Elsevier

Neuropharmacology

Volume 176, 1 October 2020, 108156
Neuropharmacology

Cannabidiol anticonvulsant effect is mediated by the PI3Kγ pathway

https://doi.org/10.1016/j.neuropharm.2020.108156Get rights and content

Highlights

  • CBD is anticonvulsant in a model of pilocarpine-induced behavioral seizures.

  • CBD reduces the pathological postictal events.

  • CB1 receptor mediates the effects of CBD.

  • PI3Kγ pathway mediates the anticonvulsant neuroprotective effects of CBD.

Abstract

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt)/mechanistic target of rapamycin (mTOR) signaling pathway has been associated with several pathologies in the central nervous system (CNS), including epilepsy. There is evidence supporting the hypothesis that the PI3Kγ signaling pathway may mediate the powerful anticonvulsant properties associated with the cannabinoidergic system. This work aims to investigate if the anticonvulsant and neuroprotective effects of cannabidiol (CBD) are mediated by PI3Kγ. In vitro and in vivo experiments were performed on C57Bl/6 wild-type (WT) and PI3Kγ-/- mice. Behavioral seizures were induced by bilateral intra-hippocampal pilocarpine microinjection. Twenty-four hours after the first behavioral seizure, animals were perfused and their brains removed and processed, for histological analysis of neurodegeneration, microgliosis and astrocytosis. Primary cultures of hippocampal neurons were used for glutamate-induced cell death assay. CDB increased latency and reduced the severity of pilocarpine-induced behavioral seizures, as well as prevented postictal changes, such as neurodegeneration, microgliosis and astrocytosis, in WT animals, but not in PI3Kγ-/-. CBD in vivo effects were abolished by pharmacological inhibition of cannabinoid receptor or mTOR. In vitro, PI3Kγ inhibition or deficiency also changed CBD protection observed in glutamate-induced cell death assay. Thus, we suggest that the modulation of PI3K/mTOR signaling pathway is involved in the anticonvulsant and neuroprotective effects of CBD. These findings are important not only for the elucidation of the mechanisms of action of CBD, which are currently poorly understood, but also to allow the prediction of therapeutic and side effects, ensuring efficacy and safety in the treatment of patients with epilepsy.

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.

References (74)

  • I.V. Lima et al.

    Postictal alterations induced by intrahippocampal injection of pilocarpine in C57BL/6 mice

    Epilepsy Behav.

    (2016)
  • I.V. Lima et al.

    PI3Kgamma deficiency enhances seizures severity and associated outcomes in a mouse model of convulsions induced by intrahippocampal injection of pilocarpine

    Exp. Neurol.

    (2015)
  • R. Mechoulam et al.

    Cannabidiol: an overview of some chemical and pharmacological aspects. Part I: chemical aspects

    Chem. Phys. Lipids

    (2002)
  • M.R. Pazos et al.

    Cannabidiol administration after hypoxia-ischemia to newborn rats reduces long-term brain injury and restores neurobehavioral function

    Neuropharmacology

    (2012)
  • R.J. Racine

    Modification of seizure activity by electrical stimulation. II. Motor seizure

    Electroencephalogr. Clin. Neurophysiol.

    (1972)
  • M.G. Sanchez et al.

    Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction

    Cell. Signal.

    (2003)
  • G. Seifert et al.

    Astrocyte dysfunction in epilepsy

    Brain Res. Rev.

    (2010)
  • I. Serra et al.

    Cannabidiol modulates phosphorylated rpS6 signalling in a zebrafish model of Tuberous Sclerosis Complex

    Behav. Brain Res.

    (2019)
  • J.P. Szaflarski et al.

    Cannabidiol improves frequency and severity of seizures and reduces adverse events in an open-label add-on prospective study

    Epilepsy Behav.

    (2018)
  • E.A. Thiele et al.

    Cannabidiol in patients with seizures associated with Lennox-Gastaut syndrome (GWPCARE4): a randomised, double-blind, placebo-controlled phase 3 trial

    Lancet

    (2018)
  • L.R. Vilela et al.

    Enhancement of endocannabinoid signaling protects against cocaine-induced neurotoxicity

    Toxicol. Appl. Pharmacol.

    (2015)
  • L.R. Vilela et al.

    Anticonvulsant effect of cannabidiol in the pentylenetetrazole model: pharmacological mechanisms, electroencephalographic profile, and brain cytokine levels

    Epilepsy Behav.

    (2017)
  • P.C. Yang et al.

    Phosphatidylinositol 3-kinase activation is required for stress protocol-induced modification of hippocampal synaptic plasticity

    J. Biol. Chem.

    (2008)
  • A. Acebes et al.

    At a PI3K crossroads: lessons from flies and rodents

    Rev. Neurosci.

    (2012)
  • T. Bisogno et al.

    Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

    Br. J. Pharmacol.

    (2001)
  • C. Blazquez et al.

    The CB(1) cannabinoid receptor signals striatal neuroprotection via a PI3K/Akt/mTORC1/BDNF pathway

    Cell Death Differ.

    (2015)
  • P.S. Buckmaster et al.

    Inhibition of the mammalian target of rapamycin signaling pathway suppresses dentate granule cell axon sprouting in a rodent model of temporal lobe epilepsy

    J. Neurosci.

    (2009)
  • P.S. Buckmaster et al.

    Rapamycin suppresses mossy fiber sprouting but not seizure frequency in a mouse model of temporal lobe epilepsy

    J. Neurosci.

    (2011)
  • P. Calabresi et al.

    Antiepileptic drugs as a possible neuroprotective strategy in brain ischemia

    Ann. Neurol.

    (2003)
  • C.T. Campbell et al.

    Cannabinoids in pediatrics

    J. Pediatr. Pharmacol. Therapeut.

    (2017)
  • M. Ceprian et al.

    Cannabidiol reduces brain damage and improves functional recovery in a neonatal rat model of arterial ischemic stroke

    Neuropharmacology

    (2016)
  • J.Y. Chen et al.

    Valproate reduces neuroinflammation and neuronal death in a rat chronic constriction injury model

    Sci. Rep.

    (2018)
  • S. Chen et al.

    Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-kappaB pathway dependent of HDAC3

    J. Neuroinflammation

    (2018)
  • J.H. Choi et al.

    Effects of PI3Kgamma overexpression in the hippocampus on synaptic plasticity and spatial learning

    Mol. Brain

    (2014)
  • L.C. Constantino et al.

    Role of phosphatidylinositol-3 kinase pathway in NMDA preconditioning: different mechanisms for seizures and hippocampal neuronal degeneration induced by quinolinic acid

    Neurotox. Res.

    (2018)
  • A.C. de Oliveira et al.

    Regulation of prostaglandin E2 synthase expression in activated primary rat microglia: evidence for uncoupled regulation of mPGES-1 and COX-2

    Glia

    (2008)
  • A.C. de Oliveira et al.

    Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia

    J. Neuroinflammation

    (2012)
  • Cited by (25)

    • Isobolographic analysis of adjunct antiseizure activity of the FDA-approved cannabidiol with neurosteroids and benzodiazepines in adult refractory focal onset epilepsy

      2023, Experimental Neurology
      Citation Excerpt :

      CBD is an antagonist or partial agonist of CB2 receptors (Pertwee, 2008; Reddy and Golub, 2016; Rosenberg et al., 2017). Thus, the overall seizure protective effect of CBD may involve multiple targets including the GABA-A receptor and the GPR55 (Kaplan et al., 2017; Vilela et al., 2017; Lima et al., 2020) (see Fig. 7B). Clinical studies have shown that CBD has a plasma half-life of 56–61 h, following twice-daily dosing for 7 consecutive days in healthy clinical controls (Taylor et al., 2018).

    • Efficacy of the FDA-approved cannabidiol on the development and persistence of temporal lobe epilepsy and complex focal onset seizures

      2023, Experimental Neurology
      Citation Excerpt :

      Hence, the typical endocannabinoid signaling is not the main target for the antiseizure effects of CBD. Other potential targets include the vanilloid receptor and the GPR55 (Kaplan et al., 2017; Vilela et al., 2017; Lima et al., 2020). Nevertheless, these potential targets of CBD could not explain its atypical U-shaped protective curves.

    • Cannabidiol in canine epilepsy

      2022, Veterinary Journal
      Citation 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.

    • Prophylactic Administration of Cannabidiol Reduces Microglial Inflammatory Response to Kainate-Induced Seizures and Neurogenesis

      2022, Neuroscience
      Citation 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).

    View all citing articles on Scopus
    View full text