In vitro human cell culture models in a bench-to-bedside approach to epilepsy.

Epilepsy is the most common chronic neurological disease, affecting nearly 1%-2% of the world's population. Current pharmacological treatment and regimen adjustments are aimed at controlling seizures; however, they are ineffective in one-third of the patients. Although neuronal hyperexcitability was previously thought to be mainly due to ion channel alterations, current research has revealed other contributing molecular pathways, including processes involved in cellular signaling, energy metabolism, protein synthesis, axon guidance, inflammation, and others. Some forms of drug-resistant epilepsy are caused by genetic defects that constitute potential targets for precision therapy. Although such approaches are increasingly important, they are still in the early stages of development. This review aims to provide a summary of practical aspects of the employment of in vitro human cell culture models in epilepsy diagnosis, treatment, and research. First, we briefly summarize the genetic testing that may result in the detection of candidate pathogenic variants in genes involved in epilepsy pathogenesis. Consequently, we review existing in vitro cell models, including induced pluripotent stem cells and differentiated neuronal cells, providing their specific properties, validity, and employment in research pipelines. We cover two methodological approaches. The first approach involves the utilization of somatic cells directly obtained from individual patients, while the second approach entails the utilization of characterized cell lines. The models are evaluated in terms of their research and clinical benefits, relevance to the in vivo conditions, legal and ethical aspects, time and cost demands, and available published data. Despite the methodological, temporal, and financial demands of the reviewed models they possess high potential to be used as robust systems in routine testing of pathogenicity of detected variants in the near future and provide a solid experimental background for personalized therapy of genetic epilepsies. PLAIN LANGUAGE SUMMARY: Epilepsy affects millions worldwide, but current treatments fail for many patients. Beyond traditional ion channel alterations, various genetic factors contribute to the disorder's complexity. This review explores how in vitro human cell models, either from patients or from cell lines, can aid in understanding epilepsy's genetic roots and developing personalized therapies. While these models require further investigation, they offer hope for improved diagnosis and treatment of genetic forms of epilepsy.

Sulforaphane Ameliorates Metabolic Changes Associated With Status Epilepticus in Immature Rats.

Status epilepticus (SE) is a common paediatric emergency with the highest incidence in the neonatal period and is a well-known epileptogenic insult. As previously established in various experimental and human studies, SE induces long-term alterations to brain metabolism, alterations that directly contribute to the development of epilepsy. To influence these changes, organic isothiocyanate compound sulforaphane (SFN) has been used in the present study for its known effect of enhancing antioxidative, cytoprotective, and metabolic cellular properties via the Nrf2 pathway. We have explored the effect of SFN in a model of acquired epilepsy induced by Li-Cl pilocarpine in immature rats (12 days old). Energy metabolites PCr, ATP, glucose, glycogen, and lactate were determined by enzymatic fluorimetric methods during the acute phase of SE. Protein expression was evaluated by Western blot (WB) analysis. Neuronal death was scored on the FluoroJadeB stained brain sections harvested 24 h after SE. To assess the effect of SFN on glucose metabolism we have performed a series of 18F-DG µCT/PET recordings 1 h, 1 day, and 3 weeks after the induction of SE. Responses of cerebral blood flow (CBF) to electrical stimulation and their influence by SFN were evaluated by laser Doppler flowmetry (LDF). We have demonstrated that the Nrf2 pathway is upregulated in the CNS of immature rats after SFN treatment. In the animals that had undergone SE, SFN was responsible for lowering glucose uptake in most regions 1 h after the induction of SE. Moreover, SFN partially reversed hypometabolism observed after 24 h and achieved full reversal at approximately 3 weeks after SE. Since no difference in cell death was observed in SFN treated group, these changes cannot be attributed to differences in neurodegeneration. SFN per se did not affect the glucose uptake at any given time point suggesting that SFN improves endogenous CNS ability to adapt to the epileptogenic insult. Furthermore, we had discovered that SFN improves blood flow and accelerates CBF response to electrical stimulation. Our findings suggest that SFN improves metabolic changes induced by SE which have been identified during epileptogenesis in various animal models of acquired epilepsy.

Specific pathogenic mutations in the M3 domain of the GluN1 subunit regulate the surface delivery and pharmacological sensitivity of NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs.

Surface Expression, Function, and Pharmacology of Disease-Associated Mutations in the Membrane Domain of the Human GluN2B Subunit.

N-methyl-D-aspartate receptors (NMDARs), glutamate-gated ion channels, mediate signaling at the majority of excitatory synapses in the nervous system. Recent sequencing data for neurological and psychiatric patients have indicated numerous mutations in genes encoding for NMDAR subunits. Here, we present surface expression, functional, and pharmacological analysis of 11 de novo missense mutations of the human hGluN2B subunit (P553L; V558I; W607C; N615I; V618G; S628F; E657G; G820E; G820A; M824R; L825V) located in the pre-M1, M1, M2, M3, and M4 membrane regions. These variants were identified in patients with intellectual disability, developmental delay, epileptic symptomatology, and autism spectrum disorder. Immunofluorescence microscopy indicated that the ratio of surface-to-total NMDAR expression was reduced for hGluN1/hGluN2B(S628F) receptors and increased for for hGluN1/hGluN2B(G820E) receptors. Electrophysiological recordings revealed that agonist potency was altered in hGluN1/hGluN2B(W607C; N615I; and E657G) receptors and desensitization was increased in hGluN1/hGluN2B(V558I) receptors. The probability of channel opening of hGluN1/hGluN2B (V558I; W607C; V618G; and L825V) receptors was diminished ~10-fold when compared to non-mutated receptors. Finally, the sensitivity of mutant receptors to positive allosteric modulators of the steroid origin showed that glutamate responses induced in hGluN1/hGluN2B(V558I; W607C; V618G; and G820A) receptors were potentiated by 59-96% and 406-685% when recorded in the presence of 20-oxo-pregn-5-en-3ß-yl sulfate (PE-S) and androst-5-en-3ß-yl hemisuccinate (AND-hSuc), respectively. Surprisingly hGluN1/hGluN2B(L825V) receptors were strongly potentiated, by 197 and 1647%, respectively, by PE-S and AND-hSuc. Synaptic-like responses induced by brief glutamate application were also potentiated and the deactivation decelerated. Further, we have used homology modeling based on the available crystal structures of GluN1/GluN2B NMDA receptor followed by molecular dynamics simulations to try to relate the functional consequences of mutations to structural changes. Overall, these data suggest that de novo missense mutations of the hGluN2B subunit located in membrane domains lead to multiple defects that manifest by the NMDAR loss of function that can be rectified by steroids. Our results provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with hypofunction of the glutamatergic system.