Cannabidiol anticonvulsant effect is mediated by the PI3Kγ pathway
Graphical abstract
Introduction
Epilepsy is a neurological disorder characterized by recurrent seizures and the neurobiological, cognitive and social consequences associated with this condition (Fisher et al., 2014). Epileptic seizures occur on a recurrent and spontaneous basis, characterized by a hypersynchronized discharge of central nervous system (CNS) neuron populations due to increased excitability and/or coupling (McNamara, 1994). Importantly, the events that occur after the status epilepticus (SE) may lead to a plethora of pathological processes that may contribute to epileptogenesis (Pitkanen et al., 2015). Therefore, it is necessary to investigate these postictal events.
Temporal Lobe Epilepsy (TLE) is the most common type of epilepsy in the adult population, accounting for about 40 % of all cases (Wiebe, 2000). There is an imbalance in glutamatergic and/or GABAergic neurotransmission, leading to an excessive glutamate release in the synaptic cleft and excitotoxicity, accompanied by gliosis, which leads to hippocampal sclerosis (Babb et al., 1991). These changes can also affect hippocampal adjacent structures, such as amygdaloid nucleus, parahippocampal gyrus and enthorrinal cortex. Patients may have focal or secondarily generalized seizures, originating from temporal lobe structures, especially in the hippocampal formation (McNamara, 1994).
Currently, there are some compounds available for the treatment of this condition. However, the available medicines are not efficient to approximately 30–50 % of TLE patients (Laxer et al., 2014). Therefore, there is an urge for the development of new therapies. In this context, cannabidiol (CBD) is a phytocannabinoid present in the herb Cannabis sativa that has been increasingly attracting therapeutic interest, particularly due to its anticonvulsive actions (Ibeas Bih et al., 2015). Despite its approval for clinical use, its mechanism of action, as well as its involvement with the endocannabinoid system, are still not completely elucidated. Some studies suggest that CBD effects may not be directly dependent on its binding to cannabinoid receptors type-1 and type-2 (CB1 and CB2 receptors) (McPartland et al., 2007; Mechoulam and Hanus, 2002), since it has low affinity as an orthosteric binder of CB1 receptor. However, it may influence the activity of the receptor via an indirect mechanism (McPartland et al., 2015), such as facilitating the receptor activation by increasing the brain levels of endocannabinoids (Bisogno et al., 2001).
The phosphatidylinositol 3-kinase (PI3K) intracellular signaling pathway is involved in a wide variety of biological processes, such as modulation of inflammatory mediators; neuronal growth, survival and differentiation; neuroprotection and synaptic plasticity (Acebes and Morales, 2012). PI3Ks act as important intracellular messengers and may activate signaling proteins involved in long-term potentiation (LTP) and long-term depression (LTD) (Sanna et al., 2002; Yang et al., 2008). Non-selective inhibitors of PI3K block LTP in the CA1 region of the hippocampus (Sanna et al., 2002), and the genetic and pharmacological blockade of PI3Kγ interfere with NMDAR-dependent LTD and hippocampal behavioral flexibility (Kim et al., 2011). Another study has demonstrated that the overexpression of the p110γ catalytic subunit in mice CA1 region of the hippocampus impairs NMDAR-dependent LTD and spatial memory, but does not affect LTP and aversive memory (Choi et al., 2014). Moreover, studies conducted by our group have demonstrated that PI3K inhibition increases production of inflammatory mediators in microglial cells cultures activated by inflammatory stimuli, and in hippocampus and cortex of animals submitted to pilocarpine injection (de Oliveira et al., 2008, 2012; Lima et al., 2015). In addition, knockout animals for PI3Kγ (PI3Kγ-/-) enzyme present a more intense SE, as well as an increase of some neuroinflammatory and neurodegenerative parameters, when compared with wild-type (WT) mice (Lima et al., 2015). Thus, PI3K may control both seizures and postictal changes, and be involved in anticonvulsive and antiepileptic drug actions.
In this context, it is possible that the neuroprotective effects of CBD and other phytocannabinoids may be mediated by the PI3K/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) pathway. Indeed, phytocanabinoids, such as delta(9)-tetrahydrocannabinol and R-(+)-methanandamide, activate the PI3K/Akt/mTOR pathway by activating both CB1 and CB2 receptors (Sanchez et al., 2003). Besides, it has recently been demonstrated that the anticonvulsive effect of CBD on the cocaine-induced seizures model was abolished by the administration of the mTOR inhibitor rapamycin, indicating the possible involvement of this intracellular signaling pathway in the CBD mediated anticonvulsive properties (Gobira et al., 2015). However, the role of CBD in seizures and postictal events, and the involvement of the PI3K/Akt/mTOR pathway on such effects remains unclear. Therefore, the aim of this study was to evaluate the role of the PI3Kγ signaling pathway in modulating the anticonvulsive and neuroprotective effects of CBD.
Section snippets
Drugs
Pilocarpine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile saline solution (0.9 % NaCl). CBD was supplied by THC-Pharma (Frankfurt, Germany) and was dissolved in sterile saline solution containing 2 % of tween 80. The CB1 receptor antagonist AM251 (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in cremophor:ethanol:saline at the proportion of 1:1:18. Valproic acid sodium salt (VPA, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile saline solution.
CBD reduces pilocarpine-induced behavioral seizure severity
First, we tested the hypothesis that CBD reduces the seizures induced by pilocarpine injection in the hippocampus. We performed a dose-response curve to evaluate the effect of CBD on the behavioral seizures induced by pilocarpine. Animals were divided in groups Pilocarpine + Vehicle (n = 11), Pilocarpine + VPA 300 (n = 10), Pilocarpine + CBD 30 (n = 10), Pilocarpine + CDB 60 (n = 12) and Pilocarpine + CBD 90 (n = 11). We evaluated latency to behavioral seizures onset, number of seizures scores
Discussion
In the present study, we investigated the involvement of the PI3Kγ enzyme on CBD protective effects against seizures and postictal alterations induced by pilocarpine. Our results indicate that the PI3K/mTOR pathway may be involved in the anticonvulsant and neuroprotective effects of CBD.
A single dose of CBD decreased the behavioral seizures intensity induced by intrahippocampal pilocarpine administration. In accordance, many studies have demonstrated the anticonvulsant effects of CBD in some
Funding sources
This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (CBB-APQ-02044-15), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 479254/2013-3, 424588/2016-1 and 310347/2018-1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
CRediT authorship contribution statement
Isabel Vieira de Assis Lima: Conceptualization, Methodology, Validation, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Visualization. Paula Maria Quaglio Bellozi: Formal analysis, Investigation, Writing - original draft. Edleusa Marques Batista: Investigation. Luciano Rezende Vilela: Investigation. Ivan Lucas Brandão: Investigation. Fabíola Mara Ribeiro: Conceptualization, Methodology, Resources. Márcio Flávio Dutra Moraes: Writing - review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowlegdements
We would like to thank Dr. M. M. Teixeira, from ICB - UFMG, Belo Horizonte, Brazil, for kindly providing PI3Kγ-/- mice. FMR, MFDM, FAM and ACPO acknowledge CNPq for the productivity fellowships.
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2022, Veterinary JournalCitation Excerpt :In this context, it would be of particular interest to address the question of whether an interference with glial cell function and proinflammatory signaling (for review see: Scarante et al., 2020) contributes to CBD’s antiseizure effects or might even mediate disease-modifying effects. Moreover, it would be of interest to further assess the potential role of an interaction of CBD with mechanistic target of rapamycin (mTOR) signaling, which has recently been suggested as a potential target with relevance for CBD’s anticonvulsant effects (Lima et al., 2020). In addition, the proposed interaction of CBD with voltage-gated sodium channels (Ghovanloo et al., 2018) should be further explored.
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2022, NeuroscienceCitation Excerpt :We evaluated the effects of commercially available CBD on seizure severity and examined its efficacy as a monotherapy using a multiple seizure paradigm. Previous studies have used various CBD doses, most commonly ranging from 30 to 200 mg/kg (Kaplan et al., 2017; Gu et al., 2019; de Assis Lima et al., 2020). The half-life of CBD in rodents is approximately 4.5 h (Deiana et al., 2012), much shorter than the 24 h half-life measured in humans (Ohlsson et al., 1986).