Chronic treatment with the phytocannabinoid Cannabidivarin (CBDV) rescues behavioural alterations and brain atrophy in a mouse model of Rett syndrome
Introduction
Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioural and physiological symptoms (Hagberg et al., 2002; Rett, 1966; Ricceri et al., 2012). One essential feature of RTT is the apparently normal perinatal development until about 6–18 months of age, when RTT patients start losing their acquired cognitive, social, and motor skills and develop a wide variety of symptoms (Hagberg, 2002). Classic RTT is caused in about 90–95% of cases by de novo mutations in the X-linked MECP2 gene, which encodes the methyl CpG-binding protein 2 (MECP2), a multifunctional protein that binds to methylated DNA and mainly acts as a key transcriptional regulator (Guy et al., 2011). Despite extensive effort in this research field, how mutations in MECP2 lead to the symptomatology of RTT is still unknown and no effective therapy is currently available for this devastating syndrome.
The endocannabinoid system (ECS) is a complex neuromodulatory system found in all vertebrate classes, involved in the regulation of numerous physiological functions (Kano et al., 2009). At the central level, ECS modulates several physiological processes and behavioural responses that are impaired in RTT (Di Marzo et al., 2015), such as social behaviour (Wei et al., 2017), anxiety and stress response (Jenniches et al., 2016) and motor control (El Manira and Kyriakatos, 2010). Moreover, ECS deregulation has been associated with many neuropsychiatric disorders such as anxiety and depression (Jenniches et al., 2016; Micale et al., 2013), Fragile X syndrome (Jung et al., 2012), schizophrenia (Clarke et al., 2017) and with neurodegenerative disorders characterized by cognitive and motor dysfunctions such as Alzheimer's, Huntington's and Parkinson's disease (Dowie et al., 2009; Maroof et al., 2014; Pisani et al., 2011). Recent data also suggest an involvement of the ECS in Autism Spectrum disorders (Chakrabarti et al., 2015).
A growing number of molecules able to directly or indirectly modulate the ECS have been identified to date. Pioneering studies increased the ECS response using agonists of the CB1 receptor, one of the two well characterized G-coupled receptors for endocannabinoids (Berrendero and Maldonado, 2002; Jiang et al., 2005; Patel et al., 2003). However, CB1 agonists may cause psychotropic side effects, similar to those reported with cannabis use in recreational settings. Such effects are now known to be due to the assimilation of Δ9-tetrahydrocannabinol (THC), the main compound of Cannabis sativa. To avoid these undesirable effects, most recent preclinical studies focussed on the identification of molecules that modulate the ECS without the psychotropic effects of THC, such as Rimonabant and URB597 (see e.g.(Griebel et al., 2005; Marco et al., 2015)). In addition, much attention has been devoted to non-psychotropic molecules from Cannabis sativa, which contains more than 120 substances (Morales et al., 2017). This has led to the identification of few non-psychotropic phytocannabinoids (phCBs) with a potential as novel drugs. These include Cannabigerol (CBG) and Cannabidiol (CBD), the second and the third most abundant chemical class types contained in Cannabis sativa respectively. In particular CBD bears a high potential in the treatment of muscular spasms and rigidity (Di Marzo, 2011), epilepsy (Chiu et al., 1979; Devinsky et al., 2017), mood disorders (Linge et al., 2016) and Alzheimer's disease (Cheng et al., 2014). Moreover, recent evidences suggest a potential application for CBD in pediatric conditions such as autistic-related syndromes (Kaplan et al., 2017) and in children with refractory epilepsy (Brodie and Ben-Menachem, 2018; Geffrey et al., 2015).
Another promising phCB is Cannabidivarin (CBDV), the n-propyl analog of CBD. Recent evidence proves that in vitro and in vivo treatment with CBDV in mouse and rat exerts anticonvulsant effects (Hill et al., 2012) and prevents neuronal hyperexcitability (Iannotti et al., 2014). However, the studies focussed on this compound are still very limited and the mechanisms of action of CDBV have not been clarified so far. Current evidences suggest that at physiologically relevant concentrations of CBDV show no affinity for CB1 and CB2 receptors (Hill et al., 2012) and presents antagonistic properties on the G protein-coupled receptor 55 (GPR55) receptor, the leading candidate for the CB3 receptor name (Anavi-Goffer et al., 2012; Iannotti et al., 2014; Marichal-Cancino et al., 2017; Turner et al., 2017). This lipid-activated G protein-coupled receptor has been suggested to regulate motor function, spatial memory and sociability (Bjursell et al., 2016; Kramar et al., 2017; Marichal-Cancino et al., 2018), behavioural domains that are compromised in RTT (De Filippis et al., 2014; Moretti et al., 2006). Moreover, antagonism of GPR55 has been recently suggested as a potential therapeutic approach for Dravet syndrome (Kaplan et al., 2017), an autistic-like syndrome with several symptoms in common with RTT.
Importantly, a clinical trial is currently listed aimed at evaluating the potential efficacy of a treatment with CBDV on children affected by Autism Spectrum Disorder (clinicaltrial.gov, NCT03202303). Based on the high translational potentiality of CBDV as an innovative therapeutic agent, in the present study MeCP2-308 hemizygous male mice, a highly validated mouse model of RTT (De Filippis et al., 2010), and wild-type littermate controls received a repeated systemic intraperitoneal (i.p.) treatment with CBDV (2, 20, 100 mg/Kg ip for 14 days). Mice were treated at 5 months of age, an early symptomatic stage at which MeCP2-308 mice already present reduced spontaneous home-cage motor activity, motor coordination impairments, and a more marked profile of d-amphetamine-released stereotyped behavioural syndrome than WT controls (De Filippis et al., 2010). A battery of behavioural analyses was carried out to evaluate treatment effects. Given CBDV antagonistic action on GPR55 (Marichal-Cancino et al., 2017), levels of this receptor were evaluated, to verify whether they are abnormal in RTT and CBDV treatment effects thereon. As markers of efficacy we also explored whether the CBDV treatment impacts the abnormal activation of the ribosomal protein (rp) S6, a downstream target of mTOR, in the brain of MeCP2-308 mice (De Filippis et al., 2014; Ricciardi et al., 2011), and the alterations in brain neurotrophins levels (Chang et al., 2006; Ricceri et al., 2011). Indeed, based on previous reports suggesting that ECS modulation in mouse brain can impact mTOR signalling (Busquets-Garcia et al., 2013; Puighermanal et al., 2012) and neurotrophins levels (Keimpema et al., 2014), we hypothesised that the CBDV treatment may normalize these RTT-related brain molecular alterations. A focus was made on the hippocampus, a brain region critically involved in regulation of relevant behavioural domains (Kaplan et al., 2017; De Filippis et al., 2014).
Section snippets
Animals
The experimental subjects were 5 month-old MeCP2-308 hemizygous male mice (RTT) and wild-type (WT) littermates (B6.129S-MeCP2tm1Heto/J from the Jackson Laboratories (USA), stock number: 005439) (De Filippis et al., 2010; Shahbazian et al., 2002), bred in our facility. Mice were weaned at postnatal day 25 and maintained in groups of 2–3 (according to sex) until 5 months of age. Temperature was maintained at 21 ± 1 °C and relative humidity at 60 ± 10%. Animals were provided ad libitum with tap
General health score
The evaluation of the general health status of the experimental mice confirmed that consistent gross phenotypic alterations can be detected in 5 month old RTT mice (Fig. 2a, RTT, veh vs WT, veh, p < 0.01 after post-hoc comparison on Genotype*Treatment interaction: F(3,60) = 2.906; p = 0.042). We found that 14 injections of CBDV at the doses of 20 and 100 mg/Kg can improve the general health status of RTT mice (Fig. 2a, RTT, veh vs RTT, CBDV 20 mg/Kg, p < 0.01 and RTT, veh vs RTT, CBDV, 100
Discussion
The present study demonstrates, for the first time, that 14 days of treatment with CBDV, a phCB extracted from Cannabis sativa, improves important aspects of the aberrant phenotype in a validated mouse model of RTT. The positive effects of CBDV treatment on RTT mice were specifically related to the general health status, the social sphere and the motor skills, phenotypic domains which are highly compromised in RTT patients (Morel and Demily, 2017; Stahlhut et al., 2017). Of note, the reduced
Conclusions
No cure is currently available for patients suffering from RTT, a devastating neurodevelopmental disorder with a huge burden for families. Present data provide evidence that the CBDV treatment rescues several behavioural and phenotypic defects in a mouse model of RTT, thus representing a potential therapeutic approach for this disorder. Moreover, GPR55 was herein for the first time suggested to be a potential target for the treatment of RTT. Even though further studies are needed to clarify the
Founding and disclosure
GW Research Limited supplied CBDV and financially supported this study.
Acknowledgements
The authors are grateful to Luigia Cancemi for animal care, Nadia Francia and Stella Falsini for administrative assistance, Vanessa Medici and Maria Cristina Talamo for technical assistance and Francesca Cirulli and Elena Ortona for providing advices and made available the laboratory for molecular analyses.
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2021, Pharmacology and TherapeuticsCitation Excerpt :Very few preclinical studies have specifically addressed the possible therapeutic potential of CBDV in animal models of ASD. Namely, CBDV has been tested in an environmentally-based animal model of ASD (Zamberletti, Gabaglio, Woolley-Roberts, et al., 2019) and in two different animal models of Rett Syndrome (RTT; Vigli et al., 2018; Zamberletti et al., 2019). The environmental model used to investigate CBDV's effects on ASD signs is based on prenatal VPA exposure in rats.