In the present study, we characterized the expression and function of the ECS to assess its potential contribution to the etiopathology of MCDs, in particular FCD Type II. Our results reveal a striking increase in CB₁ receptor expression levels in FCD Type II, and this enrichment occurs in neurons with overactive mTORC1 signaling. In addition, characterization of genomic DNA in the dysplastic brain resections showed an enrichment of three SNPs in the DAGLA gene in FCD Type II versus controls. The SNPs analyzed in other ECS element genes did not show any difference between groups. In addition, sequencing of the CNR1 coding exon did not reveal any SNP or mutation differentially present in FCD Type II versus control brains.

Presynaptic CB₁ receptors engaged by retrograde endocannabinoid messengers constitute an efficient regulatory mechanism of excessive neurotransmitter release. Hence, endocannabinoid signaling is a crucial pathway controlling neuronal activity and its activation or blockade modulates seizures and epilepsy development (Soltesz et al., 2015). Patients of temporal lobe epilepsy have decreased CB₁ receptor expression (Ludányi et al., 2008; Goffin et al., 2011). Likewise, selective loss of function of presynaptic CB₁ receptors in projection neuron populations of the mouse brain results in an imbalance of the excitatory/inhibitory tone and a higher susceptibility to seizures (Monory et al., 2006). Thus, the enrichment in CB₁ receptor expression in FCD lesions could represent a compensatory mechanism to attenuate the imbalance of excitatory/inhibitory neuronal activity. In addition, gene expression assays indicate that, at least for the hCB₁ receptor promoter employed (-3016 to +142 bp sequence), is not directly induced by overactive mTORC1 pathway, indicating that other signaling events control CB₁ receptor expression in this context.

SNPs analyses and genomic DNA sequencing of the CNR1 exon did not reveal any mutation associated with FCD Type II. Alternatively, the potential involvement of 2-AG metabolism in FCD Type II is suggested by the existence of a selective enrichment in three SNPs of the DAGLA gene. The impact of the DAGLA SNPs found in our study in 2AG production or DAGL regulation, is yet unknown as they correspond to non-coding regions. Nevertheless DAGLA transcript levels were slightly reduced in FCD when compared to control tissue. These results are in agreement with a recent study that found DAGLA polymorphisms associated with neurodevelopmental disorders and seizures (Smith et al., 2017), while CB₁ receptor associated with pain sensitivity, sleep, memory or anxiety, but not seizures. CB₁ receptors are coupled to the mTORC1 signaling pathway at early stages of neocortex formation (Díaz-Alonso et al., 2015) as well as in the adult brain (Puighermanal et al., 2009). We therefore analyzed CB₁ receptor downstream signaling in FCD-derived organotypic slices. The CB₁ agonist HU-210 was unable to further increase mTORC1 activation, reflecting that the pathway is already overactive. Remarkably, the CB₁ inverse agonist rimonabant was efficient in reducing phospho-S6 levels. This is of interest, as dampening overactive CB₁ receptor activity with rimonabant in other settings as the Fmr1 knockout mice efficiently decreases exacerbated mTORC1 activity and symptoms (Busquets-Garcia et al., 2013). Thus, these results point to a basal cannabinoid signaling tone that sustains exacerbated mTORC1 activity in FCD Type II. The ribosomal S6 protein is regulated by phosphorylation at multiple sites (Meyuhas, 2015). S6 phosphorylation at Ser240/244 is selectively mediated by S6K1/2 providing a better readout of mTORC1 upstream activation, while Ser235/236 phosphorylation is regulated by different signaling pathways (cAMP/PKA, casein kinase 1, MAPK-activated protein kinase-1 and mTORC1/S6K1/2). Initial studies revealed increased levels of phosphoS235/236-S6 protein in FCD and TSC (Baybis et al., 2004; Aronica et al., 2007). More recently, somatic mutations of MTOR signaling pathway and other upstream regulators PI3K/Akt, TSC1, TSC2, DEPDC5 have been demonstrated in FCD Type II (Jansen et al., 2015; Lim et al., 2015; Ricos et al., 2016). Hence, phosphorylation of S6 protein by different mechanisms may have different functional consequences in FCD and may differ among neural cell types (Ljungberg et al., 2006; Biever et al., 2015). Considering the finding that CB₁ receptor antagonism attenuates the phosphorylation of the ribosomal S6 protein at different amino acids (Ser235/236 and Ser240/244) it can be predicted that different CB₁ downstream signaling effectors: cyclic AMP-mediated and cAMP-independent (PI3K/Akt-mediated) contribute to its regulation. Hence, regulation of cannabinoid signaling constitutes and attractive target for various mTOR-associated disorders and symptoms -the so-called “mTORopathies”. Cannabidiol, a non-psychotomimetic cannabinoid with multiple targets, marketed as Epidiolex (GW Pharma), has demonstrated its efficacy as antiepileptic drug in refractory epilepsy including TSC, Dravet and Lennox-Gastaut syndromes, and is under clinical trial for FCDs (Devinsky et al., 2015; Hess et al., 2016; Thiele et al., 2018).

Interestingly, FCD type II is believed to be originated from the dorsal telencephalic progenitor cell compartment and their excitatory neuronal progeny (D’Gama et al., 2017). Noteworthy, in mTORopathies experimental models of MCDs unbalanced deep and upper layer neuronal development occurs as consequence of aberrant expression of neural fate determinants. Hence, a contribution of CB₁ receptor signaling to neural precursor alterations responsible for FCD Type II cannot be excluded. The consequences of CB₁ receptor activity during development and later in the adult brain can be intrinsically different. During embryonic development, CB₁ receptor controls radial glial to intermediate progenitor cell transition via mTORC1 signaling (Díaz-Alonso et al., 2015), and later, at postmitotic stages, tunes deep cortical neuronal differentiation (Mulder et al., 2008; Díaz-Alonso et al., 2012). Thus, CB₁ receptor activity during embryonic stages controls progenitor proliferation and neuronal differentiation, and alterations in cannabinoid signaling have the potential to evoke long-term neuronal plasticity and abnormalities underlying FCD neuronal hyperexcitability. In animal models, conditional ablation of the CB₁ receptor during embryonic development results in increased RhoA levels, ectopic neuron accumulations and increased seizure susceptibility (Díaz-Alonso et al., 2017). Noteworthy, RhoA knockdown prevents brain hyperexcitability and projection neuron alterations induced by CB₁ receptor ablation. Therefore, either hyper- or hypoactive endocannabinoid signaling during cortical development can be responsible for neuronal differentiation and positioning deficits contributing to MCDs. Later, in adult brain, when neuronal activity is established, the neuromodulatory function of the ECS takes place. The precise consequences of CB₁ receptor activity in the hyperexcitability neuronal circuit of the dysplastic brain remain unknown. Among other mechanisms involved in FCD, decreased hyperpolarization-activated non-specific cation currents contribute to pyramidal layer V hyperexcitability (Albertson et al., 2011). Interestingly, a particular pool of somatodendritic CB₁ receptors can regulate Ih currents and this explains in turn some of the cognitive consequences of CB₁ signaling (Maroso et al., 2016).

This study highlights the pathological implications of altered developmental cannabinoid signaling in refractory epilepsy. Characterization of epileptogenic FCD tissue from palliative surgery and dysplastic-derived organotypic cultures indicates that increased CB₁ receptor signaling may constitute a compensatory mechanism to counteract FCD Type II hyperexcitability, and its antagonism can dampen mTORC1 overactivation. We anticipate that in the near future new genetic linkage association analyses using larger cohorts of patients with pediatric epilepsy could provide further support to cannabinoid signaling deregulation as a causal mechanism underlying refractory epilepsy.