healthy mouse neocortex is neuronal (Savonenko et al., 2015). Nonetheless, conventional CB2R immunstaining techniques may have possible limitations (Ashton, 2011). Therefore, it is important that the highly sensitive and specific RNAscope technique also proved the presence of CB2R mRNA in excitatory and inhibitory neurons (but hardly in resting microglia) throughout the hippocampus of adult mice (Li and Kim, 2015a). Furthermore, these hippocampal (and medial entorhinal) CB2Rs appear to control the synaptic release of GABA (Morgan et al., 2009; Andó et al., 2012), and the deletion of CB2Rs is associated with impaired synaptic plasticity at excitatory synapses, too, in the mouse hippocampus (Li and Kim, 2015b). Not surprisingly, the CB2R KO mice exhibit memory impairments as well (Li and Kim, 2016).

Besides these indefinite physiological roles, the therapeutic potential of central CB2Rs have also been recognized. There is an increasing clinical interest in endorsing novel selective CB2R agonists to treat different illnesses (Atwood and Mackie 2010; Pertwee 2012; Han et al., 2013), including AD. Indeed, prolonged oral treatment with a CB2R selective agonist conferred anti-inflammatory effects and reduced microglial activation, which have been demonstrated to mitigate the cognitive deficits of TgAPP mice (Martín-Moreno et al., 2012). Similarly, subchronic treatment with another CB2R agonist promoted β-amyloid (Aβ) clearance, and restored synaptic plasticity, cognition, and memory in Aβ injected rats (Wu et al., 2013). These results were extended in other reports using TgAPP/PS1 mice (Aso and Ferrer 2014; Cheng et al., 2014). Additionally, the severity of Aβ pathology correlates positively with the density of the CB2R rather than the CB1R (Esposito et al., 2007; Solas et al., 2013; Savonenko et al., 2015). Therefore, to map the putative glucoregulatory potential of CB2Rs in healthy mice and in a model of β-amyloidosis, we have taken advantage of recently optimized in vitro and in vivo protocols (Lemos et al., 2012; Martín-Moreno et al., 2012). Here we report that acute CB2R activation increases glucose uptake in the brain of young and middle-aged mice both in vitro and in vivo, which highlights a novel cerebral role for the CB2R with therapeutic potential.

2. Materials and methods 2.1 Chemicals

3H-2-deoxy-D-glucose (3HDG; specific activity: 60 Ci/mmol), arachidonylethanolamide [ethanolamide 1-3H] (50 Ci/mmol) and unlabelled AEA were obtained from American Radiolabeled Chemicals, Inc. (Saint Louis, MO, USA). 2-arachidonoyl-[1,2,3-3H]glycerol (3H-2-AG; 40 Ci/mmol) was bought from PerkinElmer, Madrid, Spain). 2-NBDG was purchased from Invitrogen (Carslbad, California, USA). Non-organic reagents, HEPES and DMSO were purchased from Sigma-Aldrich

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Portugal (Sintra, Portugal) or Merck Biosciences (Darmstadt, Germany). ACEA, AM630, DuP697, GP1a, JWH133, JZL184, LY2183240, WIN55212-2 and WWL70 were obtained from Tocris Bioscience (Bristol, U.K.). All non-water soluble substances and 2-NBDG were reconstituted in DMSO at 1000× the final concentration, and stored in aliquots at -20ºC.

2.2 Animals

All studies were carried out in accordance with the EU (2010/63/EU) and FELASA guidelines, and they were approved by the Spanish and Portuguese Ministries of Agriculture, as well as the local institutional Animal Care Committees (license No. 280279-31-A and 025781). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). A total of 73 animals were used in the experiments described here.

For the experiments which are summarized in Figs. 3AB, 4 and 5, we used 12-month-old (middle-aged) C57Bl/6j mice (wild type -WT- littermates as controls), and 12-month-old C57Bl/6j transgenic 2576 mice, containing the human APP695 transgene with mutations at KM670/671NL (hereafter, TgAPP mice). TgAPP mice have β-amyloid-burden but no evident cell loss (Hsiao et al., 1996). At 12 months, TgAPP mice did not differ in weight from the WT C57Bl/6j mice (WT: 39.6±2.4 g; TgAPP: 38.6±2.3 g), yet they displayed compromised new object recognition compared to WT mice (data not shown). Data shown in Fig. 3C were obtained from 10-week-old male WT CD-1 mice and their CB1R global KO littermates (Ledent et al., 1999). All these transgenic and WT mice were genotyped from tail tip biopsies.

Data shown in Fig. 1 were obtained from young adult (8-10 week-old) male C57bl/6 mice (Charles-River, Barcelona, Spain), while to obtain embryos for cell cultures, we mated young adult C57Bl/6 mice (Charles-River).

The animals were group-housed under controlled temperature (23±2 ºC) and subjected to a fixed 12 h light/dark cycle, with free access to food and water. We applied the principles of the ARRIVE guideline in designing and performing the in vivo and in vitro pharmacological experiments (see below), as well as for data management and interpretation. All efforts were made to reduce the number of animals used and to minimize their stress and discomfort. The animals used to perform the in vitro studies were deeply anesthetized with halothane before sacrifice (no reaction to handling or tail pinching, while still breathing). 2.3 In vitro real-time fluorescent measurement of deoxyglucose uptake in hippocampal slices

Experiments were performed as before (Lemos et al., 2015). Five young C57bl/6 mice were sacrificed, and their brain was quickly removed and placed in ice-cold Krebs-HEPES solution (in mM: NaCl 113, KCl 3, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25, glucose 5.5, HEPES 10, pH 7.2). Coronal vibratome slices (300 µm) were left to recover for 1 h at room temperature in Krebs-HEPES solution gassed with 5% CO2 and 95% O2, and then gently mounted on coverslips with the hippocampus in the center, and placed in a RC-20 superfusion chamber on a PH3 platform (Warner Instruments, Harvard, UK). The slices were superfused with gassed Krebs-HEPES solution at a rate of 0.5 mL min-1 in a closed circuit, and they were then photographed with a CoolSNAP digital camera (Roper Scientific, Trenton, NJ, USA) every 30 sec over the following 30 min (using a 5× PlanNeofluar-objective [NA 0.25] on an inverted

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Axiovert 200M fluorescence microscope [Carl Zeiss, Germany], coupled to a Lambda DG-4 integrated 175 Watt light source and wavelength switching excitation system [Sutter Instrument Company, Novato, CA, USA] to allow real-time video imaging). The data was band-pass filtered for excitation (BP470/40) and emission (BP525/50).

After recording 4 images for auto-fluorescence (to establish the baseline), 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; 30 µM) was applied through the reservoir and 15 min later, JWH133 (1 µM), GP1a (100 nM) or the DMSO vehicle alone (0.1% v/v) were administered. All measurements were obtained in duplicate from each animal (see Supplementary Fig. 1 for additional details). 2.4 Cell culture preparation

Because the embryonic hippocampus is too small to provide enough cells for our assays, neuronal and astrocyte primary cultures were prepared from the neocortex as before (Lemos et al., 2015). The neocortex of E18 male and female C57Bl/6 rat embryos was digested for 15 min with 0.125% trypsin (type II-S, from porcine pancreas, Sigma-Aldrich Portugal) and 50 µg/mL DNAse (Sigma-Aldrich) in Hank’s balanced salt solution without calcium and magnesium (in mM: NaCl 137, KCl 5.36, KH2PO4 0.44, NaHCO3 4.16, Na2HPO4 0.34, D-glucose 5, pH 7.2). After dissociation, astrocytes were grown in plastic Petri dishes (p100) for 14 days in DMEM with 10% fetal bovine serum, 50 U/mL penicillin and 50 µg/mL streptomycin (Sigma-Aldrich), at 37ºC in an atmosphere of 95%/5% air/CO2, replacing half of the medium at DIV 7. The cells were then trypsinized and plated onto poly-D-lysine (100 µg/mL, Sigma-Aldrich) and laminin (10 µg/mL, Sigma-Aldrich) coated 24-well culture plates at a cell density of 150,000/cm2. Uptake assays were performed 24 h later. For neuronal cultures, cells were plated directly onto poly-D-lysine and laminin coated 24-well culture plates, at the same cell density, in DMEM plus 10% fetal bovine serum, 50 U/mL penicillin and 50 µg/mL streptomycin. Two hours after seeding, the medium was replaced by Neurobasal medium with 2% B27 supplement (GIBCO, Life technologies), 50 U/mL penicillin, 50 µg/ml streptomycin and 2 mM glutamine (Sigma-Aldrich), and cells were grown at 37ºC in an atmosphere of 95%/5% air/CO2 until they were used 14 days later. The medium was partially (40%) replaced every 4 days. On average, 15% of cells were found positive for glial fibrillar acidic protein immunostaining in randomly chosen wells (Lemos et al., 2015). 2.5 In vitro 3H-deoxyglucose uptake in neocortical neuron and astrocyte cultures

The assays were carried out according to Lemos et al. (2015). For these assays, we used 7 different astrocyte cultures and 5 neuron cultures from 7 and 5 independent preparations (pregnant dams). After 15 days in culture, the culture medium was replaced with 250 µL Krebs-HEPES assay solution (pH 7.4; 37ºC) (see above), containing either the vehicle alone (DMSO 0.1%, in half of the plate), or the CB2R-selective antagonist, AM630 (1 µM, in the other 12 wells). The cultures were incubated for 60 min, at 37ºC, and the CB2R agonists JWH133 (100 nM) (4 wells/plate) or GP1a (100 nM) (4 wells/plate), or their vehicle (DMSO 4 wells/plate), was gently added to the wells in a volume of 250 µL, together with AM630 (i.e. 3×4 wells/plate) or its vehicle (i.e. 3×4 wells/plate), and the radioactive glucose analogue 3H-2-deoxyglucose (3HDG; 16.6 nM, final concentration). After a further 30 min incubation at 37ºC, the plates were transferred to ice and the 3H was measured in an aliquot from each well (to assess the small differences in the individual 3H content in each well that pipetting and mixing solutions may introduce). The cells were then washed gently 3 times with 1 mL ice-cold Krebs-HEPES, 300 µL NaOH (0.5 M) was added to each well, and the plates were left

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on a shaker for an hour to recover the proteins and 3HDG taken up by the cells. Tritium was measured in a Tricarb β-counter (PerkinElmer, USA), and these values were adjusted for protein quantity and the 3H concentrations in each individual wells, and multiplied by 3.3×105 (the ratio between 3HDG and D-glucose in the medium).

In this experimental model, cytochalasin B (10 µM) largely inhibits glucose uptake in both cell cultures, thus confirming the specificity of the transport process (Lemos et al., 2015). Additionally, the plasma membrane Na+/K+-ATPase inhibitor, g-strophantine, also inhibited glucose uptake in this assay by 42.8±2.1% (n=3, P < 0.01; figure not shown).

2.6 In vitro 3H-deoxyglucose uptake in hippocampal slices To better characterize the glucoregulator role of CB2R signaling pharmacologically,

we moved to an in vitro glucose uptake protocol previously optimized in acute hippocampal slices which allows the effect of various treatments to be compared simultaneously in different strains in a pairwise arrangement (Lemos et al, 2012, 2015). Animals were anesthetized with halothane, sacrificed (around 14:00 o'clock each experimental day to reduce potential circadian hormonal effects), and their brain was immediately placed in ice-cold Krebs-HEPES assay. The hippocampus was cut into 450 µm-thick transverse slices with the help of a McIlwain tissue chopper, and the slices were gently separated in ice-cold carboxygenated (95% O2 and 5% CO2) assay solution, and maintained at 37ºC in a multichamber slice incubator with 50 ml of carboxygenated assay solution until the end of the experiment. Four slices were used from each animal and in each experimental condition. The CB2R-selective antagonist, AM630 or its vehicle (DMSO 0.1%) were bath-applied from min 55 of the preincubation period. WIN55212-2, JWH133, ACEA or DuP697, LY2183240, as well as JZL184 and WWL70 combined or their vehicle (DMSO 0.1%) were added from min 60 of the preincubation period, and the radioactive glucose analogue, 3H-2-deoxy-D-glucose (3HDG; final cc. 2 nM) was applied to the bath 5 minutes later. After 30 min, the slices were washed twice in ice-cold assay solution for 5 min and collected in 1 mL NaOH (0.5 M) to dissolve the slices. An aliquot of 800 µL was then assayed for 3H in a Tricarb β-counter, while the remaining solution was used to quantify the protein in a bicinchoninic acid assay (Merck Biosciences, Germany). Tritium uptake was multiplied by a factor of 2.75 × 10 to estimate the total glucose uptake (corresponding to the concentration difference between D-glucose and 3HDG in the uptake medium; Lemos et al, 2012). 2.7 Blood glucose measurements

Blood glucose from non-fasted WT and TgAPP mice was assessed before and one hour following the acute administration of JWH133 0.2 mg/kg ip. Glucose was measured in one drop of blood from the tail using Glucocard Memory meter strips (Menarini, Spain), in a range of 40-500 mg/mL. 2.8 Endocannabinoid level measurement in mouse hippocampus

Endocannabinoids were measured externally (Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, UK). Lipid extraction was performed by homogenizing the frozen hippocampi on ice in 600 µL of acetonitrile/methanol (50/50) using an Ultra-Turrax. Upon addition of 6 pmol of N-(2- hydroxyethyl-1,1,2,2-d4)-5Z,8Z-,11Z,14Z-eicosatetraenamide [deuterated (d4)-AEA; Tocris Bioscience, UK] as an internal standard, the samples were diluted to 70% water

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and centrifuged. The pellets were kept for total protein determination. Solid-phase extraction (Strata-X 33 µm columns, Phenomenex) was performed before the LC/MS analysis on a Thermo Surveyor liquid chromatographic system coupled to an electrospray ionization triple quadrupole TSQ Quantum mass spectrometer operated in positive ionization mode. Chromatographic analysis was performed on an ACE 5 µm C8 (150×2.1 mm, Hichrom) column and the analytes were eluted using an isocratic mobile phase of water/methanol/formic acid (15/85/0.5 v/v/v) at a flow rate of 200 µL/min. The column was maintained at 30°C and the sample tray at 4°C during the analysis. The extraction efficiency was > 95% and the limit of quantification was 0.01 pmol for AEA and < 25 pmol for 2-AG. The sample concentrations were determined from calibration curves and the endocannabinoid levels were expressed per mg protein (Bradford assay). 2.9 Endocannabinoid hydrolysis assay

Experiments were carried out as before (Mulder et al., 2011), and adapted to mice (Pascual et al., 2014). Cortices were mechanically homogenized in Tris–HCl (20 mM, pH 7.2) containing 0.32 M sucrose and protease inhibitors, and centrifuged to eliminate nuclei and cellular debris (1000 g, 10 min, 4º C). Protein concentrations were determined by Bradford’s colorimetric method. The 2-AG-degrading activity was assessed by incubating sample fractions (50 µg protein) in a solution containing Tris–HCl (10 mM, pH 7.2), 1 mM EDTA, fatty acid-free bovine serum albumin (1.25 mg/mL), and 3H-2-AG in a final volume of 200 µl for 15 min at 37º C. Identical conditions were used to assess AEA degrading activity using 3H-AEA ([1- H]ethanolamine, 25 mM, 40 Ci/mmol specific activity; ARC Inc.). Reactions were stopped by the addition of 400 µl chloroform:methanol (2:1, v/v) and vigorous vortexing, after centrifugation (2000 g, 10 min, 4º C), ethanolamine or glycerol, the AEA and 2-AG hydrolysis products respectively, were obtained in the upper phase, and they were transferred into scintillation vials and radioactivity measured by liquid scintillation spectroscopy.

The experimental conditions were selected from pilot experiments establishing the relationship of substrate and protein concentrations to identify optimal AEA-degrading enzymatic activity (data not shown), allowing 50% of AEA converted into ethanolamine.

Blanks were prepared identically except that the membranes were omitted or inactivated by boiling for 10 min or by the addition of chloroform:methanol (1:1, v/v) before use. No differences were observed among the different blanks. These values were subtracted from each enzymatic activity.

Enzyme activity was expressed as picomole [1,2,3- H]glycerol formed×mg protein×15 min and picomole [ H]ethanolamine formed×mg protein ×15 min respectively. 2.10 18F-fluorodeoxy-D-glucose positron emission tomography (18FDG-PET)

These experiments were carried out as described previously (Martín-Moreno et al, 2012) with only slight modifications. In the PET studies the images of basal (no treatment) uptake were acquired, and 4 days later the animals were injected with JWH133 0.2 mg/kg ip, and after 30 min, the radiotracer was injected and new PET images were acquired (see below).

PET images were acquired in fasted mice injected (i.p.) with FDG (approx. 11.1 MBq or 300 µCi/200 µl saline, PET Technology Institute, Madrid). After a 30 min period for radiotracer uptake, the animals were anesthetized by isoflurane (2%)

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inhalation, to assure immobilization during the scanning process, and they were placed on the bed of the tomograph. Images of FDG uptake were acquired over 30 min, immediately followed by a CT (computed tomography) acquisition (Albira ARS small animal PET/CT dual scanner, Oncovision, Spain). PET images were reconstructed using the ordered subset expectation maximization algorithm (OSEM, 3 iterations), applying corrections for dead time, radioactive decay, random coincidences and scattering. For the CT images a high resolution filtered back projection (FBP) algorithm was used.

Metabolic activity was quantified by matching the CT image of the skull of each animal to a common magnetic resonance (MR) mouse brain template, in which regions of interest were previously delineated. After saving the spatial transformation, it was applied to the corresponding fused PET image, to allow the correct co-registration of the PET image to the MR brain template. Results were normalized to the actual radioactivity dose injected and the body weight of each animal (Kuntner et al., 2009). All these processes were carried out with PMOD software version 3.0 (PMOD Technologies, Switzerland).

2.11 Data presentation and statistical analysis

All data are expressed as the means ± SEM of the number of independent observations (n) indicated. Normalized data were tested for normality with the Kolmogorov-Smirnov normality tests and statistical significance was calculated by one sample t-test against the hypothetical value of zero (Figs. 1C, 2). Alternatively, a paired t-test (Fig. 3C) or repeated measures of ANOVA followed by Bonferroni's post-hoc test were used, and a value of P < 0.05 was accepted as significant. 3. Results 3.1 CB2R activation rapidly enhances glucose uptake in hippocampal slices

First we assessed whether CB2R activation affects glucose uptake in superfused hippocampal slices, and the rate of fluorescence-labeled deoxyglucose (2-NBDG) accumulation was tested at 30 second resolution in such slices. The basal accumulation of 2-NBDG was most evident in astrocyte-rich areas of transverse hippocampal slices, i.e. in the in the stratum lacunosum, followed by the strata radiatum and moleculare, and the smallest signal was found in the strata pyramidale and granulare (Fig. 1A-A'), consistent with previous findings (Jakoby et al., 2014). Importantly, both of the two selective CB2R agonists, GP1a (100 nM, P < 0.01) and JWH133 (1 µM, P < 0.05), rapidly increased the velocity of 2-NBDG accumulation in these hippocampal slices (Fig. 1 B,C, and Supplementary Video).

Fig. 1 (1.5-column fitting figure)

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Time-course and sub-regional variation of the effect of CB2R agonists on the uptake of the fluorescent glucose analogue, 2-NBDG in hippocampal slices of young C57Bl/6j male mice. For technical details and additional information, see Supplementary Methods, Supplementary Fig. 1 and Supplementary Video. A,A') Representative images illustrating the distribution of the fluorescence signal, using the false color scale of blue (low signal), green (moderate signal) and yellow (strong signal). As indicated in the time-course graph (B), image (A) was taken just before the treatment (minute 15 of baseline), and image (A') was photographed close to the end of the exposure to GP1a (100 nM: see also Supplementary Video). B) JWH133 (1 µM) and GP1a (100 nM) both increased the uptake in astrocyte-rich zones of the hippocampus. The displayed curves represent the mean + S.E.M. of individual net effects (in duplicate) in each animal, which was obtained after the subtraction of the respective DMSO controls (in duplicates per animal) from the raw treatment data. Therefore, the DMSO control is represented with the dashed line crossing the zero value. C) The individual points represent the average of 2 different slices from the same mouse in duplicate (n = 5): *P < 0.05 and **P < 0.01. 3.2 CB2R activation stimulates glucose transport in cultured neurons and astrocytes

Since the use of 2-NBDG underrepresents neuronal glucose transport (Jakoby et al., 2014), we measured the effect of acute in vitro CB2R activation on glucose uptake by primary mouse cortical astrocytes and neurons using a 3HDG transport assay. In the presence of JWH133 (30 nM) over 30 min, glucose uptake augmented by 35.4±7.5% in astrocytes (n=7, P < 0.01 vs. DMSO control) and by 13.5±4.0% in neurons (n=5, P <0.05: 2 A,B). Notably, JWH133 exhibits an EC50 value close to 1 µM at the CB1R (Huffman et al., 1999). To avoid the possible non-specific co-activation of CB1R in these cell cultures, we opted for testing JWH133 here at 30 nM, i.e. 9-times above its EC50 at the CB2R but more than an order of magnitude below its EC50 at the CB1R (Huffman et al., 1999).

Similarly, GP1a at a concentration ~2700-times above its EC50 at the CB2R but ~4-fold below its EC50 at the CB1R, i.e. at 100 nM (Murineddu et al., 2006) also increased glucose uptake in astrocytes by 22.4±10.7% (n=7, P < 0.05) and by 13.7±2.4% in neurons (n=5, P < 0.01: Fig. 2 A,B). Therefore, with the chosen ligand concentrations we achieved good selectivity toward the CB2R while still observing maximal effect amplitudes. The CB2R antagonist AM630 (1 µM) did not alter glucose uptake per se in cultures of astrocytes (n=7) or neurons (n=5). However, in the presence of AM630, neither GP1a nor JWH133 significantly altered glucose uptake in either of these two cell types (P > 0.05 vs. AM630 control; astrocytes n=5, neurons n=4: Fig. 2 A,B). In summary, CB2R activation was more effective at increasing glucose uptake in astrocytes compared with neurons. As stated previously, our neuron cultures contained 15% of astrocytes, which likely contributed to the effect of the CB2R agonists.

However, this low astrocytic contamination cannot solely account for the effect amplitudes observed neuronal cultures.

Fig. 2 (1-column fitting figure)

CB2R activation stimulates hippocampal glucose uptake in astrocytes and neurons in vitro. The CB2R-selective agonists JWH133 (30 nM) and GP1a (100 nM) stimulated glucose uptake in (A) primary cortical astrocyte and (B) neuronal cultures from

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C57Bl/6 mice, as assessed using the 3HDG uptake assay (30 min). The CB2R selective antagonist, AM630 (1 µM) prevented the action of the CB2R agonists, while it had no effect on the uptake per se. D-glucose uptake is expressed in nmol mg-1 protein, mean + SEM (n=7 astrocyte and n=5 neuron cultures, performed in quadruplicate, at a density of 15×104 cells cm-2 in 24-well plates). *P < 0.05 and **P < 0.01 vs. DMSO control; n.s., not significant.

3.3 β-amyloidosis hinders endogenous CB2R activation A similar pharmacological analysis was then performed in hippocampal slices of 12-

month-old TgAPP mice and their age-matched controls. The normalized control uptake values of the TgAPP hippocampus amounted to 105.4±8.9% of the WT control uptake (i.e. +5.4±8.9% over DMSO control) when assessed in a pairwise arrangement (n=19 pairs, P > 0.05: Fig. 3 A,B). Glucose uptake was significantly enhanced by the CB2R-selective agonist JWH133 (1 µM) in both the WT (+26.3±10.0%, n=12, P < 0.05: Fig. 3A) and the TgAPP slices (+16.7±6.7%, n=12, P < 0.05: Fig. 3B). Due to the limited availability of 12-month old TgAPP mice, we made a risk-free decision when testing JWH133 at 1 µM, i.e. at the maximal concentration where good CB2R-selectivity is still achieved (Huffman et al., 1999).

Importantly, glucose uptake was also stimulated by the non-selective ("mixed") CB1R/CB2R agonist, WIN55212-2 (1 µM), in both WT (+22.4±9.2%, n=12, P < 0.05: Fig. 3A) and TgAPP slices (+17.5±7.1%, n=12, P < 0.05: Fig. 3B). While the selective CB2R antagonist, AM630 (1 µM) had no effect on glucose uptake per se in either strain (n=12), it did prevent JWH133 and WIN55212-2 from stimulating glucose uptake in both WT and TgAPP slices (n=6: Fig. 3 A,B).

Cannabinoid agonists exert their actions predominantly via CB1R activation in the mammalian brain (Katona and Freund, 2012). To check if CB1Rs are also involved in the stimulation of glucose uptake, first we tested the CB1R-selective agonist ACEA at a concentration half way between its Kd values at the CB1R and the CB2R, i.e. at 1 µM (Hillard et al., 1999). ACEA did not affect glucose uptake in the middle-aged C57Bl/6 mice (+5.3±7.9%, n=7, P > 0.05: Fig. 3A). Subsequently, we asked if CB1Rs were involved in the effect of JWH133, taken that this ligand has some affinity toward the CB1R at 1 µM (Huffman et al., 1999). Thus, we tested JWH133 (1 µM) in acute hippocampal slices of young adult CB1R KO mice and their WT littermates on the CD-1 strain. JWH133 (1 µM) significantly stimulated glucose uptake +13.4±3.7% (n=6, P < 0.05) in the WT mice and +26.7±9.0% (n=6, P < 0.05) in the CB1R KO, thus virtually, JWH133 was more twice as more effective in the absence of the CB1R. These data together with the results with ACEA also confirm the absence of acute modulation of hippocampal glucose uptake by CB1Rs.

Next, we assessed whether endogenous activation of CB2R by the major endocannabinoids, AEA and 2-AG also bolstered glucose uptake. To that end, we inhibited either fatty acid amide hydrolase (FAAH) or cyclooxygenase-2 (COX-2), two enzymes known to be involved in AEA degradation (Egertová et al., 2003; Fowler et al., 2013). The treatment of slices with DuP697 (500 nM), a selective COX-2 inhibitor (IC50 at COX-2, 10 nM; IC50 at COX-1, 800 nM; Gierse et al., 1995), augmented glucose uptake by +16.6±7.7% in the WT mice (n=6, P < 0.05: Fig. 3A) - an effect that was prevented by CB2R blockade (n=6: Fig. 3A). However, unlike in the WT, DuP697 failed to modify glucose uptake in the TgAPP mice (n=6, Fig. 3B). In contrast,

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LY2183240 (100 nM), a potent dual inhibitor of FAAH and AEA reuptake (Nicolussi et al., 2014), did not affect glucose uptake in WT mice, and therefore, it was not tested in TgAPP mice slices. The major enzymes that degrade 2-AG in the mammalian brain are monoacylglycerol lipase (MAGL) and α/β-hydrolase domain 6/12 (ABDH6/12) (Savinainen et al., 2012; Murataeva et al., 2014). The inhibition of 2-AG hydrolysis by the concurrent application of JZL184 (1 µM), a MAGL inhibitor (Long et al., 2009), and WWL70 (1 µM), an α,βDH6/12 inhibitor (Li et al., 2007), also failed to modify glucose uptake in WT mice (Fig. 3 A).

Fig. 3 (2-column fitting figure) CB2R activation stimulates glucose uptake in hippocampal slices of both middle-aged and young mice ex vivo. A) The CB2R-selective agonist, JWH133 (1 µM), the mixed CB1R/CB2R agonist, WIN55212-2 (1 µM), as well as the COX-2 inhibitor, DuP697 (500 nM) all increased glucose uptake in 12-month-old wild-type (WT) C57Bl/6j mice. All these effects were prevented by a 5 min preincubation with the CB2R-selective antagonist, AM630 (1 µM). Inhibition of other endocannabinoid metabolizing enzymes, namely, FAAH, MAGL or α,βDH6, with LY2183240, JZL184 and WWL70 (all at 1 µM) respectively, did not affect glucose uptake. B) By contrast, only JWH133 and WIN55212-2 but not DuP697 stimulated the glucose uptake in the TgAPP 2567 mice. Data represent the mean + SEM of individual measurements from 6-12 animals, with the dashed line indicating the DMSO control values: *P < 0.05 vs. DMSO-treated slices; n.s., not significant. C) JWH133 (1 µM), stimulated the uptake of glucose in acute hippocampal slices of 10 week-old CB1R KO mice and their WT littermates. Each of the connected pairs of orange squares and blue circles represent one animal. *P < 0.05 vs. DMSO-treated slices (n=6). 3.4 Lower AEA levels in the hippocampus of TgAPP mice

The inability of COX-2 inhibition to stimulate glucose uptake in a CB2R-dependent fashion in the TgAPP hippocampus suggests a loss of endocannabinoid function, which prompted us to measure endocannabinoid levels in these two strains. The hippocampal levels of AEA and 2-AG in the WT mice (Fig. 4) were similar to those reported in the rat brain (Stella et al., 1997), and while no difference in the 2-AG levels were detected between WT and TgAPP mice (n=7, P > 0.05), AEA levels in the latter were 37.8±11.4% lower than in WT (n=7, P < 0.05) (Fig. 4A,B). Accordingly, there was a 2.4±0.3 fold increase in the 2-AG/AEA ratio in TgAPP mice (n=7, P < 0.001). The degradation of 2-AG was also not statistically different between the WT and TgAPP (n=6, P > 0.05) (Fig. 4D). Interestingly, the degradation of AEA into ethanolamine by FAAH was significantly reduced in TgAPP by 33.5±8.9% (n=6, P < 0.001) (Fig. 4D).

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Fig. 4 (1-column fitting figure) AEA levels were reduced in the hippocampus of TgAPP mice. Of the two principal endocannabinoids, the levels of AEA (B) but not of 2-AG (A) were significantly lower (by 38%) in the TgAPP mice (deep blue bars) than in the WT (light blue bars), as assessed by combined liquid chromatography-mass spectrometry. Data represent the mean + SEM of individual measurements from 7 animals. 2-AG hydrolysis (C) was also unaltered in hippocampal membranes of TgAPP mice in vitro (n=6), however, AEA metabolism (D) by FAAH into ethanolamine was significantly smaller in the transgenic hippocampi in vitro (n=6). *P < 0.05, ***P < 0.001; n.s., not significant.

3.5 Effect of CB2R activation on 18FDG uptake - a PET analysis in vivo

Finally, we tested the feasibility of targeting CB2R to stimulate brain glucose uptake in middle-aged mice in vivo. We first evaluated the basal uptake of 18FDG in the brain regions typically bearing the hallmarks of the diminished 18FDG signal in the Alzheimer’s disease-affected human brain, i.e. the frontal and temporal cortices, and the hippocampus (Mosconi, 2005). Like the ex vivo data, we observed no difference in the 18FDG signal in the aforementioned areas of WT and TgAPP mice (n=9: Fig. 5 A,A',B,B',C). Four days later, the same animals were injected with a small dose of JWH133 (0.2 mg/kg, i.p.) and they were re-evaluated for 18FDG uptake. While this CB2R agonist did not alter blood glucose levels (Fig. 5 D), it did stimulate glucose uptake by an average of 31.5% in each of the three regions in the WT mice (n=4, P <0.05: Fig. 5E-G), as well as by an average of 21.3% in the TgAPP mice (n=4, P < 0.05: Fig. 5E'-G').

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Fig. 5 (1.5-column fitting figure)

Evidence for the stimulation of brain glucose uptake by a single low-dose injection of JWH133 in middle-aged mice in vivo. Basal (A1,B1) and JWH133-stimulated A2,B2) 18F-fluoro-deoxy-glucose (18FDG) uptake in the frontal and temporal cortices, and the hippocampus. A1) Representative color-coded PET of basal 18FDG uptake in 12 month old wild-type (WT) and B1) TgAPP 2567 mice. Panels A1',B1') illustrate the same images merged with the respective CT images, seen from three directions. The effect of acute JWH133 injection (0.2 mg/kg, i.p.) is shown in panels A2,A2',B1,B2'). Color code for FDG activity can be seen vertically on the left of the figure. C) Mean basal uptake values in the three regions of interest (n=9 mice). D) Acute JWH133 injection does not alter the blood glucose levels in the two strains and hence, JWH133 does not appear to influence glucose uptake in the brain through systemic mechanisms. E-G') Before-after graphs summarizing the basal uptake, and 4 days later, the JWH133-stimulated (JWH) uptake in the frontal E, E') and temporal F, F') cortices, as well as in the hippocampus G, G'), of 4 wild-type (WT, E-G) and 4 TgAPP 2567 (E'-G') mice. Uptake values were corrected for the dose injected and for body weight, *P < 0.05. 4. Discussion 4.1 A novel role for cannabinoid CB2 receptors: stimulating glucose uptake in the brain

We show here that CB2R activation in vitro, and more importantly, in vivo rapidly enhances cerebral glucose uptake by ~30% over the basal levels. These data are in concert with a recent finding that the intraperitoneal injection of the mixed CB1R/CB2R agonist, HU210 increased cerebral 18FDG PET signal by 21% in the rat (Nguyen et al., 2012). Since CB1R activation decreases mitochondrial glucose metabolism (Duarte et al., 2011) CB2Rs are likely involved in the stimulation of brain metabolism in the above study. These data support the view of the recently described antagonistic interaction between CB1Rs and CB2R in neurons (Callén et al., 2012).

Given that brain glucose availability controls cognition and memory in humans (Messier, 2004) and that central metabolic boosting alleviates the cognitive symptoms of dementia, these data suggest that CB2R agonists might represent a novel class of nootropic drugs (Branconnier, 1983). This raises the interesting hypothesis that by

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promoting AEA release, neural activity can activate central CB2Rs and thereby refuel circuits under heavy load. Indeed, it was recently found that JWH133 enhanced aversive memory consolidation in mice, while the genetic ablation or pharmacological inhibition of CB2R impaired it (García-Gutiérrez et al., 2013).

2-NBDG uptake in slices suggested a contribution of astrocytes in the effects of CB2R activation. Consistent with this, our data in cultures show that CB2R influences both astrocytic and neuronal glucoregulation. CB2R are found at low density on most cell types in the brain, including activated microglia and astrocytes controlling neuroinflammation (Ramirez et al., 2005; Martín-Moreno et al., 2012), and inhibiting astrocytoma growth (Sánchez et al., 2001). Moreover, CB2R mRNA or protein has also been found in several types of neurons, including cortical, hippocampal and midbrain neurons (Atwood and Mackie, 2010; Callén et al., 2012; den Boon et al., 2012; Zhang et al., 2014). Interestingly, CB2R expression and density in neurons apparently surpasses that in microglia or astrocytes in the mouse neocortex (Savonenko et al., 2015) and hippocampus (Li and Kim, 2015a).

The therapeutic activation of CB2R is safe and it does not trigger psychoactivity (Atwood and Mackie 2010; Pertwee 2012). Furthermore, CB2R density is known to increase in Alzheimer’s disease and Down’s syndrome, as well as in β-amyloidosis in vitro (Esposito et al., 2007; Ruiz-Valdepeñas et al., 2010; Solas et al., 2013). This probably reflects an adaptive self-defense process since CB2R activation confers neuroprotection in several experimental models of Alzheimer’s disease (Ramirez et al., 2005; Esposito et al., 2007; Ruiz-Valdepeñas et al., 2010; Martín-Moreno et al., 2012). 4.2 AEA and β-amyloidosis

The first step in common pathomechanisms toward AD pathology is excessive β-amyloid accumulation (Sperling et al., 2013; Zahs and Ashe 2013). Remarkably, the antagonistic relationship between AEA and β-amyloid appears to be bi-directional: β-amyloid toxicity is prevented by AEA in human cell lines in vitro (Milton, 2002) and conversely, β-amyloid treatment decreases AEA levels in C6 rat astroglioma cells (Esposito et al., 2007). Accordingly, AEA levels decrease in the AD brain and they are inversely correlated with β-amyloid levels (Jung et al., 2012), consistent with the lower hippocampal AEA levels in TgAPP mice. β-amyloidosis may lower AEA levels by either bolstering catabolism and/or by impairing its synthesis. COX-2 is an important enzyme in AEA metabolism (Glaser and Kaczocha, 2010; Pamplona et al., 2010), furthermore, β-amyloidosis induces COX-2 expression in astrocytes (Giovannini et al., 2002) and in AD-affected neurons. Hence, COX-2 inhibition may help to decrease the incidence or slow the progress of AD (Berk et al., 2013). In fact, in a previous work we treated TgAPP 2576 mice orally for four months with the CB2R -selective agonist JWH133 and we observed an attenuation of the AD phenotype, including recovery from memory impairment and neuroinflammation, as well as a normalization of the increase in COX-2 protein levels (Martín-Moreno et al., 2012).

Strikingly, COX-2 inhibition by the selective DuP697 antagonist facilitated glucose uptake via CB2R activation in WT but not in the TgAPP mice, probably due to the rescue of AEA from COX-2-driven tonic metabolism in WT mice (Glaser and Kaczocha, 2010; Pamplona et al., 2010). In the TgAPP mice, we found a 38% decrease of AEA, suggesting that these levels were already too low to stimulate the CB2Rs, even following COX-2 blockade. In fact, this may illustrate a deficit in synthesis, since AEA production requires the concurrent stimulation of both NMDA and acetylcholine receptors (Stella and Piomelli, 2001), both of which become hypofunctional in AD (Pavía et al., 1998; Giovannini et al., 2002). In the present work FAAH activity was

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reduced in TgAPP mice, as judged by the decrease in AEA hydrolysis, again in agreement with its reduction in AD brain, which we recently reported (Mulder et al., 2011; Pascual et al., 2014). This reduction is mimicked by pathologically relevant Aβ1- 40 peptide concentrations (Pascual et al., 2014) and may serve as to preserve AEA levels under lower AEA production, as a compensatory mechanism. Even though, FAAH inhibition did not stimulate glucose uptake, suggesting that AEA metabolism by FAAH is not a rate limiting factor for CB2R activation in the WT hippocampus. 4.3 Cannabinoid receptors and glucose uptake

Although the role of cannabinoid receptors in peripheral glucoregulation is extensively studied (Matias et al., 2008), the mechanism through which CB2R activation facilitates glucose transport in the brain awaits further interrogation. Several mechanisms could be invoked on that respect. For instance, cannabinoids stimulate AMP-activated protein kinase (Dagon et al., 2007), a major intracellular energy sensor, which leads to enhanced glucose uptake by stimulating glucose transporter (GLUT) membrane expression and transport efficiency (Rutter et al., 2003; Weisová et al., 2009). More importantly, the effect was absent in CB2R knockout mice (Dagon et al., 2007). A possible direct effect of CB2R agonists on glucose transporters remains unexplored. New synthesis of transporters would not parallel the acute pharmacological effects reported here. Therefore, we suggest that CB2R agonists may favor transporter localization at the plasma membrane. On the other hand, it is less likely that CB2Rs increased glucose uptake driven by the stimulation of its metabolism, because we found previously that WIN55212-2 slows rather than facilitates the mitochondrial oxidative metabolism of glucose via CB1R activation both in neurons and astrocytes (Duarte et al., 2012). 4.4 In conclusion, the present results provide the first direct pharmacological evidence in vitro and in vivo of a role of CB2R in central glucoregulation. Additionally, we found that glucoregulation by endogenous CB2R signalling is negatively affected by β-amyloidosis, thought to be the first pathological step in AD. Therefore, it would be interesting to perform further studies to define how CB2R mediated glucoregulation contributes to the recently discovered therapeutic potential of CB2R agonists in animal models of AD (Martín-Moreno et al., 2012; Wu et al., 2013; Aso and Ferrer 2014; Cheng et al., 2014). Acknowledgements

This work was supported by the Council of Madrid (S-BIO/0170/2006 and P2010/BMD-2349 to MLC and MAP). AMM-M received a fellowship from the Spanish Ministry of Education and Science. Authors from the University of Coimbra acknowledge funding from the following sources: NARSAD, Santa Casa da Misercórdia, DARPA (09-68-ESR-FP-010), FEDER (QREN), Programa Mais Centro under project CENTRO-07-ST24-FEDER-002006 and Programa Operacional Factores de Competitividade - COMPETE, and national funds (PTDC/SAU-OSM/105663/2008 and Pest-C/SAU/LA0001/2013-2014) via FCT – Fundação para a Ciência e a Tecnologia.

We thank Dr M. Delgado for excellent assistance in the PET studies, and Drs L. M. García-Segura and M. A. Arévalo for comments on the manuscript. We are grateful to Drs Catherine Ledent and Catherine Vroonen (IRIBHM, Brussels) for providing the CB1R KO mice and their WT littermates.

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Supplementary Material A Supplementary Video reproduces the time-course of GP1a action at 30×playback speed. Additional Supplementary Material with two supplementary figures explains the calculation of fluorescent glucose uptake on some raw data, and illustrate the three major cannabinoid agonists, GP1a, JWH133 and WIN55212-2, used in our study. Conflicts of Interest The authors have no conflict of interests to report, and received no financial support or compensation from any individual or corporate entity over the past 3 years for research or professional services, and there are no personal holdings that could be perceived as constituting a potential conflict of interest. Author Contributions AK and MLC conceived and designed the work, performed data analysis, writing and edition of the manuscript; AK, CL, BSP and AV-F performed glucose uptake in slices and in the cell cultures; ROB, PA and RJR made the cell cultures; AMM-M assessed glucose in plasma, genotyped by PCR and performed the respective data analysis; LG-G and MAP conducted the PET studies and the respective data analysis; MLC and SJP performed the endocannabinoid hydrolysis assay; and RAC interpreted results, and contributed to the writing and the edition of the manuscript, as well as to the purchase of some supplies. All authors have reviewed and commented on the manuscript. References Agudo, J., Martin, M., Roca, C., Molas, M., Bura, A.S., Zimmer, A., Bosch, F., Maldonado, R., 2010. Deficiency of CB2 cannabinoid receptor in mice improves insulin sensitivity but increases food intake and obesity with age. Diabetologia 53, 2629–2640. doi:10.1007/s00125-010-1894-6 Andó, R.D., Bíró, J., Csölle, C., Ledent, C., Sperlágh, B., 2012. The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB1 and CB2 receptors in the mouse hippocampus. Neurochem. Int. 60, 145-152. Aso, E., Ferrer, I., 2014. Cannabinoids for treatment of Alzheimer’s disease: moving toward the clinic. Front. Pharmacol. 5, 37. doi:10.3389/fphar.2014.00037 Ashton, J.C., 2011. Knockout controls and the specificity of cannabinoid CB2 receptor antibodies. Br. J. Pharmacol. 163:1113. doi: 10.1111/j.1476-5381.2010.01139.x. Atwood, B.K., Mackie, K., 2010. CB2: a cannabinoid receptor with an identity crisis. Br. J. Pharmacol. 160, 467–479. doi:10.1111/j.1476-5381.2010.00729.x Bari, M., Bonifacino, T., Milanese, M., Spagnuolo, P., Zappettini, S., Battista, N., Giribaldi, F., Usai, C., Bonanno, G., Maccarrone, M., 2011. The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release. Cell. Mol. Life Sci. CMLS 68, 833–845. doi:10.1007/s00018-010-0494-4

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Fig. 2 (1-column fitting figure) CB2R activation stimulates hippocampal glucose uptake in astrocytes and neurons in vitro. The CB2R-selective agonists JWH133 (30 nM) and GP1a (100 nM) stimulated glucose uptake in (A) primary cortical astrocyte and (B) neuronal cultures from C57Bl/6 mice, as assessed using the 3HDG uptake assay (30 min). The CB2R selective antagonist, AM630 (1 µM) prevented the action of the CB2R agonists, while it had no effect on the uptake per se. D-glucose uptake is expressed in nmol mg-1 protein, mean + SEM (n=7 astrocyte and n=5 neuron cultures, performed in quadruplicate, at a density of 15×104 cells cm-2 in 24-well plates). *P < 0.05 and **P < 0.01 vs. DMSO control; n.s., not significant. Fig. 3 (2-column fitting figure) CB2R activation stimulates glucose uptake in hippocampal slices of both middle-aged and young mice ex vivo. A) The CB2R-selective agonist, JWH133 (1 µM), the mixed CB1R/CB2R agonist, WIN55212-2 (1 µM), as well as the COX-2 inhibitor, DuP697 (500 nM) all increased glucose uptake in 12-month-old wild-type (WT) C57Bl/6j mice. All these effects were prevented by a 5 min preincubation with the CB2R-selective antagonist, AM630 (1 µM). Inhibition of other endocannabinoid metabolizing enzymes, namely, FAAH, MAGL or α,βDH6, with LY2183240, JZL184 and WWL70 (all at 1 µM) respectively, did not affect glucose uptake. B) By contrast, only JWH133 and WIN55212-2 but not DuP697 stimulated glucose uptake in the TgAPP 2567 mice. Data represent the mean + SEM of individual measurements from 6-12 animals, with the dashed line indicating the DMSO control values: *P < 0.05 vs. DMSO-treated slices; n.s., not significant. C) JWH133 (1 µM), stimulated the uptake of glucose in acute hippocampal slices of 10 week-old CB1R KO mice and their WT littermates. Each of the connected pairs of orange squares and blue circles represent one animal. *P < 0.05 vs. DMSO-treated slices (n=6). Fig. 4 (1-column fitting figure) AEA levels were reduced in the hippocampus of TgAPP mice. Of the two principal endocannabinoids, the levels of AEA (B) but not of 2-AG (A) were significantly lower (by 38%) in the TgAPP mice (deep blue bars) than in the WT (light blue bars), as assessed by combined liquid chromatography-mass spectrometry. Data represent the mean + SEM of individual measurements from 7 animals. 2-AG hydrolysis (C) was also unaltered in hippocampal membranes of TgAPP mice in vitro (n=6), however, AEA metabolism (D) by FAAH into ethanolamine was significantly smaller in the transgenic hippocampi in vitro (n=6). *P < 0.05, ***P < 0.001; n.s., not significant. Fig. 5 (1.5-column fitting figure) Evidence for the stimulation of brain glucose uptake by a single low-dose injection of JWH133 in middle-aged mice in vivo. Basal (A1,B1) and JWH133-stimulated A2,B2) 18F-fluoro-deoxy-glucose (18FDG) uptake in the frontal and temporal cortices, and the hippocampus. A1) Representative color-coded PET of basal 18FDG uptake in 12 month old wild-type (WT) and B1) TgAPP 2567 mice. Panels A1',B1') illustrate the same images merged with the respective CT images, seen from three directions. The effect of acute JWH133 injection (0.2 mg/kg, i.p.) is shown in panels A2,A2',B1,B2'). Color code

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for FDG activity can be seen vertically on the left of the figure. C) Mean basal uptake values in the three regions of interest (n=9 mice). D) Acute JWH133 injection does not alter the blood glucose levels in the two strains and hence, JWH133 does not appear to influence glucose uptake in the brain through systemic mechanisms. E-G') Before-after graphs summarizing the basal uptake, and 4 days later, the JWH133-stimulated (JWH) uptake in the frontal E, E') and temporal F, F') cortices, as well as in the hippocampus G, G'), of 4 wild-type (WT, E-G) and 4 TgAPP 2567 (E'-G') mice. Uptake values were corrected for the dose injected and for body weight, *P < 0.05.

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A Supplementary Video reproducing the time-course and sub-regional variation of 2-NBDG accumulation in a transversal hippocampal slice of young adult C57Bl/6j male mouse, under resting condition and in response to CB2R activation at a 30× playback speed. Vibratome-cut slices of 300 µm-thickness were kept at room temperature in carboxygenated modified Krebs' solution at least for one hour before use. Each time a slice was gently mounted in an RC-20 superfusion chamber on a PH3 platform. The slices were superfused with gassed Krebs-HEPES solution at a rate of 0.5 mL/min in a closed circuit, and they were then photographed with a CoolSNAP digital camera every 30 sec over the following 30 min (using a 5× PlanNeofluar-objective [NA 0.25] on an inverted Axiovert 200M fluorescence microscope, coupled to a Lambda DG-4 integrated 175 Watt light source and wavelength switching excitation system, to allow real-time video imaging). The data was band-pass filtered for excitation (BP470/40) and emission (BP525/50). After recording 7 images for autofluorescence (to establish the absolute zero signal), 2-NBDG (30 µM, 1/1000 dilution from DMSO-dissolved stock) was applied through the reservoir and 15 min later, the CB2R-selective agonist, GP1a (100 nM) was administered, as indicated by the horizontal bars. The initial 2-NBDG signal resembles a decaying oscillation which has already reported in other experimental models (O'Neil et al., 2005). Usually within 7 minutes, the accumulation of 2-NBDG achieves steady-state kinetics, allowing establishing the predrug baseline. For additional explanations, technical details and calculations, see Materials and methods, body text and legend to Figure 1, as well as the Supplementary Material.

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• CB2R activation stimulates glucose uptake in the mouse brain in vivo • CB2R activation rapidly stimulates glucose uptake in brain slices and cell

cultures • COX-2 inhibition also stimulates hippocampal glucose uptake via CB2R

activation • β-amyloidosis decreases anandamide levels, preventing the effect of COX-2

blockade