Keywords

7.1 Introduction

The drug target hypothesis indicates that the lack of efficacy of antiseizure medications (ASM) in drug-resistant epilepsy (DRE) is a consequence of structural or functional alterations of their targets (Remy and Beck 2006). This hypothesis is postulated based on the insensitivity of voltage-gated sodium channels (VGSC) to carbamazepine observed in the resected hippocampus of DRE patients and experimental models of epilepsy (Remy et al. 2003a; Vreugdenhil and Wadman 1999). This chapter discusses several mechanisms that support the drug target hypothesis in DRE, including altered receptor subunit expression, epigenetic changes, receptor mosaicism, and lipid raft expression.

7.2 Voltage-Gated Sodium Channels

Voltage-gated sodium channels (VGSC) are transmembrane proteins activated by changes in the potential that allow sodium transportation along an electrochemical gradient when they open (De Lera Ruiz and Kraus 2015). In the resected cortex of patients with DRE, VGSC mRNA expression is upregulated, which may contribute to the increased neuronal excitability associated with epilepsy (Lombardo et al. 1996). In addition, evidence shows decreased mRNA and protein expression of the β4 subunit of VGSC in hippocampal tissue resected from patients with DRE. Altered expression of the β4 subunit could decrease the frequency and duration of the VGSC resting state and, thus, be implicated in the loss of ASMs efficacy (Sheilabi et al. 2020).

In the pilocarpine-induced epilepsy model, a significant reduction of the VGSC-blocking effects induced by phenytoin and lamotrigine in the hippocampus was observed in animals. This observation was more evident in brain tissue with epileptiform activity at higher discharge frequencies (Remy et al. 2003b). Increased VGSC mRNA expression was observed following kainic acid-induced status epilepticus in the rat hippocampus. This altered expression could produce hippocampal hyperexcitability (Bartolomei et al. 1997). After pilocarpine-induced status epilepticus, the activation window of VGSC in the dentate gyrus increases, a condition that could amplify neuronal depolarization. This alteration is associated with decreased expression of β2 (early) and β1 (late) subunits (Ellerkmann et al. 2003). The β1 subunit modulates the kinetics of VGSC closure (Brackenbury and Isom 2011; Hull and Isom 2018). Significantly, mutations in this subunit dramatically decreased the blocking effect of phenytoin (Lucas et al. 2005) and carbamazepine (Uebachs et al. 2010, 2012) in animal models and cell lines. They have also been associated with generalized epilepsy (Meadows et al. 2002; Wallace et al. 1998) and epileptic encephalopathies (Hull et al. 2020). This evidence suggests time-dependent alterations in the expression of the β-subunit of VGSC that may lead to poor response to ASMs.

7.3 GABAA Receptors

GABAA receptors are pentameric proteins composed of different subunits. The endogenous ligand of these receptors is gamma amino butyric acid (GABA), the primary inhibitory neurotransmitter of the central nervous system. When GABA activates GABAA receptors at postsynaptic sites, phasic inhibition occurs, whereas activation of GABAA receptors at extrasynaptic sites mediates tonic inhibition (Sigel and Steinmann 2012). Various ASMs, such as benzodiazepines, barbiturates, and topiramate, act on these GABAA receptors (Greenfield 2013). In hippocampal tissue resected from DRE patients, GABAA receptors expression is diminished in areas of significant cellular loss. Interestingly, surviving neurons show similar or increased expression of GABAA α1 and α2 subunits, suggesting a relative increase in receptors per neuron containing these subunits (Ghit et al. 2021; Loup et al. 2000). Since drug-binding sites are located between the α-β and α-ɣ interfaces, alterations in the expression of these subunits are associated with changes in drug selectivity (Sigel and Steinmann 2012). Positron emission tomography (PET) evaluations revealed focal variations in GABAA receptor-binding sites in the hippocampus of patients with DRE. A decrease in binding is detected during the days following a seizure (Bouvard et al. 2005), suggesting a diminished therapeutic response to benzodiazepines during the post-ictal and interictal periods. These changes could be associated with cell loss and altered subunit composition of GABAA receptors, decreasing their sensitivity to ligands (Lamusuo et al. 2000). Interestingly, in vitro analysis of the temporal cortex of patients with DRE revealed increased benzodiazepine binding and increased γ2 subunit mRNA, which was more evident in patients with lower seizure frequency. These findings lead to suggest that patients with low seizure frequency may present enhanced inhibitory effects mediated by the benzodiazepine binding, an effect not evident in patients with high seizure frequency and drug-resistant phenotype. This study supports that GABA receptor expression and function alterations correlate with clinical factors (Rocha et al. 2015).

Fifty percent of the animals with epilepsy due to pilocarpine-induced status epilepticus showed a poor response to phenobarbital. Since hippocampal damage and plasma phenobarbital concentration were similar between responder and nonresponder rats, the lack of response to this ASM could be related to target alterations (Bankstahl et al. 2012). The loss of phenobarbital efficacy in a model of temporal lobe epilepsy model induced by electrical stimulation of the basolateral amygdala was associated with low expression of GABAA subunits, particularly the α1 subunit in all the areas analyzed. In contrast, the expression of α2, α5, β2/3, and γ2 subunits was significantly decreased in the hippocampus. However, these conditions did not correlate with neuronal loss (Bethmann et al. 2008). Consistent with previous evidence, decreased mRNA expression of α1 and β3 subunits in the dentate gyrus of rats with epilepsy was associated with a diminished response to benzodiazepine modulation (Brooks-Kayal et al. 1998). These data support that alterations in the expression of the α and β subunits of the GABAA complex could underlie the loss of efficacy of ASMs that modulate GABAergic neurotransmission in DRE.

7.4 Other Receptors Involved in Drug-Resistant Epilepsy

PET studies revealed that the binding of [18F]-FCWAY, a ligand with a high affinity for 5-HT1A receptors, is lower in regions close to the epileptic focus (Toczek et al. 2003). In contrast, functional binding assay revealed that the ability to activate 5-HT1A receptor-coupled Gi proteins is more significant in the hippocampus of patients with drug-resistant temporal lobe epilepsy. These observations suggest overactivation despite the low binding of 5-HT1A receptor-associated transductional mechanisms (Cuellar-Herrera et al. 2014).

DRE is also associated with changes in the opioid system (Burtscher and Schwarzer 2017). Some autoradiographic studies showed that mu-opioid receptor binding is reduced in the neocortical tissue surrounding the epileptic focus of patients with DRE (Ondarza et al. 2002). The neocortex of patients with drug-resistant temporal lobe epilepsy (TLE) shows enhanced mu-opioid receptor binding but low DAMGO-stimulated Gi protein (Rocha et al. 2009). In patients with epilepsy, elevated mu receptor mRNA expression and binding without changes in the DAMGO-stimulated Gi protein are observed in the hippocampus (Cuellar-Herrera et al. 2012). Therefore, these studies support that mu-opioid receptors mediate the changes in expression and transductional mechanisms.

Another system that has gained relevance in recent decades is the endocannabinoid system. At the central level, the endocannabinoid system modulates inhibitory and excitatory neurotransmission through CB1 and CB2 receptors and other noncanonical receptors (Cristino et al. 2020). PET studies in TLE patients revealed that the availability of CB1 receptors in the neocortex ipsilateral to the epileptic focus is increased. This observation is more evident in patients with a shorter latency to the last seizure and a high frequency of ictal events before scanning (Goffin et al. 2011). The efficacy of CB1 receptor-induced G-protein signaling is, indeed, higher in the neocortex and hippocampus in patients with drug-resistant mesial TLE and more evident in patients with no evidence of mood disorders (Rocha et al. 2020). The hippocampal and cortical microvasculature of patients with DRE showed a high efficiency in activating Gαi/o-coupled proteins mediated by CB1 and CB2 receptors, a situation that does not correlate with their protein expression (Nuñez-Lumbreras et al. 2021). These findings support the idea that alterations of different receptors or their signaling cascades could explain the drug-resistant phenotype in epilepsy.

7.5 Conditions that Reduce ASMs Effectiveness

7.5.1 Desensitization

Desensitization is the decrease in response after persistent exposure to drug agonist (Caimmi et al. 2019). Therefore, dose increases do not produce the expected effect (Gupta et al. 2018). Desensitization is particularly relevant in epilepsy since it may result from the chronic administration of ASMs (Thijs et al. 2019). It has been reported that in cell lines expressing moderate levels of the human 5-HT1A receptor, stimulation of this receptor with prototypical agonists (5-HT, F13714, befiradol, and NLX-101) reaches a saturation point. In contrast, when this receptor is overexpressed, stimulation with the same agonists increases 5-HT1A receptor activation. However, increasing the concentration of the agonists results in the loss of the intrinsic activity of these receptors until any response occurs (Newman-Tancredi et al. 2019). The use of full agonists of GABA receptors tends to cause desensitization, which can lead to tolerance and dependence (Janković et al. 2021). Similarly, 2-day exposure to zolpidem, a positive allosteric modulator of GABAA receptors, induces uncoupling of GABA and benzodiazepine from their binding sites in rat cerebellar granule cells (Vlainić et al. 2012). Moreover, autoradiography and [3H]-muscimol binding experiments demonstrated two agonist-binding sites on GABAA receptors: a low-affinity one, which requires micromolar concentrations of GABA to be activated, and a high affinity one, where nanomolar concentrations of GABA are sufficient to interact with it; however, the muscimol-functional response decreases, probably as a manifestation of the desensitized form of these receptors (Chandra et al. 2010; Jembrek and Vlainic 2015). The preference for the sensitive or desensitized state is associated with structural changes in the receptor and may be affected by the drug exposure time. In this regard, prolonged exposure to GABA and other GABA agonists such as piperidine-4-sulfonic acid, muscimol, β-alanine, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol causes significant desensitization of GABAA receptors expressed in Xenopus oocytes (Akk et al. 2011).

GABAA receptor desensitization is associated with DR-MTLE patients (Ragozzino et al. 2005). In this regard, GABA current was less efficient in oocytes injected with isolated neocortex membranes from DR-MTLE patients and in neocortical slices from these patients due to repetitive applications of GABA. The desensitization of GABAA receptors was reduced by levetiracetam exposure in the range of 0.5 to 100 μM (Palma et al. 2007). Further studies should be carried out to determine the optimal administration of the ASM able to induce therapeutic effects without inducing side effects such as desensitization.

7.5.2 Receptor Downregulation

The availability of receptors at the cell membrane depends on several processes, starting with the expression of the genes that encode them. Expression of these genes leads to protein synthesis of the receptors, which are subsequently transported to the cell membrane and are susceptible to activation after binding to their ligands (Tipton and Russek 2022). In epilepsy, reduced receptor expression compromises the effect of ASMs (Tipton and Russek 2022).

Epileptic activity in preclinical models has been associated with the downregulation of GABAA receptor subunits and scaffold proteins (González et al. 2013). GABAA receptor downregulation is also induced following chronic GABA exposure (Roca et al. 1990). Studies have reported that rats subjected to subchronic treatment with diazepam and vigabatrin show lower latency to pentylenetetrazol-induced seizures. This effect is associated with poor binding of the benzodiazepine receptor in several brain areas, indicating a differential decrease of GABA receptors (Rocha 2008). The glutamate transporter GLT-1, a Na+-dependent transporter involved in removal glutamate from extracellular space, is downregulated in the brain of patients with Rasmussen encephalitis (He et al. 2020). Therefore, downregulation of either receptor, ion channels, or transporters associated with the control of excitatory neurotransmission could be actively participating in the expression of the pharmacoresistant phenotype of epilepsy.

7.5.3 Internalization

Internalization of receptors or ion channels that are the pharmacological targets of ASMs results in low efficacy of these drugs and failure to control seizures. Internalization of receptors and ion channels occurs physiologically in cells. However, changes in the microenvironment, such as disease, receptor overactivation, and the presence of drugs, can increase the internalization rate (Estadella et al. 2020). Moreover, GABAA receptors’ internalization occurs acutely and chronically during epileptogenesis (Blair et al. 2004). In addition, GABAA receptor internalization is associated with losing response to benzodiazepines in convulsive status epilepticus in pediatric patients (Singh et al. 2020).

Receptor internalization is a stepwise process. First, as receptors are phosphorylated, they become inactive, limiting their response to agonists. Subsequently, receptors are endocytosed by a clathrin-dependent or clathrin-independent pathway involving other proteins such as RhoA and ARF6 (Estadella et al. 2020; Gong et al. 2008). The receptor endocytosis process takes a few minutes. Once internalized, receptors can remain sequestered for hours (Wang and Marvizón 2002). When receptors are internalized, two processes can occur: the receptors can be recycled and repositioned on the cell membrane or degraded. When the former occurs, the receptors return to the cell surface and, after dephosphorylation, revert to their activatable form. The kinetics of this receptor recycling process is estimated to last between 60 to 250 min (Bhattacharyya et al. 2002; Wang and Marvizón 2002). Therefore, even when ASMs are available in the brain microenvironment, they would not be able to activate the signaling pathways necessary to control seizures. Consequently, this situation may contribute to drug resistance in epilepsy.

7.6 Changes in Receptor Signaling

7.6.1 PIP2 Modifies the Response to ASMs

Different second messengers are involved in transductional mechanisms following ligand binding to the G protein-coupled receptors (GPCR). Second messengers comprise hydrophobic, hydrophilic, and gaseous molecules that transduce extracellular signals into the cell to induce a physiological effect. Hydrophobic second messengers include bioactive lipids such as sphingolipids, arachidonic acid derivatives, diacylglycerol, and phosphoinositides, which bind to the cell plasma membrane (Newton et al. 2016). Phosphoinositides are acidic phospholipids containing an inositol sugar head and differ from each other depending on the number of phosphate groups on the inositol sugar head (Falkenburger et al. 2010). As such, this family includes phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5 trisphosphate (PIP3), among others. Each member of the phosphoinositide family plays a different role in biological processes, from cell growth to ion channel regulation (Suh and Hille 2008).

PIP2 represents the major plasma membrane phosphoinositide interacting with allosteric-binding sites to control the function of several ion channels, such as inwardly rectifying (Kir) and voltage-dependent (Kv) K+ channels (Balla 2013). The interaction between PIP2 and Kv7 channels is, in fact, essential for stabilizing the open state of the Kv7 channel and the flow of potassium ions (Suh and Hille 2008; Sun and MacKinnon 2020). In this regard, Kv 7.2 (KCNQ2) and Kv 7.3 (KCNQ3) channels are significant components of M-currents that prevent excessive hyperexcitability by increasing the firing threshold and interspike interval (Lawrence et al. 2006; Soldovieri et al. 2011; Tzingounis and Nicoll 2008; Wang et al. 1998). M-currents could be suppressed by activation of Gq protein-coupled receptors (GqPCRs) through decreased PIP2 levels, as PIP2 is the lipid substrate for phospholipase C (PLC) (Suh and Hille 2008; Zhang et al. 2003) (Fig. 7.1).

Fig. 7.1
A diagram of the ion channel depicts activators that activate K v 7 when no G q P C R activation occurs, resulting in nerve fiber excitability, and potassium ions get in through the channels. When G q P C R is active, the activators do not act on K v 7. Epilepsy reduces excitability.

GqPCR modulation of Kv7 channel activity by modifying PIP2 levels. In the absence of Gq protein-coupled receptor (GqPCR) activation (left panel), phosphatidylinositol 4,5-bisphosphate (PIP2) binds to voltage-dependent potassium channels (Kv). The binding of PIP2 to Kv7 channels increases M-currents that prevent neuronal hyperexcitability and regulate the function of Kv7 channel activators. In contrast, binding of an agonist to GqPCR (right panel) triggers phospholipase C (PLC) activity. PLC degrades PIP2 into diacylglycerol (DAG) and inositol trisphosphate (IP3). Increased degradation of PIP2 reduces M-currents and thus promotes hyperexcitability. Furthermore, status epilepticus reduces PIP2 levels, and epilepsy impairs lipid metabolism. However, it is still unknown whether epilepsy directly decreases PIP2 levels or whether antiseizure medications (ASMs) act through the modulation of lipid metabolism. (Figure created using BioRender)

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M-current suppression is associated with seizures, epileptogenesis, and epileptic syndromes (Ambrosino et al. 2015; Biervert et al. 1998; Greene and Hoshi 2017; Miceli et al. 2015). Furthermore, mutations in Kv7.2 channels affect PIP2 binding inducing abnormal expression and function, which may increase neuronal excitability and underlie KCNQ2-mediated epileptic encephalopathy (Kim et al. 2018). Indeed, Kv7.2 mutation is associated with early-onset epileptic encephalopathy and suppression-burst enhances Kv7/M channel activity (Devaux et al., 2016). Thus, KCNQ2 and KCNQ3 mutations are associated with severe epilepsy syndromes, such as Ohtahara syndrome characterized by drug-resistant seizures, psychomotor retardation, and EEG changes (Soldovieri et al., 2014; Köhling & Wolfart, 2016).

Therefore, lipid metabolism is crucial for Kv channel activity and subsequent modulation of neuronal excitability. In this context, PIP2-dependent interaction between voltage-sensing and pore-forming components has been reported to be essential for the action of retigabine, a KCNQ3 channels activator used to treat diseases characterized by hyperexcitability (Kim et al. 2017). Furthermore, zinc pyrithione (ZnPy), a drug more effective on KCNQ2 than on KCNQ3, undergoes a pharmacological shift when PIP2 levels decrease (Zhou et al. 2013). In other words, PIP2 depletion promotes KCNQ3 to become more sensitive to ZnPy and loses sensitivity to retigabine. Additionally, valproic acid has been shown to exert its antiepileptic effects, at least in part, by preventing M-current suppression and decreasing phosphorylation of Kv7.2 at Ser558 through inhibition of AKAP79/150 palmitoylation (Greene et al. 2018; Kay et al. 2015). Valproic acid also reduces pentylenetetrazole-induced seizure activity in cultured rat primary hippocampal neurons and entorhinal cortex–hippocampal slices by restoring PIP3 and PIP2 levels (Chang et al. 2014).

Finally, it has been proposed that kainic acid induces Ca2+ influx in vitro by a TRPV1-related mechanism that depends on the interaction of PIP2 with the receptor (Mohandass et al. 2020). Accordingly, it is possible to suggest that some effects of ASMs depend on PIP2 levels and their proper interaction with ion channels. Therefore, any change in PIP2 metabolism may contribute to the lack of efficacy of some anticonvulsant drugs. In this regard, lipid biosynthesis and metabolism are dysregulated in a Kv1.1 knockout-induced epileptic mouse model (Johnson et al. 2020) and the hippocampus of patients with temporal lobe epilepsy (Ajith et al. 2021); meanwhile, bicuculline-induced status epilepticus leads to accelerated degradation of PIP2 (van Rooijen et al. 1986). Based on the above lines of evidence, it is possible to suggest that the changes in lipid biosynthesis and metabolism observed during epilepsy could be responsible for the decreased functional response to certain drugs. However, further studies are still required to address the relationship between antiseizure medications and levels of bioactive lipids, such as phosphoinositides.

7.6.2 Lipid Rafts Modify ASMs Effects

Lipid rafts are cell membrane regions enriched with specific lipids, such as glycolipids, sphingolipids, and cholesterol, which play regulatory roles in synaptic transmission, action potential propagation, and membrane signaling (Levental and Veatch 2016). Within lipid rafts, different constitutive membrane receptors can interact with each other, modulating their signaling (Simons and Toomre 2000). Furthermore, the composition and organization of lipid rafts can modify the signaling pathways activated by the receptors located within them (Levental and Veatch 2016). Therefore, disturbances in lipid raft composition could alter cell functions and the expected response to a drug (Regen 2020). Evidence shows an association between changes in lipid raft composition and the development of different neurological diseases such as Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, depression, and epilepsy (Grassi et al. 2020; Schrattenholz and Soskic 2006).

Lipid rafts are involved in cellular excitability. Electrophysiological and fluorescence analysis in cortical neurons showed that activation of NMDA receptors closer to sodium-calcium exchangers (NCX) within lipid rafts results in main cytosolic calcium entry. Conversely, the destruction of lipid rafts increases the distance between NMDA receptors and NCX and weakens overall cytosolic calcium accumulation (Sibarov et al. 2018). Furthermore, activation of NMDA receptors in cultured hippocampal neurons recruits AMPA receptors to raft domains on the cell surface, increasing the probability of being activated upon glutamate exposure. In contrast, the disruption of lipid rafts reduces the surface expression of AMPA receptors (Hou et al. 2008). The propensity of AMPA receptors to be activated within lipid rafts may reduce the efficacy of ASMs targeting these receptors, as their signaling is biased (Pike 2003).

GABAA,B receptors are also susceptible to entering lipid rafts during the epileptogenic process, whereas inhibiting cholesterol synthesis produces a shift of GABAA,B receptors to non-raft domains (Huo et al. 2009). Translocation of GABAA,B receptors to lipid rafts reduces signal transduction mediated by their activation, affecting the effect of ASMs such as vigabatrin and benzodiazepines (Bouwman et al. 2007; Perescis et al. 2019). Notably, drugs such as pentobarbital reduce NMDA and GABAA receptors’ association with lipid rafts (Sierra-Valdez et al. 2016). Conversely, in vitro experiments revealed that disruption of lipid rafts can potentiate the effect of diazepam on the GABAA receptor (Nothdurfter et al. 2013). At present, as the involvement of lipid rafts in drug resistance in epilepsy is unknown, further research is needed.

On the other hand, it is known that it is relevant to indicate that P-glycoprotein, a transporter associated with drug-resistant phenotype, is overexpressed in neurons of patients with pharmacoresistant epilepsy (Lazarowski et al., 2004). When expressed in rafts and non-raft membrane domains, the P-glycoprotein interacts with protein partners regulating the membrane activity and affects trafficking in the cell (Orlowski et al., 2006). Further studies are necessary to elucidate the role of P-glycoprotein overexpression in lipid rafts of neurons of patients with drug-resistant epilepsy.

7.6.3 Oligomer Receptor Complexes in Drug-Resistant Epilepsy

Protein oligomerization is an association between two or more protein subunits to form a high-order complex (Ward et al. 2013). Protein aggregates are composed of the same protein subunit (homo-oligomers) or different protein subunits (hetero-oligomers) (Ali and Imperiali 2005). Since the formation of homo- and hetero-oligomeric complexes modifies the response to drugs in various neuropathologies, it has become a new topic of study (Fuxe et al. 2014). Several studies have shown that receptor oligomerization affects pharmacological and cell signaling responses (González-Maeso 2011). Under various conditions, the physiological role of oligomeric receptors in learning processes and cognitive functions, among others (Borroto-Escuela et al. 2017), could become pathological and lead to disease (Pérez de la Mora et al. 2022).

The formation of oligomeric complexes modifies not only the structure of the receptors but also their functionality (Ginés et al. 2000). The functional implication of receptor oligomeric complexes lies simultaneously in the signaling changes after activating their elements. Evidence has shown that dimer formation modifies the normal signaling of its component receptors (Parmentier 2015). In this regard, it is known that activation of the cannabinoid receptor CB1 leads to the phosphorylation of Gi proteins, inhibiting cyclic adenosine monophosphate (cAMP) synthesis. Similarly, the dopaminergic receptor D2 also activates the Gi protein pathway, with a consequent decrease in cAMP in the cell. Interestingly, the CB1 receptor and the D2 receptor can form a heterodimeric CB1/D2 complex that stabilizes the active state of the CB1 receptor with increased Gs-protein coupling (Fig. 7.2). This stimulates the CB1/D2 complex and increases cAMP concentration. In other words, the effect of CB1/D2 complex activation is opposite to that produced by the activation of the CB1 and D2 receptors separately (Hudson et al. 2010).

Fig. 7.2
An illustration depicts 3 types of receptors. The first contains protein molecules with inhibitory and excitatory properties. The heteroreceptors are made up of G P C R 1 and G P C R 2 molecules, which have excitatory actions. Mosaic receptors use ion channels and proteins to communicate.

Heteroreceptor and mosaic receptor formation may contribute to drug-resistant epilepsy. Pharmacological targets, such as G-protein-coupled receptors (GPCR) or ion channels, can associate to form heteroreceptors. Heteroreceptors change the intracellular signaling of antiseizure medications (ASM). These signaling changes involve silencing inhibitory pathways and shifting activation routes that can produce excitatory effects and, consequently, could favor the drug-resistant phenotype of patients with epilepsy. (Figure created using BioRender)

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Moreover, dimer formation can also modify signaling by recruiting proteins such as β-arrestin. Heterodimerization of μ and δ opioid receptors leads to constitutive recruitment of β-arrestin2. Consequently, downstream signaling pathways, such as ERK1/2 phosphorylation patterns, are altered compared to those observed with individual μ and δ receptors (Rozenfeld and Devi 2007).

Several molecular studies have shown that Na+ channel α-subunits can interact as dimers (Clatot et al. 2017). Oligomerization between mutant and wild-type VGSC α-subunits leads to an impaired gating probability (Clatot et al. 2018). The β3 subunit of the VGSC heterooligomer reduces the effect of carbamazepine (Sokolov et al. 2018). Heterodimerization of the Q2 and Q3 subunits of voltage-gated potassium channels increases the susceptibility of the channel to retigabine (Li et al. 2020). In addition, mutations in the GABAA receptor complex increase the probability of this receptor oligomerization and facilitate seizure activity (Wang et al. 2016). Homo-receptor and hetero-receptor complexes regulate each other, and their imbalance is associated with neurological disorders (Borroto-Escuela et al. 2017). In DRE, the lack of efficacy of ASMs may be related to dynamic oligomer formation (Borroto-Escuela and Fuxe 2019).

Another type of oligomeric complexes is mosaics, which refers to the physical receptor–receptor association within the intermembrane space (Agnati et al. 1980). These mosaics can be composed of homo- or heteroreceptors (Agnati et al. 2005a; Fuxe et al. 2008, 2009). Some examples of heteroreceptors have been established between κ- and δ-opioid receptors, adenosine A1 and A2A with dopaminergic D1 and D2 receptors, respectively (Borroto-Escuela and Fuxe 2019; Ginés et al. 2000).

Activation of mosaic receptors alters the function of other receptors (horizontal network) or intracellular transduction signals (vertical network) of different receptors (Agnati et al. 1980, 2005b). In addition, heterodimerization of adenosine A1 and A2A receptors allows adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission, providing a switching mechanism whereby low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release (Ciruela et al. 2006).

Based on NMDA-D2 heteroreceptor expression in the striatum, D2 receptor activation can convert glutamatergic-induced long-term potentiation into long-term depression (Higley and Sabatini 2010). In Parkinson’s disease, alterations in synaptic signaling are associated with the expression of dopaminergic receptor heteroreceptors with adenosine, neurotensin, or different dopamine receptor subtypes (Borroto-Escuela et al. 2017). Different heteroreceptors (serotoninergic and metabotropic glutamate receptors, neurotensin and dopamine receptors, and adenosine and dopamine receptors) are associated with the pathophysiology of psychosis and schizophrenia (González-Maeso et al. 2008; Tanganelli et al. 2012). Dopamine D2-adenosine A2A heteroreceptors lead to acute locomotor changes and cocaine-induced sensitization (Filip et al. 2006). In addition, cocaine-induced psychostimulation is associated with the formation of heteroreceptor complexes between dopamine D2 receptors and NR2B subunits of the NMDA receptor in the neostriatum (Liu et al. 2006). In patients with different neuropathologies (e.g., depression), activation of fibroblast growth factor 1 and 5-hydroxytryptamine 1A heteroreceptor complexes could lead to the neuroprotective effect (Borroto-Escuela et al. 2012).

Patients with DRE are characterized by nonresponsiveness to medication, even when it is directed to more than one pharmacological target (Kwan et al. 2010). The expression of homo- and hetero-oligomer complexes and mosaic receptors with consequent changes in the effects of specific targets may explain the resistance to multiple ASMs with different mechanisms of action in patients with DRE. If this is the case, it is essential to establish new pharmacological strategies based on homo- and heterooligomer complexes rearrangement in synaptic and extra-synaptic regions in individuals with epilepsy (Borroto-Escuela and Fuxe 2019).

Proteins regulate cellular processes by forming homo- or hetero-oligomerizations in the cellular environment. Dysregulation of these processes may lead to the expression of the pharmacoresistant phenotype in epilepsy (Singh and Jois 2018). Future studies should determine whether the DRE condition is associated with the dynamic expression of mosaic receptors.

7.7 Epigenetic Changes in ASM Targets

DRE is characterized by the failure of adequate trials of two or more ASMs (alone or in combination) to achieve sustained seizure freedom (Kwan et al. 2010). The loss of efficacy of ASMs with distinct mechanisms of action indicates simultaneous changes in several targets. In this section, we present information supporting epigenetic changes to explain different mechanisms of target resistance.

Epigenetics has been defined as “changes in gene function that are heritable and do not depend on changes in the DNA sequence” (Deans and Maggert 2015; Holliday 1994). The nucleosome core comprises 147 bp of DNA wrapping 1.65 times the histone octamer and includes 14 contact points between histones and DNA. This structure facilitates packaging function and ensures stability under physiological conditions. However, it is essential to note that it is highly dynamic and that several protein complexes interact to regulate its configuration. Histone tails and globular domains are subject to several posttranslational modifications; one of the most studied is the acetylation of histones 3 and 4, which is associated with active transcription. Histone acetylation is performed by histone acetyltransferases, which acetylate lysine residues and change the net charge of nucleosomes, thereby loosening DNA-histone interactions. Notably, the biological function of gene expression is determined by the number of modified lysine residues and not by specific acetylated regions (Egger et al. 2004; Li et al. 2007).

Histone deacetylases (HDAC 1-11) are the enzymes responsible for the deacetylation of lysine residues and, thus, promote transcriptional repression. Increased expression of class I HDAC has been described in the resected temporal cortex of patients with DRE. Class I HDAC is notably expressed in neurons but not glia (Huang et al. 2012). Its overexpression has been associated with impaired memory formation and spatial learning, decreased dendritic arborization, diminished synaptic plasticity, and suppression of the expression of genes that regulate neuronal activity and synaptic remodeling (Guan et al. 2009).

In animal models of epilepsy and human tissue from patients with DRE, increased HDAC2 expression has been observed in the temporal neocortex (Huang et al. 2012). HDAC2 downmodulates the face of the benzodiazepine-sensitive GABAA α1 subunit (Enna 2007; Zhang et al. 2019) and the GluA2 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (Huang et al. 2002). Notably, the GluA2 subunit is responsible for calcium impermeability and modulates trafficking and tetramerization of the AMPA receptor (Greger et al. 2003; Wright and Vissel 2012). GluA2 subunit mutations are associated with several neurodevelopmental abnormalities and epileptic encephalopathies (Salpietro et al. 2019). This evidence suggests that epilepsy causes deacetylation of the GluA2 promoter, which decreases GluA2 subunit expression, leading to calcium-permeable AMPA receptors and, thus, enhancing glutamate neurotransmission (Pellegrini-Giampietro et al. 1997).

DNA methylation is a dynamic and reversible epigenetic change that induces gene silencing (Egger et al. 2004). DNA methyltransferases (DNMT) are a family of highly conserved enzymes that catalyze the addition of methyl groups to the CpG islands of promoter regions present in genomic DNA (Lyko 2018). Overexpression of DNMT1 and DNMT3 was reported in resected neocortical tissue from DRE patients (Zhu et al. 2012). DNMT1 conserved DNA methylation, whereas DNMT3 induced de novo DNA methylation (Lyko 2018). These results were consistent with those observed in autopsy brain samples from schizophrenia patients in which both DNMT1 and DNMT3 mRNAs were upregulated in GABAergic neurons (Zhubi et al. 2009).

Genome-wide methylation analysis of resected hippocampal tissue from DRE patients revealed 119 hypermethylated and 27 hypomethylated genes compared to autopsy samples. Gene ontology of the altered genes revealed alterations in developmental processes, cell development, differentiation, and death. Hypermethylated genes expressed in neurons in DRE patients included calcium, potassium, and sodium channels, proteins associated with neuronal maturation, and proteins related to synaptic transmission (Miller-Delaney et al. 2015).

Noncoding RNA transcripts do not encode proteins but modulate transcription by targeting transcriptional activators or repressors through regulating chromatin structure. Among the noncoding RNA, microRNAs (miRNAs) are typically between 21-28 nucleotides in length and act by targeting homologous sequences in the genome (Chen and Rajewsky 2007; Goodrich and Kugel 2006). Individual miRNAs are fine-tuning gene modulators as they can interfere with the expression of hundreds of proteins, although their effects are generally modest (Brennan and Henshall 2020).

In hippocampal tissue resected from patients with DRE, Dicer protein expression was reduced in samples with sclerosis but unchanged in those with moderate hippocampal pathology. Dicer is a protein that matures miRNAs, and its downregulation in DRE patients was associated with lower expression of several miRNAs, including miRNA-92a (McKiernan et al. 2012). Interestingly, miRNA-92a can downregulate AMPA receptor expression (Letellier et al. 2014). In epilepsy, downregulation of Dicer expression would induce a decrease in miRNA-92a and, thus, an increase in AMPA receptor expression, which could lead to hyperexcitability.

Overexpression of miRNA-134 has also been found in hippocampal tissue resected from DRE patients (Jimenez-Mateos et al. 2012; Reschke et al. 2017). These data suggest that miRNA-134 overexpression would impair synaptic plasticity. Overexpression of miRNA-134 was detected in hippocampal tissue from mice previously submitted to status epilepticus (Jimenez-Mateos et al. 2012, 2015; Reschke et al. 2017). Interestingly, miRNA-134 knockdown had an antiepileptogenic effect associated with decreased neuronal death and astrogliosis (Jimenez-Mateos et al. 2012). Similarly, miRNA-134 knockdown produced antiepileptogenic effects in rats subjected to electrical stimulation of the perforant pathway (Reschke et al. 2017). These data reveal that miRNA-134 is associated with the pathogenesis of seizures and epilepsy and is involved in brain excitability.

Furthermore, miRNA-155 overexpression in resected hippocampal tissue from DRE patients correlated with increased seizure frequency, sclerosis, and reduced probability of post-surgery seizure freedom (Huang et al. 2018). A similar increase of miRNA-155 was found in the hippocampus of rats with epilepsy (Huang et al. 2018). Notably, miRNA-155 was overexpressed 2.5-fold in the cerebrospinal fluid of patients whose epilepsy surgery did not modify the occurrence of seizures. Bioinformatic analysis revealed that miRNA-155 inhibits SCN1A gene expression (Zhang et al. 2018). The SCN1A gene encodes the pore-forming α-subunit of VGSC NAV1.1. The α-subunit forms the ion-conducting pore and contains voltage sensors that dictate part of its kinetics. This channel’s mutation has been associated with reduced excitability of GABAergic neurons, which preferentially express Nav1.1 (Catterall et al. 2010; Ragsdale 2008). This evidence suggests that epilepsy-induced increased expression of miRNA-155 may result in low SCN1A expression, thus reducing GABAergic neurotransmission.

7.8 Conclusions

Changes in ASMs targets involve structure, expression, oligomer formation, and membrane localization modifications. In addition, changes at the intracellular signaling level may also be involved, reducing the effects of ASMs, or even changing their pharmacological effects and facilitating neuronal excitability. Both structural and signaling alterations could explain why some patients develop the drug-resistant phenotype in epilepsy. It is essential to consider these alterations and search for new therapeutic strategies to control the pharmacoresistant condition in epilepsy.