1 Cannabidivarin completely rescues cognitive deficits and delays
2 neurological and motor defects in male Mecp2 mutant mice
3
4 Zamberletti E.1, Gabaglio M.1, Piscitelli F.2, Brodie J.S.3, Woolley-Roberts M.3,
5 Barbiero I.1, Tramarin M.1, Binelli G.1, Landsberger N.4, Kilstrup-Nielsen C.1,
6 Rubino T.1, Di Marzo V.2, Parolaro D.1,5
7
8 1Dept. of Biotechnology and Life Sciences (DBSV), University of Insubria,
9 Varese, Italy; 2Endocannabinoid Research Group, Institute of Biomolecular
10 Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy; 3GW
11 Research Ltd, Cambridge (UK); 4Dept. of Medical Biotechnology and
12 Translational Medicine, University of Milan, Italy; 5Zardi Gori Foundation, Milan
13 (Italy).
14
15 Corresponding author:
16 Daniela Parolaro
17 DBSV, University of Insubria and Zardi Gori Foundation
18 Tel. +39 0331339417
19 Fax. +39 0331339459
20 Email [email protected]
1
1 Abstract
2 Background: Recent evidence suggests that 2-week treatment with the non-
3 psychotomimetic cannabinoid cannabidivarin (CBDV) could be beneficial towards
4 neurological and social deficits in early symptomatic Mecp2 mutant mice, a model
5 of Rett syndrome (RTT).
6 Aim: To provide further insights into the efficacy of CBDV in Mecp2-null mice we
7 used a lifelong treatment schedule (from 4 to 9 weeks of age) and evaluated its
8 effect on recognition memory and neurological defects in both early and
9 advanced stages of the phenotype progression.
10 Methods: CBDV 0.2, 2, 20, 200 mg/kg/day was administered to Mecp2-null mice
11 from 4 to 9 weeks of age. Cognitive and neurological defects were monitored
12 during the whole treatment schedule. Biochemical analyses were carried out in
13 brain lysates from 9-week-old wildtype and knockout mice to evaluate brain-
14 derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) levels
15 as well as components of the endocannabinoid system.
16 Results: CBDV rescues recognition memory deficits in Mecp2 mutant mice and
17 delays the appearance of neurological defects. At the biochemical level, it
18 normalizes BDNF/IGF1 levels and the defective PI3K/AKT/mTOR pathway in
19 Mecp2 mutant mice at an advanced stage of the disease. Mecp2 deletion
2
1 upregulates CB1 and CB2 receptor levels in the brain and these changes are
2 restored after CBDV treatment.
3 Conclusions: CBDV administration exerts an enduring rescue of memory
4 deficits in Mecp2 mutant mice, an effect that is associated with the normalization
5 of BDNF, IGF-1 and rpS6 phosphorylation levels as well as CB1 and CB2
6 receptor expression. CBDV delays neurological defects but this effect is only
7 transient.
8 Keywords: cannabidivarin; Mecp2 mutant mice; endocannabinoids; BDNF;
9 IGF-1
10
11 Short title: CBDV partially relieves Mecp2-null mouse phenotype
12
13
14
15
16
17
18
19
3
1 1. Introduction
2 Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder with
3 a prevalence rate of 1 in 10,000 females. RTT patients have apparently normal
4 perinatal development until about 6–18 months of age, after which they undergo
5 a period of rapid regression, characterized by the appearance of autistic features,
6 cognitive regression, stereotypic hand movements and loss of language. RTT
7 girls also have seizures during childhood, breathing arrhythmias, develop
8 scoliosis and lose mobility between ages 1 and 4 years (Chahrour and Zoghbi,
9 2007; Hagberg, 2002; Neul et al., 2014).
10 Mutations affecting the methyl-CpG-binding protein 2 (MeCP2) represent the
11 almost exclusive cause of the disorder (Amir et al., 1999). Given the location of
12 the MECP2 gene on the X chromosome, males with MECP2 mutations are more
13 severely affected and rarely survive infancy while females, owing to X
14 chromosome inactivation, are mosaics with cells that express either the wild-type
15 (WT) or mutant version of MeCP2 (Chahrour and Zoghbi, 2007).
16 Mecp2 deficiency in mice closely mimics the clinical features of the human
17 disorder, including motor and neurological defects and breathing abnormalities
18 (Ricceri et al., 2008), possibly reflecting the high conservation of the MeCP2
19 amino acid sequence and the parallel dynamics of MeCP2 expression during
20 brain development in the two species. Thus, despite differences in brain structure
4
1 and developmental timing between humans and mice, the molecular
2 consequences of the MeCP2 mutation appear to be similar in the two species,
3 and mouse models represent an essential tool for testing potential treatment
4 strategies for RTT.
5 Converging evidence points to a role for endocannabinoid signaling dysregulation
6 in the pathophysiology of neurodevelopmental conditions including social
7 dysfunction and autism spectrum disorder (ASD) (Karhson et al., 2016; Wei et
8 al., 2017; Zamberletti et al., 2017). Remarkably, improvement in social
9 functioning and anxiety-like behavior was consistently reported following
10 pharmacological modulation of different components of the endocannabinoid
11 system in genetic and environmental models of autism (Busquets-Garcia et al.,
12 2013; Gomis-González et al., 2016; Hosie et al., 2018; Kerr et al., 2016; Servadio
13 et al., 2016; Jung et al., 2012; Wei et al., 2016). In line with animal data, recent
14 human findings support a link between altered endocannabinoid activity and
15 autism (Karhson et al., 2018) possibly suggesting that compounds acting on the
16 endocannabinoid system could be beneficial in alleviating ASD symptoms.
17 Phytocannabinoids are terpenophenolic constituents of the cannabis sativa plant
18 that, in addition to their effects on the endocannabinoid system, exhibit a range
19 of neuromodulatory, neuroprotective, anti-oxidant and anti-inflammatory
20 properties, including effects on biochemical pathways that could contribute to
5
1 achieve an overall therapeutic effect in major neurological disorders (Campos et
2 al., 2016; Fernandez-Ruiz et al., 2013; Ligresti et al., 2016; Nagarkatti et al.,
3 2009). Delta-9-tetrahydrocannabinol (THC) is the major psychotomimetic
4 component of cannabis whose euphoric effects are mediated by the activation of
5 CB1 receptors in the central nervous system; however, this mechanism is
6 responsible for many untoward adverse effects, whose risk is greater with early
7 use and high doses (Volkow et al., 2014), thus discouraging its use among
8 children and high-risk groups. Unlike THC, other phytocannabinoids with weak or
9 no psychotropic activity seem to hold great yet unexplored promise as therapeutic
10 agents for complex and multifaceted neurodevelopmental diseases. Cannabidiol
11 (CBD) and cannabidivarin (CBDV) are both effective in ameliorating epilepsy
12 (Brodie and Ben-Menachem, 2017) and motor dysfunctions (Iannotti et al., 2018),
13 two conditions frequently observed also in RTT patients (Steffenburg et al., 2001;
14 Huppke et al., 2007), supporting their potential as treatment options for RTT.
15 Accordingly, a recent paper showed that a 2-week long treatment schedule with
16 CBDV could be beneficial towards neurological and social deficits in early
17 symptomatic Mecp2 mutant mice, a model for RTT, possibly via its action at
18 GPR55 receptors (Vigli et al., 2018). Both CBD and CBDV activate and
19 desensitize TRPV1 cation channels and modulate anandamide uptake.
20 Additionally, CBDV also modulates the levels of the endocannabinoid 2-AG (De
6
1 Petrocellis et al., 2011; 2012), which is crucially involved in synaptic plasticity and
2 cognitive processes (Zhang et al., 2014).
3 To gain more insight into the possible efficacy of CBDV in the context of RTT, in
4 this study we provided additional pieces of data by testing the effect of similar
5 doses of CBDV in another animal model of RTT, namely Mecp2-null mice, using
6 a lifelong treatment schedule (from 4 to 9 weeks of age). CBDV effects on
7 recognition memory and neurological deficits were monitored throughout the
8 entire animals’ lifespan. At an advanced stage of the phenotype progression (9-
9 week old mice), biochemical analysis were performed to assess whether CBDV
10 could modulate neurochemical abnormalities that are found in the brain of Mecp2-
11 null mice. In particular, we investigated CBDV’s effect on brain-derived
12 neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1) and their common
13 downstream PI3K/Akt/mTOR signaling pathway whose reduced levels have been
14 implicated in the disease progression (Castro et al., 2014; Li and Pozzo-Miller,
15 2014; Ricciardi et al., 2011) and we analyzed nuclear volumes in the layer V of
16 the medial prefrontal cortex (mPFC) and CA1 region of the hippocampus, whose
17 reduction represents a consistent feature of Mecp2 deficiency at the cellular level
18 (Gadalla et al., 2013; Giacometti et al., 2007). Finally, given that altered
19 endocannabinoid activity contributes to the pathogenesis of several psychiatric
20 conditions including anxiety-related disorders, intellectual disabilities, major
7
1 motor disorders, seizures and autism (Campolongo and Trezza, 2012;
2 Fernández-Ruiz and Gonzáles, 2005; Katona, 2015; Zamberletti et al., 2017), we
3 investigated the status of components of the endocannabinoid system, in terms
4 of endocannabinoid levels, enzymatic machinery and CB1, CB2 receptors.
5 2. Experimental procedures
6 2.1.1 Animals
7 We used the CD1 Mecp2-null mouse strain described in Cobolli Gigli et al. (2016).
8 All experimental procedures were carried out in male Mecp2 WT and KO mice,
9 housed in groups of 4-5 in clear plastic cages (33.1x15.9x13.2 cm) on a 12 h
10 light-dark cycle (lights on 8:00 AM) and in a temperature- (22 ± 2°C) and humidity-
11 controlled environment (50 ± 10%) with a plastic tube for environmental
12 enrichment and free access to food and water. Experimental procedures were
13 performed in accordance with the guidelines released by the Italian Ministry of
14 Health (D.L. 2014/26) and the European Community directives regulating animal
15 research (2010/63/EU). Protocols were approved by the Italian Minister for
16 Scientific Research and all efforts were made to minimize the number of animals
17 used and their suffering.
18 2.1.2 Breeding procedure and genotyping
19 Wild-type CD1 males and Mecp2+/- heterozygous CD1 females aged between 2
20 and 4 months were mated for four days. Dams gave birth and raised their litters
8
1 until weaning. Litters were not culled and no selection other than genotype was
2 made. Mouse genotypes were determined through PCR on genomic DNA
3 purified from tail biopsy. Biopsies were obtained within the third and fourth week
4 of life. Forward PCR primer sequences: 5’-CCATGCGATAGGCTTGATGA for the
5 identification of the null allele, 5’-GACCCCTTGGGACTGAAGTT for the wt allele.
6 Common reverse primer sequence: 5’-CCACCCTCCAGTTTGGTTTA . The
7 obtained PCR products were: a single band of 450 base pairs (bp) for the Mecp2-
8 null mice; a single band of 400 bp for the wt animals and the two bands for
9 heterozygous females.
10 2.2 Treatment
11 A total of 112 littermate male mice from 4 different cohorts were used in the study.
12 Based on the available litters at weaning, animals were randomly divided into 10
13 treatment groups: 22 for WT-vehicle, 10 for WT-CBDV 0.2 mg/kg, 13 for WT-
14 CBDV 2 mg/kg, 12 for WT-CBDV 20 mg/kg, 3 for WT-CBDV 200 mg/kg, 11 for
15 KO-vehicle, 6 for KO-CBDV 0.2 mg/kg, 13 for KO-CBDV 2 mg/kg, 14 for KO-
16 CBDV 20 mg/kg and 8 for KO-CBDV 200 mg/kg.
17 Purified CBDV (96.4%; CBD 3.6%) was provided by GW Research (Cambridge,
18 UK), stored at -20°C and freshly prepared daily by dissolution in ethanol, kolliphor
19 EL and saline (2:1:17). The treatments started at PND 28 (week 4) and lasted
20 until PND 67 (week 9). Mice received a daily intraperitoneal injection of CBDV (or
9
1 vehicle) at the doses of 0.2, 2, 20 or 200 mg/kg between 9:00 am and 11:00 am
2 based on previous pharmacokinetic (Deiana et al., 2012) and in vivo (Vigli et al.,
3 2018) studies.
4 2.3 Behavioral studies
5 Behavioral testing was carried out by two observers blind to the treatment
6 conditions. The timeline of the experiments is reported in supplementary figure
7 S1.
8 2.3.1 Score test
9 Neurological defects in Mecp2-null mice were evaluated using observational
10 scoring for hindlimb clasping, gait, breathing, tremor, mobility and general
11 condition. Starting from PND 28, Mecp2 WT and KO mice were scored every
12 other day to evaluate the effect of CBDV treatment on motor symptoms,
13 neurological signs and general condition. Score data were expressed as the
14 weekly average of four-day observations in each corresponding week. Each of
15 the six symptoms was scored from 0 to 2 (0 corresponds to the symptom being
16 absent or the same as in the WT animal; 1 when the symptom was present; 2
17 when the symptom was severe) as previously described (Guy et al., 2007;
18 Szczesna et al., 2014; see Supplementary Methods for a detailed description).
19 Whenever a mouse scored 2 out of 2 in the three criteria tremor, breathing,
10
1 general condition, or lost 20% of its body weight during the experiment, it was
2 killed by cervical dislocation.
3 2.3.2 Novel object recognition (NOR) test
4 The test was performed on PND 41, 56 and 66. The experimental apparatus used
5 for the NOR test was an open-field box (43x43x32 cm) made of Plexiglas, placed
6 in a dimly illuminated room. Animals performed each test individually as in
7 Zamberletti et al. (2015). Each animal was placed in the arena and allowed to
8 explore two identical previously unseen objects for 10 minutes (familiarization
9 phase). The test phase was performed after an inter-trial interval of 30 minutes.
10 During the test phase, the time spent exploring the familiar object (Ef) and the
11 new object (En) was recorded separately by two observers blind to the groups
12 and the discrimination index was calculated as follows: (En-Ef)/(En+Ef) x 100.
13 Objects were changed in subsequent testing sessions of the NOR test.
14 2.4 Biochemical studies
15 2.4.1 Western blot
16 Mice having all the same age (9 weeks old) were sacrificed by cervical dislocation
17 24 hours after the last CBDV (or vehicle) injection. The brains were quickly
18 removed, frozen in liquid nitrogen and stored at -80°C.
19 50 µg of total protein lysates from hemisected brains were run on a SDS-
20 polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride
11
1 (PVDF) membranes and blocked for 2 hours before incubation overnight at 4°C
2 with specific primary antibodies (listed in supplementary Table S1). Bound
3 antibodies were detected with horseradish peroxidase (HRP) conjugated
4 secondary anti-rabbit or anti-goat antibody (1:2000-1:5000; Chemicon
5 International, Temecula, CA). The blots were normalized against mouse anti-β-
6 actin monoclonal antibody (1:10000; Sigma Aldrich, Italy), rabbit polyclonal anti-
7 totalAKT (1:1000; Abcam, Cambridge, UK), rabbit polyclonal anti-totalS6 (1:1000;
8 Cell Signaling, Danvers, MA) or rabbit polyclonal anti-totalERK1/2 (1:1000; Cell
9 Signaling, Danvers, MA). Bound antibodies were visualized using Clarity Western
10 ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and bands were
11 detected with a GBOX XT camera (Syngene, Cambridge, UK). Optical density of
12 the bands was quantified using Image Pro Plus 7.0 software (MediaCybernetics,
13 Bethesda, MD, USA), normalized and expressed as arbitrary units.
14 2.4.2 Lipid extraction and endocannabinoid measurement
15 Hemisected brains were dounce-homogenized and extracted with acetone
16 containing internal deuterated standards for anandamide (AEA),
17 palmitoylethanolamide (PEA), oleoylethanolamide (OEA) and 2-
18 arachidonoylglycerol (2-AG) quantification by isotope dilution LC-MS, i.e. 5 pmol
19 d8-AEA, and 50 pmol d4-PEA, d4-OEA, and d5-2-AG, (Cayman Chemical),
20 respectively. The lipid-containing organic phases were then purified by open bed
12
1 chromatography on silica and fractions were obtained by eluting the column with
2 99:1, 90:10, and 50:50 (v/v) chloroform/methanol. Fractions eluted with
3 chloroform/methanol 90:10 were collected, the excess solvent evaporated with a
4 rotating evaporator, and aliquots analyzed by isotope dilution-LC/atmospheric
5 pressure chemical ionization/MS carried out in the selected ion monitoring mode
6 by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu
7 (LCMS-2020) quadrupole mass spectrometer via a Shimadzu atmospheric
8 pressure chemical ionization interface. MS detection was performed by using
9 values of m/z 356 and 348 (molecular ions + 1 for d8-AEA and AEA), m/z 304
10 and 300 (molecular ions + 1 for d4-PEA and PEA), m/z 330 and 326 (molecular
11 ions + 1 for d4-OEA and OEA) and m/z 384 and 379 (molecular ions +1 for d5-2-
12 AG and 2-AG). AEA, PEA, OEA, and 2-AG levels were therefore calculated on
13 the basis of their area ratios with the internal deuterated standard signal areas.
14 Lipid amounts expressed as pmol were then normalized per gram or milligram of
15 wet tissue.
16 2.4.3 Immunohistochemistry
17 Frozen brain sections were obtained using a Leica cryostat CM1510 set to 20μm
18 thickness and a −20°C chamber temperature. Sagittal sections were collected on
19 gelatin-coated slides (4 sections per slide, 200μm apart) and permeabilized with
20 100% methanol at -20°C for 30 minutes. After three washes in 0.05% Triton X-
13
1 100 in TBS, sections were blocked with 3% normal goat serum in TBS for 1 hour
2 at room temperature and then incubated overnight 4°C with rabbit anti-phospho-
3 rpS6 (Ser240/244) (1:50) from Cell Signaling Technology (Danvers, MA, USA)
4 diluted in blocking solution. After blocking peroxidase activity with 0.3% H2O2 in
5 TBS for 15 min, sections were washed in TBS and incubated for 4 hours at room
6 temperature with goat anti-rabbit horseradish peroxidase (HRP) conjugated
7 secondary antibody diluted 1:100 (Vector Labs, Burlingame, CA, USA). The
8 sections were then incubated in chromogen 3,3’-diaminobenzidine
9 tetrahydrochloride (DAB) for 5 min. The reaction was stopped in PBS, sections
10 were dehydrated and cover slipped.
11 Digital Images were captured using Retiga R1 CCD camera (QImaging, Surrey,
12 BC, Canada) attached to an Olympus BX51 (Tokyo, Japan) polarizing/light
13 microscope. Ocular imaging software (QImaging) was used to import images
14 from the camera. Images of layer V neurons in the medial prefrontal cortex were
15 acquired by first delineating the brain sections and the regions of interest (ROI)
16 at low magnification (×4 objective) and the ROI were further refined under a ×40
17 objective. Labeled cells were counted manually in every ten 20μm-thick sagittal
18 section (200μm apart) within the ROI using a 300 × 300 μm grid as reference.
19 Three mice per each experimental group (four sections/mouse) were analyzed
20 blinded.
14
1 2.4.4 Nuclear size measurements
2 For DAPI staining, slides were pre-incubated in 80% Methanol at 4°C for 5 min,
3 washed in TBS for 10 min and then incubated in a working solution 1 to 10 in
4 PBS from a 0.01% stock solution in the dark for 30 minutes at room temperature.
5 After several washes in PBS, sections were dehydrated and cover slipped.
6 Images of layer V neurons in the medial prefrontal cortex and of CA1 pyramidal
7 cell layer were acquired using a ×20 objective after having defined the outlines of
8 the regions of interest at low magnification (×4 objective). Outlines of nuclei were
9 manually traced at ×20 image magnification using ImageJ software (NIH,
10 Bethesda, MD, USA). Four slices of tissue from three animals per each
11 experimental group (30–75 cells/animal) were analyzed blind to the treatment
12 groups. Only in-focus neurons that were completely within the field of view and
13 whose signal was not occluded by overlying cells were measured.
14 2.5 Statistics
15 The Shapiro–Wilk test was first used to determine if the data were normally
16 distributed. Qualitative non-normally distributed score data were analyzed non-
17 parametrically using Pearson's Chi-square test with Yates correction. NOR data
18 and biochemical results were expressed as mean ± SEM and analyzed by two-
19 way repeated measures and main effects ANOVA, respectively, by the GLM
20 package of Statistica 7.0. Bonferroni’s post-hoc test was used to control for
15
1 multiple comparisons. Kaplan-Meier survival curves were generated and
2 compared using a Dunnet-type log-rank method. The level of statistical
3 significance was set at p<0.05. Calculations were carried out using Statistica 7.0
4 and R (R Core Team, 2013) Statistical Software.
5 3. Results
6 3.1 Behavioral data
7 3.1.1 Effect of chronic CBDV treatment on motor and neurological sign
8 progression in Mecp2-deficient male mice
9 Figure 1A shows the effect of chronic CBDV treatment on tremors, hind limb
10 clasping, breathing, gait, mobility and general condition in KO mice over time. No
11 symptoms were present in Mecp2 KO mice until 4 weeks of age (data not shown).
12 Either WT mice treated with vehicle or CBDV did not show any symptom (score
13 = 0) during the entire period of observation (data not shown).
14 Male Mecp2 KO mice exhibited tremors beginning from 5 weeks of age. CBDV
2 15 administration significantly reduced tremors in Mecp2 KO mice at 5 (χ [8]3.
2 16 =12.765, p=0.012) and 6 (χ [8] =16.422, p=0.036) weeks of age, but not later.
17 Doses of 2, 20 and 200 mg/kg showed similar efficacy (p<0.001) whereas the
18 lowest dose of CBDV tested, 0.2 mg/kg, was ineffective.
19 Mecp2 KO mice showed a significant increase in hind limb clasping starting from
20 6 weeks of age. CBDV treatment significantly attenuated hind limb clasping in KO
16
2 1 mice at 6 weeks of age (χ [8]=22.814, p=0.0036). This effect was reached at
2 doses of 2 (p>0.05), 20 (p<0.01) and 200 mg/kg (p<0.05). In contrast, CBDV was
3 ineffective at the lowest dose tested, 0.2 mg/kg.
4 Breathing and gait abnormalities were evident in Mecp2 KO mice starting from 7
2 2 5 weeks of age. Breathing (χ [8]=22.405, p=0.0042) and gait (χ [8]=16.275,
6 p=0.039) were improved by CBDV in Mecp2 KO mice at 7 weeks of age. CBDV
7 treatment attenuated breathing abnormalities at doses of 2 (p<0.01), 20 (p<0.05)
8 and 200 (p<0.05) mg/kg compared to KO-vehicle mice. Similarly, gait alterations
9 were significantly reduced by CBDV treatment at doses of 2 (p<0.01), 20 (p<0.01)
10 and 200 (p<0.001) mg/kg. Again, the lowest dose tested was completely
11 ineffective.
12 Impaired mobility and deterioration of general condition in KO mice was observed
13 starting from 7 weeks of age. CBDV administration at all doses tested did not
14 affect both parameters in KO mice during the whole treatment schedule.
15 To gain an overall impression of CBDV treatment in Mecp2 KO mice over time,
16 individual behavior scores were combined into a total symptom score for each
17 mouse (figure 1B). Total symptom score was significantly increased in KO mice
18 treated with vehicle starting from 5 weeks of age compared to WT mice and
19 continued to increase until the end of the observation period. Significant effects
2 2 20 were observed on total symptom score at 6 (χ [8]=19.617, p=0.0037) and 7 (χ
17
1 [8]=21.732, p=0.0041) weeks of age. Treatment with CBDV 2, 20 and 200 mg/kg
2 showed similar efficacy in attenuating motor and neurological deficits in KO mice.
3 Indeed, they significantly improved total symptom score in KO mice at 6
4 (p<0.001) and 7 (p<0.001) weeks of age, although CBDV’s beneficial effect was
5 lost at later stages of the disease progression (i.e. 8 and 9 weeks of age). In
6 contrast, CBDV 0.2 mg/kg did not affect the progression of symptoms over the
7 entire observation period.
8 3.1.2 Effect of chronic CBDV treatment on survival and body weight in Mecp2-
9 deficient male mice
10 Survival data refer to all mice that underwent behavioral assessment. Figure 2A
11 shows the percentage survival of Mecp2 KO and WT mice treated with CBDV or
12 vehicle. The results are plotted as Kaplan–Meier survival curves and expressed
13 as the percentage of surviving mice over total mice with respect to time for each
14 experimental group. No lethality was observed in WT mice treated with either
15 vehicle or CBDV. In contrast, survival in KO mice treated with vehicle was only
16 38.5% by 9 weeks of age. All doses of CBDV showed a trend to increase survival
17 rates in Mecp2 KO mice at the end of the observation period compared to vehicle-
18 treated littermates (KO-CBDV 0.2 mg/kg: 66.6%; KO-CBDV 2 mg/kg: 61.5%; KO-
19 CBDV 20 mg/kg: 71.4%; KO-CBDV 200 mg/kg: 87.5%), although this effect
18
1 reached statistical significance only at the highest dose tested, 200 mg/kg
2 (p=0.0433; log Rank test).
3 Regarding body weight, as expected, Mecp2 KO mice were significantly leaner
4 than WT littermates (figure 2B). Chronic CBDV administration did not recover
5 body weight in KO mice, although a post-hoc test did not reveal significant
6 differences between WT-vehicle and KO mice treated with CBDV 2/20/200
7 mg/kg.
8 3.1.3 Effect of chronic CBDV treatment on recognition memory deficits in Mecp2-
9 deficient male mice
10 Figure 3 shows the effect of chronic CBDV administration on short-term
11 recognition memory at PND 41, 56 and 66. No differences in exploratory behavior
12 and locomotor activity were observed in WT and KO mice during the training
13 session of the recognition memory test (Supplementary Figure S2). Exploration
14 times of the novel object and the familiar object in the test phase are reported in
15 Supplementary Figure S3. Repeated measures ANOVA with time as the
16 dependent variable revealed significant main effects of genotype (F[1-244]=111.5,
17 p<0.0001), treatment (F[4-244]=47.77, p<0.0001) and genotype x treatment
18 interaction (F[8-244]=4.152, p=0.0440) on recognition memory. A cognitive
19 impairment in recognition memory was consistently present in KO mice and was
20 maintained until adulthood. KO animals showed significant reductions of the
19
1 discrimination index at PND 41 (p<0.001), 56 (p<0.001) and 66 (p<0.001)
2 compared to WT mice. At each time point, administration of CBDV 2, 20 and 200
3 mg/kg significantly counteracted the cognitive impairment in recognition memory
4 present in KO mice (p<0.001). In contrast, chronic CBDV treatment at the lowest
5 dose tested, 0.2 mg/kg, did not rescue the cognitive impairment in KO mice.
6 3.2 Biochemical data
7 3.2.1 Effect of chronic CBDV treatment on BDNF, IGF-1 protein levels and
8 downstream PI3K/AKT/mTOR and ERK1/2 pathways in hemisected brains of
9 Mecp2-deficient male mice
10 CBDV at all doses tested did not affect the expression of all the considered
11 biochemical markers when given chronically to WT mice.
12 Statistical analysis showed significant effects of genotype (F[1,38]=5.677,
13 p=0.0220), treatment (F[4,38]=2.677, p=0.0455) and genotype x treatment
14 interaction (F[4,38]=9.997, p=0.0081) on BDNF expression. BDNF protein levels
15 were significantly reduced in Mecp2 KO mice treated with vehicle or CBDV 0.2
16 mg/kg with respect to WT littermates (p<0.01; p<0.05 respectively). In contrast,
17 2, 20 and 200 mg/kg CBDV significantly restored BDNF levels in KO mice, to a
18 similar degree (p<0.05; Figure 4A).
19 Two-way ANOVA analysis revealed significant effects of genotype (F[1,38]=4.773,
20 p=0.0032) and genotype x treatment interaction (F[4,38]=2.709, p=0.0444) on IGF-
20
1 1 levels. IGF-1 levels were significantly reduced in Mecp2 KO mice treated with
2 vehicle compared to WT animals (p<0.05). CBDV significantly increased IGF-1
3 levels in KO mice at doses of 2 (p<0.05), 20 (p<0.05) and 200 (p<0.01) mg/kg
4 (Figure 4A).
5 A significant genotype x treatment interaction (F[4,26]=6.574, p=0.0009) and a
6 trend for genotype effect (F[1,26]=3.967, p=0.0570) were found on pAKT levels
7 (figure 4B). pAKT was significantly reduced in Mecp2 KO mice treated with
8 vehicle compared to WT-vehicle animals (p<0.05). Chronic CBDV administration
9 dose dependently recovered pAKT levels in Mecp2 deficient mice, reaching
10 statistical significance at doses of 20 (p<0.01) and 200 (p<0.001) mg/kg.
11 Significant effects of genotype (F[1,29]=23.83, p<0.0001) and genotype x treatment
12 interaction (F[4,29]=2.702, p=0.0500) on rpS6 phosphorylation were also
13 observed. Mecp2 deletion significantly reduced rpS6 phosphorylation within the
14 adult brain (p<0.001). Chronic CBDV treatment significantly increased rpS6
15 phosphorylation levels in KO mice compared to vehicle-treated mutants at doses
16 of 2 (p<0.05), 20 (p<0.01) and 200 (p<0.05) mg/kg.
17 Immunohistochemical analysis revealed significant effects of genotype
18 (F[1,20]=12.07; p=0.0024), treatment (F[4,20]=3.042; p=0.0413) and genotype x
19 treatment interaction (F[4,20]=7.156; p=0.0010) on rpS6 phosphorylation in layer V
20 neurons of the medial prefrontal cortex (figure 4C). Indeed, rpS6 phosphorylation
21
1 was significantly decreased in Mecp2 KO mice compared to WT animals
2 (p<0.05); CBDV administration significantly enhanced rpS6 phosphorylation in
3 mutant mice at doses of 20 (p<0.05) and 200 (p<0.01) mg/kg without affecting its
4 levels in WT animals. A trend towards an enhancement of rpS6 phosphorylation
5 was also present in KO mice after administration of the 2 mg/kg dose (p=0.0717)
6 whereas the 0.2 mg/kg dose was completely ineffective.
7 Finally, neither genotype nor CBDV treatment affected ERK1/2 phosphorylation
8 (figure 4B).
9 3.2.2 CBDV effect on nuclear areas in Layer V neurons of the medial prefrontal
10 cortex and in the CA1 layer of the hippocampus
11 In the mPFC, the mean nuclear area of KO neurons was 35.5% smaller than that
12 of WT neurons (WT-vehicle = 69.98 ± 2.091 μm2, KO-vehicle = 45.10 ± 1.208
13 μm2; p<0.001, Figure 5A). CBDV treatment, at all doses tested, did not affect
14 nuclear size in KO neurons (CBDV 0.2 mg/kg = 40.48 ± 1.598 μm2, CBDV 2
15 mg/kg = 47.71 ± 1.779 μm2, CBDV 20 mg/kg = 48.44 ± 1.782 μm2, CBDV 200
16 mg/kg = 53.08 ± 2 μm2). In the CA1 layer of the hippocampus, the mean nuclear
17 area of KO neurons was 32.3% smaller than that of WT neurons (WT-vehicle =
18 63.5 ± 2.237 μm2, KO-vehicle = 43.0 ± 1 μm2; p<0.001, Figure 5B). A partial
19 rescue of hippocampal neurons nuclear size was observed in KO mice after
20 chronic CBDV administration at doses of 2, 20 and 200 mg/kg (CBDV 2 mg/kg =
22
1 53.46 ± 1.721 μm2, CBDV 20 mg/kg = 52.55 ± 1.982 μm2, CBDV 200 mg/kg =
2 51.66 ± 1.650 μm2), but not 0.2 mg/kg (CBDV 0.2 mg/kg = 47.26 ± 1.862 μm2).
3 3.2.3 Effect of chronic CBDV treatment on cannabinoid CB1 and CB2 receptors
4 in Mecp2-deficient male mice
5 Two-way ANOVA analysis revealed significant effects of genotype and genotype
6 x treatment interaction on CB1 (F[1,32]=19.89, p<0.0001; F[4,32]=3.429, p=0.0193)
7 and CB2 (F[1,32]=4.662, p=0.0384; F[4,32]=3.834, p=0.0118) receptors (figure 6).
8 CB1 levels were significantly increased in Mecp2 KO mice treated with vehicle
9 compared to WT-vehicle littermates (p<0.001). Chronic CBDV treatment
10 significantly reduced CB1 receptor expression in KO mice at doses of 2 (p<0.05),
11 20 (p<0.05) and 200 (p<0.05) mg/kg. CBDV at all doses tested did not affect CB1
12 receptor levels when chronically administered to WT animals. CB2 receptor
13 expression was significantly enhanced in Mecp2 KO mice treated with vehicle
14 compared to WT-vehicle controls (p<0.001). Chronic CBDV administration
15 significantly rescued CB2 receptor expression in KO mice at doses of 2
16 (p<0.001), 20 (p<0.001) and 200 (p<0.001) mg/kg, without affecting its levels in
17 WT littermates.
18 3.2.4 Effect of chronic CBDV treatment on endocannabinoid contents in Mecp2-
19 deficient male mice
23
1 No statistically significant differences in AEA, OEA PEA and 2-AG levels were
2 found between the two genotypes, although a trend for the increase of AEA
3 (p=0.0613) and OEA (p=0.0817) was noticed in KO mice (figure 7A). Statistical
4 analysis revealed significant effects of genotype x CBDV treatment interaction on
5 AEA (F[4,20]=5.859, p=0.045) and 2-AG (F[4,20]=5.375, p=0.041) levels as well as
6 non-significant trend on OEA (F[4,20]=8.787, p=0.067) content. CBDV
7 administration at doses of 2 (p<0.05), 20 (p<0.001) and 200 (p<0.01) mg/kg
8 significantly increased AEA levels in Mecp2 KO mice with respect to WT-vehicle
9 mice. A similar effect of CBDV was also observed on OEA levels. Indeed, CBDV
10 at 2 (p<0.01), 20 (p<0.001) and 200 (p<0.05) mg/kg significantly enhanced OEA
11 content. Chronic CBDV administration significantly reduced 2-AG levels in the
12 brain of KO mice at doses of 20 (p<0.05) and 200 (p<0.01) mg/kg compared to
13 vehicle-treated animals. CBDV treatment also showed a trend toward reduction
14 of 2-AG levels in WT mice. Finally, CBDV treatment did not affect PEA content in
15 the brain of both WT and KO animals.
16 3.2.5 Effect of chronic CBDV treatment on endocannabinoid synthetic and
17 degrading enzymes in Mecp2-deficient male mice
18 Two-way ANOVA analysis revealed significant effects on NAPE-PLD (genotype,
19 F[1,32]=5.724, p=0.0228; genotype x treatment interaction, F[4,32]=3.227,
20 p=0.0247), FAAH (genotype, F[1,32]=11.14, p=0.0022; treatment, F[4,32]=1.782,
24
1 p=0.1576) and DAGLα (treatment, F[4,32]=11.75, p<0.0001) levels in the brains of
2 KO mice (figure 7B). The expression of the main AEA synthetic enzyme NAPE-
3 PLD was significantly increased in male Mecp2 KO mice after chronic treatment
4 with CBDV at the highest dose tested (p<0.01). In contrast, CBDV treatment at
5 the lowest doses did not affect NAPE-PLD levels both in Mecp2 WT and KO mice.
6 Deletion of Mecp2 resulted in a significant reduction of FAAH levels (p<0.05) that
7 were not rescued by chronic CBDV administration. In addition, chronic treatment
8 with CBDV showed a trend towards a reduction in FAAH expression in WT mice
9 at doses of 0.2 and 20 mg/kg. CBDV treatment also affected the expression of
10 DAGLα, a key enzyme in the biosynthesis of the endocannabinoid 2-AG. In fact,
11 chronic CBDV administration significantly reduced DAGLα protein levels at all
12 doses tested both in WT (p<0.01) and in KO (p<0.001) mice. No effect of either
13 genotype or CBDV treatment was observed on the 2-AG degrading enzyme MAG
14 lipase.
15 3.2.6 Protein levels of BDNF, p-rpS6, endocannabinoid receptors and enzymes
16 in the brain of pre-symptomatic 4-week-old mice
17 In order to establish whether the expression of BDNF, p-rpS6 as well as
18 endocannabinoid receptors and synthetic/degrading enzymes was altered in an
19 apparently asymptomatic phase of the phenotype progression, we performed
20 Western blot analysis in brain lysates from 4-week-old WT and KO mice, i.e.
25
1 before starting CBDV treatment. As reported in supplementary figure S4, both
2 BDNF and rpS6 phosphorylation levels were significantly reduced in 4-week-old
3 Mecp2-null mice compared to WT animals by about 48.3% and 35.9%
4 respectively. In contrast, no alteration in the expression of CB1 and CB2
5 receptors was present at this stage of the phenotype progression. Similarly, no
6 changes were found in the main endocannabinoid synthetic and degrading
7 enzymes, although a trend towards a reduction in MAG lipase expression was
8 present (p=0.0805).
9 4. Discussion
10 Chronic administration of the non-psychotomimetic phytocannabinoid CBDV
11 completely recovers recognition memory deficits in Mecp2 KO animals and this
12 improvement persists at advanced stages of the phenotype progression.
13 Furthermore, CBDV prolongs survival and delays the appearance of neurological
14 and motor signs in Mecp2 KO mice in a time window between 6 and 7 weeks of
15 age. This last effect is in line with previous findings by Vigli et al. (2018) showing
16 that 14 days of treatment with similar doses of CBDV improves the general health
17 status, the social sphere and the motor skills in MeCP2-308 male mice. However,
18 our study provides additional information supporting that CBDV’s beneficial effect
19 on motor and neurological signs is only transient and is lost when a longer
20 treatment schedule (5 weeks long) is applied. Thus, the effect of CBDV might be
26
1 explained better as a delay in the onset of the neurological defects rather than a
2 reduction in their intensity at a specific age.
3 From a pharmacological point of view, CBDV does not show a linear dose-
4 response curve but it elicits an “all-or-none” response, as doses of 2, 20 and 200
5 mg/kg show similar efficacy in ameliorating the phenotype of Mecp2 mutant mice,
6 whereas the lowest dose tested, 0.2 mg/kg, is completely ineffective. Although a
7 steep dose-response effect might exist between 0.2 and 2 mg/kg, CBDV’s non-
8 linear dose response curve could be a consequence of drug tolerance following
9 repeated exposure or target desensitization. However, a final explanation must
10 await the identification of the specific target responsible for CBDV’s effects in this
11 model. In addition, we cannot rule out the possibility that CBD content in our
12 preparation, which is primarily CBDV (96.4%), but also contains low levels of CBD
13 (3.6%), might have affected CBDV pharmacokinetics. In fact, different dose-
14 response curves can be observed when cannabinoids are administered in
15 isolation or in combinations that contain also low levels of other
16 phytocannabinoids (Gallily et al., 2015; Zuardi et al., 2017).
17 Also, we cannot exclude that CBD could participate in the overall effects of drug
18 treatment reported in this study, as significant neuroprotective effects of this
19 phytocannabinoid were reported in conditions including depression, anxiety,
20 stroke and neurodegenerative diseases even at very low doses (Campos et al.,
27
1 2017; Crippa et al., 2018) and low doses of CBD improved cognition, motor
2 activity and BDNF levels in animal models (Avraham et al.,2011; Magen et al.,
3 2010).
4 Biochemical studies performed at an advanced stage of the phenotype shows
5 that CBDV treatment simultaneously elevates the levels of BDNF and IGF-1 in
6 the brain of clearly symptomatic Mecp2 KO mice at behaviorally efficacious
7 doses.
8 Expression of BDNF is decreased in mouse models of RTT (Chang et al., 2006;
9 Chen et al., 2003; Li and Pozzo-Miller, 2014; Martinowich et al., 2003) and
10 enhancement of BDNF levels in vivo relieves some symptoms of the mutant
11 phenotype (Chang et al., 2006; Deogracias et al., 2012; Johnson et al., 2012;
12 Kline et al., 2010; Kron et al., 2014; Ogier et al., 2007; Roux et al., 2012; Schmid
13 et al., 2012). Unfortunately, the therapeutic potential of BDNF is limited by its poor
14 efficiency at crossing the blood–brain barrier (Pardridge et al., 1994). Hence,
15 possible therapeutic strategies must rely on the identification of agents capable
16 of indirectly stimulating BDNF levels. Remarkably, our data support CBDV
17 potential to modulate BDNF levels in Mecp2-null mice.
18 CBDV treatment also increases IGF-1 levels in the brain of mutant mice. IGF-1
19 is another neurotrophic factor that holds great promise as therapeutic agent in
20 RTT. Like BDNF, IGF-1 is widely expressed in the brain during development
28
1 (Dyer et al., 2016). In mouse models, administration of both IGF-1 and its
2 truncated form (1-3)IGF-1 reverses many of the features of the RTT phenotype
3 (Castro et al., 2014; Chen and Russo-Neustadt, 2007; Della Sala et al., 2016;
4 Tropea et al., 2009) and early studies in RTT patients have demonstrated the
5 tolerability and safety of IGF-1 as a potential treatment (Khwaja et al., 2014; Pini
6 et al., 2016; Pini et al., 2014; Pini et al., 2012).
7 Although both BDNF and IGF-1 signal via PI3K/AKT and MAPK/ERK pathways
8 to affect neuronal maturation and survival (Tropea et al., 2006; Yoshii and
9 Constantine-Paton, 2007; Zheng and Quirion, 2004), there is direct evidence of
10 the involvement of the AKT pathway in RTT. Reduced phosphorylation of rpS6 in
11 Mecp2 deficient mice is specific to signaling via AKT and not ERK1/2 kinases
12 (Ricciardi et al., 2011). Worth noting, CBDV-mediated increases of BDNF and
13 IGF-1 are associated with the normalization of their common downstream
14 AKT/mTOR signaling pathway whereas no effects of either genotype or CBDV
15 treatment are observed on ERK1/2 expression. Interestingly, reduced
16 phosphorylation of rpS6 in Mecp2 deficient mice is most intense in the mPFC, in
17 line with recent studies supporting a contribution of mPFC hypofunction to the
18 development of RTT-like phenotypes (Howell et al., 2018; Sceniak et al., 2016).
19 In particular, excitatory hypoconnectivity in the mPFC has been linked to cognitive
20 impairments in Mecp2 mutant mice (Howell et al., 2018) possibly suggesting that
29
1 CBDV’s ability to normalize rpS6 phosphorylation in the mPFC could contribute
2 to its beneficial effect on memory impairments. Consistent with this hypothesis,
3 the normalization of PI3K/AKT/mTOR pathway after CBDV administration is
4 observed at a time point when CBDV is still recovering memory deficits but is no
5 longer effective towards motor and neurological signs (i.e. 9 weeks of age).
6 As reduction of nuclear volumes represents a consistent feature of Mecp2
7 deficiency at the cellular level (Gadalla et al., 2013; Giacometti et al., 2007), we
8 assessed whether CBDV treatment could affect this parameter. Analysis of
9 nuclear volumes indicates that CBDV did not rescue nuclear size in the layer V
10 of the mPFC and only a partial effect was observed in the CA1 area of the
11 hippocampus of Mecp2 mutant mice, suggesting CBDV’s inability to recover this
12 morphological alteration at least at the end of the treatment schedule.
13 A previous study found increased levels of GPR55 receptor in RTT mouse
14 hippocampus (Vigli et al., 2018), hence we here checked for possible changes
15 on other components of the endocannabinoid system. Interestingly, the
16 expression of brain CB1 and CB2 receptors is up-regulated in Mecp2 deficient
17 mice and chronic CBDV treatment at doses of 2, 20 and 200 mg/kg significantly
18 normalized the levels of both receptors at this advanced stage of the phenotype
19 progression. Given that CBDV binds to CB1 and CB2 receptors with very weak
20 affinity (Amada et al., 2013; Hill et al., 2012a; Hill et al., 2013; Iannotti et al., 2014;
30
1 Rosenthaler et al., 2014), we hypothesize that normalization of CB1 receptor
2 expression following CBDV treatment in Mecp2 KO mice might occur indirectly
3 as a consequence of its ability to modulate endocannabinoid signaling, namely
4 by increasing AEA levels and hence down-regulating cannabinoid receptor
5 expression. Indeed, chronic CBDV administration at doses of 2, 20 and 200
6 mg/kg increased AEA levels and elevates OEA content in Mecp2 mutant mice,
7 effects that are associated with reduced FAAH expression and, only for the
8 highest dose tested, i.e. 200 mg/kg, with enhancement of NAPE-PLD expression.
9 Furthermore, CBDV greatly reduced 2-AG levels both in WT and KO mice. This
10 effect might be exerted at least in part via reduction of DAGLα expression,
11 although the doses that were more efficacious at reducing 2-AG levels were also
12 the ones least efficacious at exerting this effect. It is possible that CBDV reduces
13 2-AG levels also via its previously reported ability to inhibit DAGLα activity in vitro
14 (Bisogno et al., 2003; De Petrocellis et al., 2011). Thus, in our experimental
15 conditions CBDV is associated with enhancement of AEA and OEA levels likely
16 through inhibition of FAAH and reduction of 2-AG, possibly in part through
17 inhibition of DAGLα.
18 Up-regulation of CB1 and CB2 receptors and down-regulation of FAAH levels
19 were also present in naïve 9-week-old Mecp2 KO mice (see supplementary figure
31
1 S5), indicating that these changes did not arise from chronic handling procedure
2 (Sciolino et al., 2010).
3 It is not possible from our data to draw any conclusion as to whether alterations
4 of the endocannabinoid system in Mecp2 mutant mice sustain the phenotype or
5 represent a compensatory mechanism. Similarly, further studies are needed in
6 order to assess whether CBDV effects on the components of the
7 endocannabinoid (AEA and 2-AG) and endocannabinoid-related (OEA) signaling
8 systems are a mere consequence or, instead, contribute to its therapeutic effect.
9 The observation that, unlike BDNF and p-rpS6, endocannabinoid receptors and
10 endocannabinoid enzymatic machinery are not altered in the brain of apparently
11 asymptomatic 4-week-old Mecp2 KO mice possibly suggests that the expression
12 of the different components of the endocannabinoid system could be dynamically
13 regulated during phenotype progression in Mecp2 KO mice. However, any
14 conclusion on the role of the endocannabinoid system in this animal model must
15 await the investigation of the effects of drugs specifically targeting the single
16 components of the system both at baseline and following CBDV treatment.
17 Two major limitations should be taken into account when interpreting results from
18 this study. First, Western blot experiments were carried out on hemibrains rather
19 than discrete brain regions and future studies must test the molecular alterations
20 in different brain structures to determine whether these changes are universal or
32
1 brain region-specific. Second, all the neurochemical characterizations have been
2 performed at the end of CBDV treatment, when the compound was still effective
3 in recovering cognitive deficits but no longer beneficial towards neurological and
4 motor signs. Thus, it cannot be excluded that different modifications could have
5 been observed as a consequence of CBDV administration if the same analysis
6 would have been carried out at earlier time points (i.e. at 7 weeks of age when
7 CBDV showed efficacy also towards neurological and motor signs). Accordingly,
8 when biochemical investigations were performed at an early symptomatic stage
9 of the disease they failed to reveal any defect on BDNF and IGF1 levels as well
10 as on the phosphorylation level of the rpS6 in the brain of MeCP2-308 mice
11 treated with either vehicle or CBDV (Vigli et al., 2018). Importantly, our data
12 obtained at this advanced stage of the disease highlight that CBDV treatment can
13 normalize BDNF/IGF1 levels as well as the defective PI3K/AKT/mTOR pathway
14 in Mecp2 mutant mice, possibly contributing to the restoration of cognitive deficits.
15 Despite these limitations, this paper demonstrate that chronic CBDV
16 administration exerts an enduring and complete rescue of recognition memory
17 deficits in Mecp2 mutant mice, an effect that is associated with the normalization
18 of BDNF, IGF-1 and rpS6 phosphorylation levels as well as CB1 and CB2
19 receptor expression. Regarding neurological and motor signs, our data support
20 previous findings indicating that CBDV can ameliorate neurological and motor
33
1 signs during an early symptomatic phase of the disease (Vigli et al., 2018) but
2 highlight that this effect is only transient and is lost at advanced stages of the
3 phenotype progression.
4 As a whole, available evidence encourages the investigation of the molecular
5 mechanism(s) by which CBDV can affect behavioral and molecular parameters
6 in this animal model as well as a more specific evaluation of CBDV’s efficacy in
7 female models of RTT-like symptoms.
8 Funding
9 This work was funded by GW Research Ltd. (Cambridge, UK). NL and CKN are
10 indebted to the Italian association of parents proRETT research for their
11 continuous economical support and faith in our work. EZ has a postdoctoral
12 fellowship from Zardi Gori Foundation (Milan, Italy).
13 Declaration of conflicting interests
14 JSB and MW are employees of GW Research Ltd. (Cambridge, UK). DP, FP, TR
15 and VD receive research grants from GW Research Ltd. All other authors declare
16 no conflicts of interest.
17 References
18 Amada N, Yamasaki Y, Williams CM, Whalley BJ (2013) Cannabidivarin
19 (CBDV) suppresses pentylenetetrazole (PTZ)-induced increases in epilepsy-
20 related gene expression. PeerJ 1: e214.
34
1 Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY
2 (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding
3 methyl-CpG-binding protein 2. Nat Genet 23(2): 185-188.
4 Avraham Y, Grigoriadis N, Poutahidis T, Vorobiev L, Magen I, Ilan Y,
5 Mechoulam R, Berry E (2011) Cannabidiol improves brain and liver function
6 in a fulminant hepatic failure-induced model of hepatic encephalopathy in
7 mice. Br J Pharmacol 162(7):1650-1658.
8 Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, Matias I,
9 Schiano-Moriello A, Paul P, Williams EJ, Gangadharan U, Hobbs C, Di
10 Marzo V, Doherty P (2003) Cloning of the first sn1-DAG lipases points to the
11 spatial and temporal regulation of endocannabinoid signaling in the brain. J
12 Cell Biol 163(3): 463-468.
13 Busquets-Garcia A, Gomis-González M, Guegan T, Agustín-Pavón C, Pastor
14 A, Mato S, Pérez-Samartín A, Matute C, de la Torre R, Dierssen M,
15 Maldonado R, Ozaita A (2013) Targeting the endocannabinoid system in the
16 treatment of fragile X syndrome. Nat Med 19(5):603-607.
17 Campolongo P, Trezza V (2012) The endocannabinoid system: a key
18 modulator of emotions and cognition. Front Behav Neurosci 6:73.
19 Campos AC, Fogaça MV, Scarante FF, Joca SRL, Sales AJ, Gomes FV,
20 Sonego AB, Rodrigues NS, Galve-Roperh I, Guimarães FS (2017) Plastic
35
1 and Neuroprotective Mechanisms Involved in the Therapeutic Effects of
2 Cannabidiol in Psychiatric Disorders. Front Pharmacol 8:269.
3 Campos AC, Fogaca MV, Sonego AB, Guimaraes FS (2016) Cannabidiol,
4 neuroprotection and neuropsychiatric disorders. Pharmacol Res 112: 119-
5 127.
6 Chahrour M, Zoghbi HY (2007) The story of Rett syndrome: from clinic to
7 neurobiology. Neuron 56(3): 422-437.
8 Chang Q, Khare G, Dani V, Nelson S, Jaenisch R (2006) The disease
9 progression of Mecp2 mutant mice is affected by the level of BDNF
10 expression. Neuron 49(3): 341-348.
11 Chen MJ, Russo-Neustadt AA (2007) Running exercise- and antidepressant-
12 induced increases in growth and survival-associated signaling molecules are
13 IGF-dependent. Growth Factors 25(2): 118-131.
14 Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R,
15 Greenberg ME (2003) Derepression of BDNF transcription involves calcium-
16 dependent phosphorylation of MeCP2. Science 302(5646): 885-889.
17 Cobolli Gigli C, Scaramuzza L, Gandaglia A, Bellini E, Gabaglio M, Parolaro
18 D, Kilstrup-Nielsen C, Landsberger N, Bedogni F (2016) MeCP2 Related
19 Studies Benefit from the Use of CD1 as Genetic Background. PLoS One
20 11(4): e0153473.
36
1 Crippa JA, Guimarães FS, Campos AC, Zuardi AW (2018) Translational
2 Investigation of the Therapeutic Potential of Cannabidiol (CBD): Toward a
3 New Age. Front Immunol 9:2009.
4 De Petrocellis L, Ligresti A, Moriello AS, Allara M, Bisogno T, Petrosino S,
5 Stott CG, Di Marzo V (2011) Effects of cannabinoids and cannabinoid-
6 enriched Cannabis extracts on TRP channels and endocannabinoid
7 metabolic enzymes. Br J Pharmacol 163(7): 1479-1494.
8 De Petrocellis L, Orlando P, Moriello AS, Aviello G, Stott C, Izzo AA, Di
9 Marzo V (2012) Cannabinoid actions at TRPV channels: effects on TRPV3
10 and TRPV4 and their potential relevance to gastrointestinal inflammation.
11 Acta Physiol (Oxf) 204(2):255-266.
12 Deiana S, Watanabe A, Yamasaki Y, Amada N, Arthur M, Fleming S,
13 Woodcock H, Dorward P, Pigliacampo B, Close S, Platt B, Riedel G (2012)
14 Plasma and brain pharmacokinetic profile of cannabidiol (CBD),
15 cannabidivarine (CBDV), Delta(9)-tetrahydrocannabivarin (THCV) and
16 cannabigerol (CBG) in rats and mice following oral and intraperitoneal
17 administration and CBD action on obsessive-compulsive behaviour.
18 Psychopharmacology (Berl) 219(3): 859-873.
19 Della Sala G, Putignano E, Chelini G, Melani R, Calcagno E, Michele Ratto
20 G, Amendola E, Gross CT, Giustetto M, Pizzorusso T (2016) Dendritic Spine
37
1 Instability in a Mouse Model of CDKL5 Disorder Is Rescued by Insulin-like
2 Growth Factor 1. Biol Psychiatry 80(4): 302-311.
3 Deogracias R, Yazdani M, Dekkers MP, Guy J, Ionescu MC, Vogt KE, Barde
4 YA (2012) Fingolimod, a sphingosine-1 phosphate receptor modulator,
5 increases BDNF levels and improves symptoms of a mouse model of Rett
6 syndrome. Proc Natl Acad Sci U S A 109(35): 14230-14235.
7 Dyer AH, Vahdatpour C, Sanfeliu A, Tropea D (2016) The role of Insulin-Like
8 Growth Factor 1 (IGF-1) in brain development, maturation and
9 neuroplasticity. Neuroscience 325: 89-99.
10 Fernández-Ruiz J, Gonzáles S (2005) Cannabinoid control of motor function
11 at the basal ganglia. Handb Exp Pharmacol (168):479-507.
12 Fernandez-Ruiz J, Sagredo O, Pazos MR, Garcia C, Pertwee R, Mechoulam
13 R, Martinez-Orgado J (2013) Cannabidiol for neurodegenerative disorders:
14 important new clinical applications for this phytocannabinoid? Br J Clin
15 Pharmacol 75(2): 323-333.
16 Gadalla KK, Bailey ME, Spike RC, Ross PD, Woodard KT, Kalburgi SN,
17 Bachaboina L, Deng JV, West AE, Samulski RJ, Gray SJ, Cobb SR (2013)
18 Improved survival and reduced phenotypic severity following AV9/MECP2
19 gene transfer to neonatal and juvenile male Mecp2 knockout mice. Mol Ther
20 21(1): 18-30.
38
1 Gallily R, Yekhtin Z, Hanuš LO (2015) Overcoming the Bell‐Shaped Dose‐
2 Response of Cannabidiol by Using Cannabis Extract Enriched in
3 Cannabidiol. Pharmacology & Pharmacy, 6, 75‐85.
4 Giacometti E, Luikenhuis S, Beard C, Jaenisch R (2007) Partial rescue of
5 MeCP2 deficiency by postnatal activation of MeCP2. Proc Natl Acad Sci USA
6 104(6): 1931-1936.
7 Gomis-González M, Busquets-Garcia A, Matute C, Maldonado R, Mato S,
8 Ozaita A (2016) Possible Therapeutic Doses of Cannabinoid Type 1
9 Receptor Antagonist Reverses Key Alterations in Fragile X Syndrome Mouse
10 Model. Genes (Basel). 7(9). pii: E56.
11 Guy J, Gan J, Selfridge J, Cobb S, Bird A (2007) Reversal of neurological
12 defects in a mouse model of Rett syndrome. Science 315(5815): 1143-1147.
13 Hagberg B (2002) Clinical manifestations and stages of Rett syndrome. Ment
14 Retard Dev Disabil Res Rev 8(2): 61-65.
15 Hill AJ, Mercier MS, Hill TD, Glyn SE, Jones NA, Yamasaki Y, Futamura T,
16 Duncan M, Stott CG, Stephens GJ, Williams CM, Whalley BJ (2012a)
17 Cannabidivarin is anticonvulsant in mouse and rat. Br J Pharmacol 167(8):
18 1629-1642.
39
1 Hill AJ, Williams CM, Whalley BJ, Stephens GJ (2012b) Phytocannabinoids
2 as novel therapeutic agents in CNS disorders. Pharmacol Ther 133(1): 79-
3 97.
4 Hill TD, Cascio MG, Romano B, Duncan M, Pertwee RG, Williams CM,
5 Whalley BJ, Hill AJ (2013) Cannabidivarin-rich cannabis extracts are
6 anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism.
7 Br J Pharmacol 170(3): 679-692.
8 Hosie S, Malone DT, Liu S, Glass M, Adlard PA, Hannan AJ, Hill-Yardin EL
9 (2018) Altered Amygdala Excitation and CB1 Receptor Modulation of
10 Aggressive Behavior in the Neuroligin-3R451C Mouse Model of Autism.
11 Front Cell Neurosci 12:234.
12 Howell CJ, Sceniak MP, Lang M, Krakowiecki W, Abouelsoud FE, Lad SU, Yu
13 H, Katz DM (2018) Activation of the Medial Prefrontal Cortex Reverses
14 Cognitive and Respiratory Symptoms in a Mouse Model of Rett Syndrome.
15 eNeuro 4(6).
16 Iannotti FA, Hill CL, Leo A, Alhusaini A, Soubrane C, Mazzarella E, Russo E,
17 Whalley BJ, Di Marzo V, Stephens GJ (2014) Nonpsychotropic plant
18 cannabinoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate and
19 desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro:
40
1 potential for the treatment of neuronal hyperexcitability. ACS Chem Neurosci
2 5(11): 1131-1141.
3 Iannotti FA, Pagano E, Moriello AS, Alvino FG, Sorrentino NC, D'Orsi L,
4 Gazzerro E, Capasso R, De Leonibus E, De Petrocellis L, Di Marzo V (2018)
5 Effects of non-euphoric plant cannabinoids on muscle quality and
6 performance of dystrophic mdx mice. Br J Pharmacol. 2018 Aug 3. doi:
7 10.1111/bph.14460. [Epub ahead of print]
8 Johnson RA, Lam M, Punzo AM, Li H, Lin BR, Ye K, Mitchell GS, Chang Q
9 (2012) 7,8-dihydroxyflavone exhibits therapeutic efficacy in a mouse model of
10 Rett syndrome. J Appl Physiol (1985) 112(5): 704-710.
11 Jung KM, Sepers M, Henstridge CM, Lassalle O, Neuhofer D, Martin H,
12 Ginger M, Frick A, DiPatrizio NV, Mackie K, Katona I, Piomelli D, Manzoni OJ
13 (2012) Uncoupling of the endocannabinoid signalling complex in a mouse
14 model of fragile X syndrome. Nat Commun 3:1080.
15 Karhson DS, Hardan AY, Parker KJ (2016) Endocannabinoid signaling in
16 social functioning: an RDoC perspective. Transl Psychiatry 6(9): e905.
17 Karhson DS, Krasinska KM, Dallaire JA, Libove RA, Phillips JM, Chien AS,
18 Garner JP, Hardan AY, Parker KJ (2018) Plasma anandamide
19 concentrations are lower in children with autism spectrum disorder. Mol
20 Autism 9:18.
41
1 Katona I (2015) Cannabis and Endocannabinoid Signaling in Epilepsy.
2 Handb Exp Pharmacol 231: 285-316.
3 Kerr DM, Gilmartin A, Roche M (2016) Pharmacological inhibition of fatty acid
4 amide hydrolase attenuates social behavioural deficits in male rats prenatally
5 exposed to valproic acid. Pharmacol Res 113(Pt A):228-235.
6 Khwaja, O. S., Ho, E., Barnes, K. V., O'Leary, H. M., Pereira, L. M.,
7 Finkelstein, Y., Nelson, C. A., 3rd, Vogel-Farley, V., DeGregorio, G., Holm, I.
8 A., Khatwa, U., Kapur, K., Alexander, M. E., Finnegan, D. M., Cantwell, N.
9 G., Walco, A. C., Rappaport, L., Gregas, M., Fichorova, R. N., Shannon, M.
10 W., Sur, M., Kaufmann, W. E., 2014. Safety, pharmacokinetics, and
11 preliminary assessment of efficacy of mecasermin (recombinant human IGF-
12 1) for the treatment of Rett syndrome. Proc Natl Acad Sci U S A 111, 4596-
13 4601.
14 Kron, M., Lang, M., Adams, I. T., Sceniak, M., Longo, F., Katz, D. M., 2014. A
15 BDNF loop-domain mimetic acutely reverses spontaneous apneas and
16 respiratory abnormalities during behavioral arousal in a mouse model of Rett
17 syndrome. Dis Model Mech 7, 1047-1055.
18 Li, W., Pozzo-Miller, L., 2014. BDNF deregulation in Rett syndrome.
19 Neuropharmacology 76 Pt C, 737-746.
42
1 Ligresti, A., De Petrocellis, L., Di Marzo, V., 2016. From Phytocannabinoids
2 to Cannabinoid Receptors and Endocannabinoids: Pleiotropic Physiological
3 and Pathological Roles Through Complex Pharmacology. Physiol Rev 96,
4 1593-1659.
5 Magen I, Avraham Y, Ackerman Z, Vorobiev L, Mechoulam R, Berry EM
6 (2010) Cannabidiol ameliorates cognitive and motor impairments in bile-duct
7 ligated mice via 5-HT1A receptor activation. Br J Pharmacol 159(4):950-957.
8 Martinowich, K., Hattori, D., Wu, H., Fouse, S., He, F., Hu, Y., Fan, G., Sun,
9 Y. E., 2003. DNA methylation-related chromatin remodeling in activity-
10 dependent BDNF gene regulation. Science 302, 890-893.
11 Nagarkatti, P., Pandey, R., Rieder, S. A., Hegde, V. L., Nagarkatti, M., 2009.
12 Cannabinoids as novel anti-inflammatory drugs. Future Med Chem 1, 1333-
13 1349.
14 Neul, J. L., Lane, J. B., Lee, H. S., Geerts, S., Barrish, J. O., Annese, F.,
15 Baggett, L. M., Barnes, K., Skinner, S. A., Motil, K. J., Glaze, D. G.,
16 Kaufmann, W. E., Percy, A. K., 2014. Developmental delay in Rett syndrome:
17 data from the natural history study. J Neurodev Disord 6, 20.
18 Ogier, M., Wang, H., Hong, E., Wang, Q., Greenberg, M. E., Katz, D. M.,
19 2007. Brain-derived neurotrophic factor expression and respiratory function
43
1 improve after ampakine treatment in a mouse model of Rett syndrome. J
2 Neurosci 27, 10912-10917.
3 Pardridge, W. M., Kang, Y. S., Buciak, J. L., 1994. Transport of human
4 recombinant brain-derived neurotrophic factor (BDNF) through the rat blood-
5 brain barrier in vivo using vector-mediated peptide drug delivery. Pharm Res
6 11, 738-746.
7 Pini, G., Congiu, L., Benincasa, A., DiMarco, P., Bigoni, S., Dyer, A. H.,
8 Mortimer, N., Della-Chiesa, A., O'Leary, S., McNamara, R., Mitchell, K. J.,
9 Gill, M., Tropea, D., 2016. Illness Severity, Social and Cognitive Ability, and
10 EEG Analysis of Ten Patients with Rett Syndrome Treated with Mecasermin
11 (Recombinant Human IGF-1). Autism Res Treat 2016, 5073078.
12 Pini, G., Scusa, M. F., Benincasa, A., Bottiglioni, I., Congiu, L., Vadhatpour,
13 C., Romanelli, A. M., Gemo, I., Puccetti, C., McNamara, R., O'Leary, S.,
14 Corvin, A., Gill, M., Tropea, D., 2014. Repeated insulin-like growth factor 1
15 treatment in a patient with rett syndrome: a single case study. Front Pediatr
16 2, 52.
17 Pini, G., Scusa, M. F., Congiu, L., Benincasa, A., Morescalchi, P., Bottiglioni,
18 I., Di Marco, P., Borelli, P., Bonuccelli, U., Della-Chiesa, A., Prina-Mello, A.,
19 Tropea, D., 2012. IGF1 as a Potential Treatment for Rett Syndrome: Safety
20 Assessment in Six Rett Patients. Autism Res Treat 2012, 679801.
44
1 R Core Team, 2013. R: A language and environment for statistical
2 computing. R foundation for Statistical Computing, Vienna, Austria.
3 Ricceri, L., De Filippis, B., Laviola, G., 2008. Mouse models of Rett
4 syndrome: from behavioural phenotyping to preclinical evaluation of new
5 therapeutic approaches. Behav Pharmacol 19, 501-517.
6 Ricciardi, S., Boggio, E. M., Grosso, S., Lonetti, G., Forlani, G., Stefanelli, G.,
7 Calcagno, E., Morello, N., Landsberger, N., Biffo, S., Pizzorusso, T.,
8 Giustetto, M., Broccoli, V., 2011. Reduced AKT/mTOR signaling and protein
9 synthesis dysregulation in a Rett syndrome animal model. Hum Mol Genet
10 20, 1182-1196.
11 Rosenthaler, S., Pohn, B., Kolmanz, C., Huu, C. N., Krewenka, C., Huber, A.,
12 Kranner, B., Rausch, W. D., Moldzio, R., 2014. Differences in receptor
13 binding affinity of several phytocannabinoids do not explain their effects on
14 neural cell cultures. Neurotoxicol Teratol 46, 49-56.
15 Roux, J. C., Zala, D., Panayotis, N., Borges-Correia, A., Saudou, F., Villard,
16 L., 2012. Modification of Mecp2 dosage alters axonal transport through the
17 Huntingtin/Hap1 pathway. Neurobiol Dis 45, 786-795.
18 Sceniak, M. P., Lang, M., Enomoto, A. C., James Howell, C., Hermes, D. J.,
19 Katz, D. M., 2016. Mechanisms of Functional Hypoconnectivity in the Medial
20 Prefrontal Cortex of Mecp2 Null Mice. Cereb Cortex 26, 1938-1956.
45
1 Schmid, D. A., Yang, T., Ogier, M., Adams, I., Mirakhur, Y., Wang, Q.,
2 Massa, S. M., Longo, F. M., Katz, D. M., 2012. A TrkB small molecule partial
3 agonist rescues TrkB phosphorylation deficits and improves respiratory
4 function in a mouse model of Rett syndrome. J Neurosci 32, 1803-1810.
5 Sciolino, N. R., Bortolato, M., Eisenstein, S. A., Fu, J., Oveisi, F., Hohmann,
6 A. G., Piomelli, D., 2010. Social isolation and chronic handling alter
7 endocannabinoid signaling and behavioral reactivity to context in adult rats.
8 Neuroscience 168, 371-386.
9 Servadio M, Melancia F, Manduca A, di Masi A, Schiavi S, Cartocci V,
10 Pallottini V, Campolongo P, Ascenzi P, Trezza V (2016) Targeting
11 anandamide metabolism rescues core and associated autistic-like symptoms
12 in rats prenatally exposed to valproic acid. Transl Psychiatry 6:e902.
13 Szczesna, K., de la Caridad, O., Petazzi, P., Soler, M., Roa, L., Saez, M. A.,
14 Fourcade, S., Pujol, A., Artuch-Iriberri, R., Molero-Luis, M., Vidal, A., Huertas,
15 D., Esteller, M., 2014. Improvement of the Rett syndrome phenotype in a
16 MeCP2 mouse model upon treatment with levodopa and a dopa-
17 decarboxylase inhibitor. Neuropsychopharmacology 39, 2846-2856.
18 Tropea, D., Giacometti, E., Wilson, N. R., Beard, C., McCurry, C., Fu, D. D.,
19 Flannery, R., Jaenisch, R., Sur, M., 2009. Partial reversal of Rett Syndrome-
46
1 like symptoms in MeCP2 mutant mice. Proc Natl Acad Sci U S A 106, 2029-
2 2034.
3 Tropea, D., Kreiman, G., Lyckman, A., Mukherjee, S., Yu, H., Horng, S., Sur,
4 M., 2006. Gene expression changes and molecular pathways mediating
5 activity-dependent plasticity in visual cortex. Nat Neurosci 9, 660-668.
6 Vigli, D., Cosentino, L., Raggi, C., Laviola, G., Woolley-Roberts, M., De
7 Filippis, B., 2018. Chronic treatment with the phytocannabinoid
8 Cannabidivarin (CBDV) rescues behavioural alterations and brain atrophy in
9 a mouse model of Rett syndrome. Neuropharmacology 140, 121-129.
10 Volkow ND, Baler RD, Compton WM, Weiss SR (2014) Adverse health
11 effects of marijuana use. N Engl J Med. 370(23):2219-2227.
12 Wei D, Allsop S, Tye K, Piomelli D (2017) Endocannabinoid signaling in the
13 control of social behavior. Trends Neurosci 40(7): 385-396.
14 Wei D, Dinh D, Lee D, Li D, Anguren A, Moreno-Sanz G, Gall CM, Piomelli D
15 (2016) Enhancement of Anandamide-Mediated Endocannabinoid Signaling
16 Corrects Autism-Related Social Impairment. Cannabis Cannabinoid Res 1(1):
17 81-89.
18 Yoshii, A., Constantine-Paton, M., 2007. BDNF induces transport of PSD-95
19 to dendrites through PI3K-AKT signaling after NMDA receptor activation. Nat
20 Neurosci 10, 702-711.
47
1 Zamberletti E, Gabaglio M, Parolaro D (2017) The Endocannabinoid System
2 and Autism Spectrum Disorders: Insights from Animal Models. Int J Mol Sci
3 18(9). pii: E1916.
4 Zamberletti, E., Gabaglio, M., Prini, P., Rubino, T., Parolaro, D., 2015.
5 Cortical neuroinflammation contributes to long-term cognitive dysfunctions
6 following adolescent delta-9-tetrahydrocannabinol treatment in female rats.
7 Eur Neuropsychopharmacol 25, 2404-2415.
8 Zhang, J., Hu, M., Teng, Z., Tang, Y., Chen, C., 2014. Synaptic and
9 Cognitive Improvements by Inhibition of 2-AG Metabolism Are through
10 Upregulation of MicroRNA-188-3p in a Mouse Model of Alzheimer's Disease.
11 J Neurosci 34, 14919-14933.
12 Zheng, W. H., Quirion, R., 2004. Comparative signaling pathways of insulin-
13 like growth factor-1 and brain-derived neurotrophic factor in hippocampal
14 neurons and the role of the PI3 kinase pathway in cell survival. J Neurochem
15 89, 844-852.
16 Zuardi AW, Rodrigues NP, Silva AL, Bernardo SA, Hallak JEC, Guimarães
17 FS, Crippa JAS (2017) Inverted U-Shaped Dose-Response Curve of the
18 Anxiolytic Effect of Cannabidiol during Public Speaking in Real Life. Front
19 Pharmacol 8:259.
20
48
1 Figure legends
2 Figure 1: Effect of chronic CBDV (0.2, 2, 20, 200 mg/kg/day, weeks 5-9)
3 treatment on the Mecp2 KO phenotype. (A) “Score Test” for tremor, hindlimb
4 clasping, breathing, gait, mobility and general condition in Mecp2 KO mice.
5 Animals were scored (from 0 to 2) every other day and score data represent the
6 weekly average of four-day observations in each corresponding week for each
7 symptom considered. (B) Total symptom scores in vehicle- and CBDV-treated
8 Mecp2 KO mice. Data represent mean ± SEM of 22 WT-vehicle, 10 WT-CBDV
9 0.2 mg/kg, 13 WT-CBDV 2 mg/kg, 12 WT-CBDV 20 mg/kg, 3 WT-CBDV 200
10 mg/kg, 11 KO-vehicle, 6 KO-CBDV 0.2 mg/kg, 13 KO-CBDV 2 mg/kg, 14 KO-
11 CBDV 20 mg/kg, 8 KO-CBDV 200 mg/kg and were analyzed using Pearson’s Chi
12 square test with Yates correction. *p<0.05, **p<0.01, ***p<0.001 vs KO-vehicle
13 mice.
14 Figure 2: (A) Effect of chronic CBDV (0.2, 2, 20, 200 mg/kg/day, weeks 5-9)
15 treatment on survival in Mecp2 KO mice. Kaplan-Meier survival curves of CBDV-
16 treated Mecp2 KO mice compared to KO-vehicle littermates (p=0.0183, Kaplan–
17 Meier log-rank test). The table summarizes the n number of KO mice in each
18 considered week. (B) Area under the curve (AUC) for body weight recorded daily
19 during the whole treatment schedule. Data are expressed as mean ± SEM 22
20 WT-vehicle, 10 WT-CBDV 0.2 mg/kg, 13 WT-CBDV 2 mg/kg, 12 WT-CBDV 20
49
1 mg/kg, 3 WT-CBDV 200 mg/kg, 11 KO-vehicle, 6 KO-CBDV 0.2 mg/kg, 13 KO-
2 CBDV 2 mg/kg, 14 KO-CBDV 20 mg/kg, 8 KO-CBDV 200 mg/kg and were
3 analyzed using a two-way ANOVA followed by Tukey’s multiple comparison test.
4 *p<0.05 vs WT-vehicle mice.
5 Figure 3: Effect of chronic CBDV (0.2, 2, 20, 200 mg/kg/day, weeks 5-9)
6 treatment on recognition memory in WT and KO mice as measured through the
7 NOR test at PND 41, 56 and 66. Data are expressed as mean ± S.E.M. of 22 WT-
8 vehicle, 10 WT-CBDV 0.2 mg/kg, 13 WT-CBDV 2 mg/kg, 12 WT-CBDV 20 mg/kg,
9 3 WT-CBDV 200 mg/kg, 11 KO-vehicle, 6 KO-CBDV 0.2 mg/kg, 13 KO-CBDV 2
10 mg/kg, 14 KO-CBDV 20 mg/kg, 8 KO-CBDV 200 mg/kg and were analyzed by
11 two-way repeated measures ANOVA (genotype and treatment as between
12 factors and time as within factor) followed by Tukey’s multiple comparison test.
13 *p<0.05, **p<0.01, ***p<0.001 vs WT-vehicle; °°°p<0.001 vs KO-vehicle.
14 Figure 4: Effect of chronic CBDV treatment on protein levels of (A) BDNF and
15 IGF-1, and (B) p-Akt (Ser473), its downstream effector p-rpS6 (Ser240/244), and
16 ERK1/2 phosphorylation in the brains of Mecp2 WT and KO mice. Measurements
17 were carried out on total protein lysates from hemisected brains. Representative
18 blot images correspond to the results of one experiment out of four. Data are
19 expressed as mean ± SEM of 3-5 animals per group and were analyzed by two-
20 way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, **p<0.01,
50
1 ***p<0.001 vs WT-vehicle; °p<0.05, °°p<0.01; °°°p<0.001 vs KO-vehicle. (C) n.
2 of p-rpS6(Ser240/244)-positive neurons in layer V of the mPFC as quantified
3 through immunohistochemistry. Data represent mean ± SEM of three animals per
4 each experimental group (four slices /animal) and were analyzed by two-way
5 ANOVA followed by Tukey’s multiple comparison test. *p<0.05 vs WT-vehicle;
6 °p<0.05, °°p<0.01 vs KO-vehicle. (D) Representative high magnification (40x)
7 views of p-rpS6 (Ser240/244) immunolabeling in layer V neurons of the mPFC in
8 9-week-old Mecp2 WT and KO animals either treated with vehicle of CBDV
9 (Scale bar = 50μm).
10 Figure 5: Nuclear volume measurements of DAPI-labeled nuclei in the layer V of
11 the mPFC (A) and (B) CA1 region of the hippocampus in Mecp2 KO and WT
12 mice after chronic CBDV administration. Bar plots show mean nuclear volumes
13 ± SEM of three animals per each experimental group (four slices/30-75
14 cells/animal). Statistical analysis was carried out by two-way ANOVA and Tukey’s
15 multiple comparison test. *p<0.05, **p<0.01, ***p<0.001 vs WT-vehicle; °p<0.05
16 vs KO-vehicle.
17 Figure 6: Effect of chronic CBDV treatment on (A) protein levels of CB1 and CB2
18 cannabinoid receptors in hemisected brains of Mecp2 WT and KO mice. (B)
19 Representative blot images correspond to the results of one experiment out of
20 four. Data are expressed as mean ± SEM of 3-6 animals per group and were
51
1 analyzed by two-way ANOVA followed by Tukey’s multiple comparison test.
2 *p<0.05, **p<0.01, ***p<0.001 vs WT-vehicle; °p<0.05, °°p<0.01; °°°p<0.001 vs
3 KO-vehicle.
4 Figure 7: Effect of chronic CBDV treatment on (A) AEA, 2-AG, PEA and OEA
5 contents and (B) protein levels of the main endocannabinoid synthetic and
6 degrading enzymes NAPE-PLD, DAGLα, FAAH and MAGL in hemisected brains
7 of Mecp2 WT and KO mice. Representative blot images correspond to the results
8 of one experiment out of four. Data are expressed as mean ± SEM of 3-6 animals
9 per group and analyzed by two-way ANOVA followed by Tukey’s multiple
10 comparison test. *p<0.05, **p<0.01, ***p<0.001 vs WT-vehicle; °p<0.05,
11 °°p<0.01; °°°p<0.001 vs KO-vehicle.
52
Page 59 of 70 Journal of Psychopharmacology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 1: Effect of chronic CBDV (0.2, 2, 20, 200 mg/kg/day, weeks 5-9) treatment on the Mecp2 KO 46 phenotype. (A) “Score Test” for tremor, hindlimb clasping, breathing, gait, mobility and general condition in 47 Mecp2 KO mice. Animals were scored (from 0 to 2) every other day and score data represent the weekly average of four-day observations in each corresponding week for each symptom considered. (B) Total 48 symptom scores in vehicle- and CBDV-treated Mecp2 KO mice. Data represent mean ± SEM of 22 WT- 49 vehicle, 10 WT-CBDV 0.2 mg/kg, 13 WT-CBDV 2 mg/kg, 12 WT-CBDV 20 mg/kg, 3 WT-CBDV 200 mg/kg, 50 11 KO-vehicle, 6 KO-CBDV 0.2 mg/kg, 13 KO-CBDV 2 mg/kg, 14 KO-CBDV 20 mg/kg, 8 KO-CBDV 200 51 mg/kg and were analyzed using Pearson’s Chi square test with Yates correction. *p<0.05, **p<0.01, 52 ***p<0.001 vs KO-vehicle mice. 53 812x1058mm (72 x 72 DPI) 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Journal of Psychopharmacology Page 60 of 70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 2: (A) Effect of chronic CBDV (0.2, 2, 20, 200 mg/kg/day, weeks 5-9) treatment on survival in Mecp2 46 KO mice. Kaplan-Meier survival curves of CBDV-treated Mecp2 KO mice compared to KO-vehicle littermates 47 (p=0.0183, Kaplan–Meier log-rank test). The table summarizes the n number of KO mice in each considered week. (B) Area under the curve (AUC) for body weight recorded daily during the whole treatment schedule. 48 Data are expressed as mean ± SEM 22 WT-vehicle, 10 WT-CBDV 0.2 mg/kg, 13 WT-CBDV 2 mg/kg, 12 WT- 49 CBDV 20 mg/kg, 3 WT-CBDV 200 mg/kg, 11 KO-vehicle, 6 KO-CBDV 0.2 mg/kg, 13 KO-CBDV 2 mg/kg, 14 50 KO-CBDV 20 mg/kg, 8 KO-CBDV 200 mg/kg and were analyzed using a two-way ANOVA followed by Tukey’s 51 multiple comparison test. *p<0.05 vs WT-vehicle mice. 52 53 812x1058mm (72 x 72 DPI) 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Page 61 of 70 Journal of Psychopharmacology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 Figure 3: Effect of chronic CBDV (0.2, 2, 20, 200 mg/kg/day, weeks 5-9) treatment on recognition memory 24 in WT and KO mice as measured through the NOR test at PND 41, 56 and 66. Data are expressed as mean 25 ± S.E.M. of 22 WT-vehicle, 10 WT-CBDV 0.2 mg/kg, 13 WT-CBDV 2 mg/kg, 12 WT-CBDV 20 mg/kg, 3 WT- 26 CBDV 200 mg/kg, 11 KO-vehicle, 6 KO-CBDV 0.2 mg/kg, 13 KO-CBDV 2 mg/kg, 14 KO-CBDV 20 mg/kg, 8 27 KO-CBDV 200 mg/kg and were analyzed by two-way repeated measures ANOVA (genotype and treatment as between factors and time as within factor) followed by Tukey’s multiple comparison test. *p<0.05, 28 **p<0.01, ***p<0.001 vs WT-vehicle; °°°p<0.001 vs KO-vehicle. 29 30 829x418mm (72 x 72 DPI) 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Journal of Psychopharmacology Page 62 of 70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 4: Effect of chronic CBDV treatment on protein levels of (A) BDNF and IGF-1, and (B) p-Akt (Ser473), 46 its downstream effector p-rpS6 (Ser240/244), and ERK1/2 phosphorylation in the brains of Mecp2 WT and 47 KO mice. Measurements were carried out on total protein lysates from hemisected brains. Representative blot images correspond to the results of one experiment out of four. Data are expressed as mean ± SEM of 48 3-5 animals per group and were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. 49 *p<0.05, **p<0.01, ***p<0.001 vs WT-vehicle; °p<0.05, °°p<0.01; °°°p<0.001 vs KO-vehicle. (C) n. of 50 p-rpS6(Ser240/244)-positive neurons in layer V of the mPFC as quantified through immunohistochemistry. 51 Data represent mean ± SEM of three animals per each experimental group (four slices /animal) and were 52 analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05 vs WT-vehicle; 53 °p<0.05, °°p<0.01 vs KO-vehicle. (D) Representative high magnification (40x) views of p-rpS6 (Ser240/244) immunolabeling in layer V neurons of the mPFC in 9-week-old Mecp2 WT and KO animals 54 either treated with vehicle of CBDV (Scale bar = 50μm). 55 56 812x1058mm (72 x 72 DPI) 57 58 59 60 http://mc.manuscriptcentral.com/jop Page 63 of 70 Journal of Psychopharmacology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Journal of Psychopharmacology Page 64 of 70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 5: Nuclear volume measurements of DAPI-labeled nuclei in the layer V of the mPFC (A) and (B) CA1 46 region of the hippocampus in Mecp2 KO and WT mice after chronic CBDV administration. Bar plots show 47 mean nuclear volumes ± SEM of three animals per each experimental group (four slices/30-75 cells/animal). Statistical analysis was carried out by two-way ANOVA and Tukey’s multiple comparison test. *p<0.05, 48 **p<0.01, ***p<0.001 vs WT-vehicle; °p<0.05 vs KO-vehicle. 49 50 194x254mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Page 65 of 70 Journal of Psychopharmacology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 Figure 6: Effect of chronic CBDV treatment on (A) protein levels of CB1 and CB2 cannabinoid receptors in 30 hemisected brains of Mecp2 WT and KO mice. (B) Representative blot images correspond to the results of 31 one experiment out of four. Data are expressed as mean ± SEM of 3-6 animals per group and were analyzed 32 by two-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001 vs WT- vehicle; °p<0.05, °°p<0.01; °°°p<0.001 vs KO-vehicle. 33 34 91x63mm (300 x 300 DPI) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Journal of Psychopharmacology Page 66 of 70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 Figure 7: Effect of chronic CBDV treatment on (A) AEA, 2-AG, PEA and OEA contents and (B) protein levels 32 of the main endocannabinoid synthetic and degrading enzymes NAPE-PLD, DAGLα, FAAH and MAGL in 33 hemisected brains of Mecp2 WT and KO mice. Representative blot images correspond to the results of one 34 experiment out of four. Data are expressed as mean ± SEM of 3-6 animals per group and analyzed by two- 35 way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001 vs WT-vehicle; 36 °p<0.05, °°p<0.01; °°°p<0.001 vs KO-vehicle. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jop Page 67 of 70 Journal of Psychopharmacology
1 2 3 Supplementary Methods 4 5 Score test 6 7 Mecp2 WT and KO mice were scored every other day to evaluate the effect of CBDV 8 treatment on Hindlimb clasping, gait, breathing, tremor, mobility and general condition. Each 9 of the six symptoms was scored from 0 to 2 (0 corresponds to the symptom being absent or 10 11 the same as in the WT animal; 1 when the symptom was present; 2 when the symptom was 12 severe). Specifically: For tremor: the mouse was observed while standing on the flat palm 13 of the hand. 0 = no tremor; 1 = intermittent mild tremor; 2 = continuous tremor or intermittent 14 violent tremor. For hind limb clasping: 0 = WT; hind limbs splay outward when suspended 15 16 by the tail; 1 = one hind limb is pulled into the body or forelimbs are stiff and splayed outward 17 without motion; and 2 = one hind limb is pulled into the body and forelimbs are stiff and 18 splayed outward without motion and might form a widened bowl shape or both hind limbs 19 are pulled into the body with or without abnormal forelimb posture. For breathing: movement 20 21 of flanks were observed whileFor the animalPeer was Reviewstanding still. 0 = normal breathing; 1 = periods 22 of regular breathing interspersed with short periods of more rapid breathing or with pauses 23 in breathing; 2 = very irregular breathing-gasping or panting. For mobility: the mouse was 24 observed when placed on bench, then when handled gently and scored as follows: 0 = as 25 26 WT; 1 = reduced movement when compared with WT: extended freezing period when first 27 placed on bench and longer periods spent immobile; 2 = no spontaneous movement when 28 placed on the bench; mouse can move in response to a gentle prod or a food pellet placed 29 nearby. For general condition: the mouse was observed for indicators of general well-being 30 31 such as coat condition, eyes and body stance. 0 = clean shiny coat, clear eyes, and normal 32 stance; 1 = eyes dull, coat dull/ungroomed, and somewhat hunched stance; 2 = eyes crusted 33 or narrowed, piloerection, and hunched posture. For gait: 0 = as WT; 1 = hind limbs spread 34 wider than WT when ambulating and/or a lowered pelvis when ambulating; and 2 = lack of 35 36 full strides by hind limbs resulting in a dragging of hindquarters. 37 38 39 Table S1 40 41 DILUITION SUPPLIER 42 Rabbit polyclonal anti-BDNF 1:1000 Millipore, Italy 43 Goat polyclonal anti-IGF-1 1:1000 Millipore, Italy 44 Rabbit polyclonal anti-pAKT Ser473 1:1000 Cell Signaling, Danvers, MA 45 Rabbit polyclonal anti-phospho-rpS6 1:1000 Cell Signaling, Danvers, MA 46 Rabbit polyclonal anti-pERK1/2 1:1000 Cell Signaling, Danvers, MA 47 Cayman Chemical, Ann Arbor, Rabbit polyclonal anti-cannabinoid CB1 receptor (CB1) 1:1000 48 MI 49 Cayman Chemical, Ann Arbor, Rabbit polyclonal anti-cannabinoid CB2 receptor (CB2) 1:1000 50 MI 51 Rabbit polyclonal anti-N-acyl-phosphatidylethanolamine- Cayman Chemical, Ann Arbor, 1:3000 52 hydrolysing phospholipase D (NAPE-PLD) MI 53 Cayman Chemical, Ann Arbor, Rabbit polyclonal anti-fatty acid amide hydrolase (FAAH) 1:2000 54 MI 55 Goat polyclonal anti-diacylglycerol Lipase α (DAGLα) 1:1000 Abcam, Cambridge, UK 56 Cayman Chemical, Ann Arbor, Rabbit polyclonal anti-monoacylglycerol lipase (MAGL) 1:1000 57 MI 58 59 60 Table S1: List of primary antibodies used for Western blot analysis
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1 2 3 Supplementary figures 4 5 Figure S1 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 Figure S1: Timeline of the experiments 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 Figure S2 4 5 Familiarization phase 6 A 35 7 Object 1 Locomotor activity 30 WT-vehicle 8 Object 2 50 9 WT-CBDV 0.2 mg/kg 25 WT-CBDV 2 mg/kg
) 40
10 s
( 20 WT-CBDV 20 mg/kg
s 11 e 30 WT-CBDV 200 mg/kg r e m
15 t i KO-vehicle e
12 t
m 20 13 10 KO-CBDV 0.2 mg/kg KO-CBDV 2 mg/kg 14 10 5 KO-CBDV 20 mg/kg 15 0 0 KO-CBDV 200 mg/kg 16 le g g g g le g g g g ic /k /k /k /k ic /k /k /k /k 17 h g g g g h g g g g e m m m m e m m m m -v 2 2 0 -v 2 2 0 0 T . 2 0 . 2 0 18 0 V 0 O 0 V 2 W V D V 2 K V D V D B D V D B D V 19 C B D C B D B - C B - C B C T - B -C O - C - T C O - 20 T W - O K O W W T K K 21 W ForK Peer Review 22 23 Familiarization phase 24 B 25 Locomotor activity 25 Object 1 WT-vehicle 50 26 20 Object 2 WT-CBDV 0.2 mg/kg WT-CBDV 2 mg/kg 27 40
) WT-CBDV 20 mg/kg
s 15
28 ( s 30 WT-CBDV 200 mg/kg r e e
29 t KO-vehicle m e
i 10 t 30 m 20 KO-CBDV 0.2 mg/kg 31 KO-CBDV 2 mg/kg 5 10 32 KO-CBDV 20 mg/kg KO-CBDV 200 mg/kg 0 33 0 34 le g g g g le g g g g ic /k /k /k /k ic /k /k /k /k h g g g g h g g g g 35 e m m m m e m m m m -v 2 2 0 0 -v 2 2 0 0 T . 2 0 . 2 0 36 0 V 2 O 0 V 2 W V D V K V D V B D V B D V D B D D B D 37 B -C B B -C B C -C C -C - T C - O -C T W T - O K O 38 W T K O W W K K 39 40 41 Familiarization phase 42 C 30 43 Object 1 Locomotor activity 44 Object 2 50 WT-vehicle 45 WT-CBDV 0.2 mg/kg 20 ) 40 WT-CBDV 2 mg/kg 46 s (
WT-CBDV 20 mg/kg e s 30 47 r WT-CBDV 200 mg/kg e m t i e 48 t KO-vehicle 10 m 20 49 KO-CBDV 0.2 mg/kg KO-CBDV 2 mg/kg 50 10 KO-CBDV 20 mg/kg 51 KO-CBDV 200 mg/kg 0 0 52 le g g g g le g g g g ic /k /k /k /k ic /k /k /k /k h g g g g h g g g g 53 e m m m m e m m m m -v 2 2 0 0 -v 2 2 0 0 T . 2 0 . 2 0 54 0 V 2 O 0 V 2 W V D V K V D V B D V B D V D B D D B D 55 B -C B B -C B C -C C -C - T C - O -C T W T - O K O 56 W T K O W W K K 57 58 Figure S2: Exploration times and locomotor activity in vehicle- and CBDV-treated Mecp2 WT and KO during 59 the familiarization phase of the NOR test at PND 41 (A), 56 (B) and 66 (C). Left panels show the time spent 60 exploring the two identical objects during the 10-minute session; right panels report total distance moved. Data are expressed as mean ± S.E.M. and analyzed using Student t test or two-way ANOVA.
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1 2 3 Figure S3 4 5 6 A Short-term memory - PND 41 7 50
) novel object
8 c e
s 40 familiar object
9 (
10 e m
i 30 t
11 n
12 o * i
t 20 *
13 a r ** ** ** o ** 14 l 10 * ** p
15 x e 16 0 e 2 2 0 0 e 2 2 0 0 17 l . 2 0 l . 2 0 ic 0 V 2 ic 0 V 2 h V D V h V D V 18 e D B D V e D B D V -v B C B D -v B C B D T - C B - C B 19 -C T - C O -C O - C W T W T - K O K O - W T K O 20 W W K K 21 For Peer Review 22 B 23 Short-term memory - PND 56 24 50
) novel object
25 c e familiar object s 40
26 (
27 e m
i 30 28 t
n
29 o i 20 t ** * 30 a r * *
o ** 31 l 10 *** p *** ** 32 x e 33 0 e 2 2 0 0 e 2 2 0 0 34 l . 2 0 l . 2 0 ic 0 V 2 ic 0 V 2 h V D V h V D V 35 e D B D V e D B D V -v B C B D -v B C B D T - C B - C B 36 -C T - C O -C O - C W T W T - K O K O - W T K O 37 W W K K 38 39 C 40 Short-term memory - PND 66 41 42 50 ) novel object c
43 e
s 40 familiar object (
44 e
45 m
i 30 t 46 n o
47 i
t 20 a
48 r ** *** o
l * 10 * ** 49 p * *** *** x
50 e 51 0 e 2 2 0 0 e 2 2 0 0 l . 2 0 l . 2 0 52 ic 0 V 2 ic 0 V 2 h V D V h V D V e B D V e B D V 53 v D B D v D B D - B -C B - B -C B T C T -C O C -C - T -C - O -C 54 W T W T K O K O W W K K O 55 W K 56 57 58 Figure S3: Exploration times of the novel object and the familiar object in vehicle- and CBDV-treated Mecp2 59 WT and KO during the test phase of the NOR test at PND 41 (A), 56 (B) and 66 (C). Data are expressed as 60 mean ± S.E.M. and analyzed using Student t test. ***p<0.001, **p<0.01, *p<0.05 vs familiar object.
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1 2 3 Figure S4 4 5 A B 6 7 BDNF p-rpS6 CB1 receptor CB2 receptor 8 1.0 9 0.5 0.6 0.8 10 0.8 s
0.4 s s s t t t t i t 0.6 i i 11 i c t n t n t n n c a 0.4 c U c U U a
U 12 0.6
a
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0.3 / y y
* y y / 6 r / r / r 13 r 0.4 F a S 2 a 1 a a r
* r r p N r B t t 0.4 B t r t i 14 0.2 i i i D - C C b b
b 0.2 b p B r r r 15 r 0.2 A A A 16 A 0.1 0.2 17 0.0 0.0 18 0.0 0.0 WT KO WT KO WT KO WT KO 19 20 21 For Peer Review 22 23 24 25 26 C D 27 NAPE PLD FAAH 28 DAGL 1.2 0.4 MAGL 29 0.5 1.0 1.0 t 30 s s t
c 0.3 t i t
i 0.4
s 0.8 a s n t
31 c t t n i i
0.8 t c a / U n n c U
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0.3 0.6 / L r r 0.2 / H y 0.6 y a P r r a 33 L
* r A r L a a t G t r E r i A i t G t 0.2 0.4 A i
34 i P b F b 0.4 A r b b r M A r r 0.1 D A
35 A N A A 0.2 36 0.2 0.1 37 0.0 0.0 0.0 0.0 38 WT KO WT KO WT KO WT KO 39 40 41 42 43 44 45 Figure S4: Protein levels of (A) BDNF and p-rpS6, (B) CB1 and CB2 receptors, (C) NAPE-PLD and FAAH, 46 (D) DAGLα and MAGL in hemisected brains of pre-symptomatic 4-weeks-old Mecp2 WT and KO mice as 47 measured by means of Western blot analysis. Data are expressed as mean ± S.E.M. of 4 WT and 3 KO 48 mice per group and were analyzed using unpaired Student t test. *p<0.05 vs WT. 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 Figure S5 4 5 6 7 8 CB1 receptor CB2 receptor FAAH 9 0.6 * 2.0 0.4 10 11 s s s t t 1.5 * t 0.3 i i i 12 t t t n n n 0.4 c c c a U U U a
13
a y y y / / r r / 14 r * 1.0 0.2 * H a a 2 1 a r r r A B B t t 15 t i i i A C C b b b
0.2 F
16 r r r
A 0.5 A 0.1 17 A 18 19 0.0 0.0 0.0 20 WT KO WT KO WT KO 21 For Peer Review 22 23 24 25 26 27 28 29 30 Figure S5: Protein levels of cannabinoid CB1 and CB2 receptors and FAAH in hemisected brains of naïve 9- 31 weeks-old Mecp2 WT and KO mice (no handling) as measured by means of Western blot analysis. Data are 32 expressed as mean ± S.E.M. of 4 mice per group and analyzed using unpaired Student t test. *p<0.05 vs 33 WT. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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