Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca²⁺) channels and intracellular Ca²⁺ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca²⁺ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca²⁺ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca²⁺ concentration and Ca²⁺ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca²⁺ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca²⁺ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development.

Focal cortical dysplasia (FCD) type II is an epileptogenic malformation of the neocortex, as well as a leading cause of drug-resistant focal epilepsy in children and young adults. The synaptic dysfunctions leading to intractable seizures in this disease appear to have a tight relationship with the immaturity of GABAergic neurotransmission. The likely outcome would include hyperpolarizing responses upon activation of GABA_ARs. In addition, it is well-established that neuroinflammation plays a relevant role in the pathogenesis of FCD type II. Here, we investigated whether IL-1β, a prototypical pro-inflammatory cytokine, can influence GABAergic neurotransmission in FCD brain tissues. To this purpose, we carried out electrophysiological recordings on Xenopus oocytes transplanted with human tissues and performed a transcriptomics analysis. We found that IL-1β decreases the GABA currents amplitude in tissue samples from adult individuals, while it potentiates GABA responses in samples from pediatric cases. Interestingly, these cases of pediatric FCD were characterized by a more depolarized E_GABA and an altered transcriptomics profile, that revealed an up-regulation of chloride cotransporter NKCC1 and IL-1β. Altogether, these results suggest that the neuroinflammatory processes and altered chloride homeostasis can contribute together to increase the brain excitability underlying the occurrence of seizures in these children.

Tuberous sclerosis complex (TSC) is a rare multi-system genetic disorder characterized by a high incidence of epilepsy and neuropsychiatric manifestations known as tuberous-sclerosis-associated neuropsychiatric disorders (TANDs), including autism spectrum disorder (ASD) and intellectual disability (ID). MicroRNAs (miRNAs) are small regulatory non-coding RNAs that regulate the expression of more than 60% of all protein-coding genes in humans and have been reported to be dysregulated in several diseases, including TSC. In the current study, RNA sequencing analysis was performed to define the miRNA and isoform (isomiR) expression patterns in serum. A Receiver Operating Characteristic (ROC) curve analysis was used to identify circulating molecular biomarkers, miRNAs, and isomiRs, able to discriminate the development of neuropsychiatric comorbidity, either ASD, ID, or ASD + ID, in patients with TSC. Part of our bioinformatics predictions was verified with RT-qPCR performed on RNA isolated from patients’ serum. Our results support the notion that circulating miRNAs and isomiRs have the potential to aid standard clinical testing in the early risk assessment of ASD and ID development in TSC patients.

Background: The genetic disorder tuberous sclerosis complex (TSC) is frequently accompanied by the development of neuropsychiatric disorders, including autism spectrum disorder and intellectual disability, with varying degrees of impairment. These co-morbidities in TSC have been linked to the structural brain abnormalities, such as cortical tubers, and recurrent epileptic seizures (in 70–80% cases). Previous transcriptomic analysis of cortical tubers revealed dysregulation of genes involved in cell adhesion in the brain, which may be associated with the neurodevelopmental deficits in TSC. In this study we aimed to investigate the expression of one of these genes – cell-adhesion molecule contactin-3. Methods: Reverse transcription quantitative polymerase chain reaction for the contactin-3 gene (CNTN3) was performed in resected cortical tubers from TSC patients with drug-resistant epilepsy (n = 35, age range: 1–48 years) and compared to autopsy-derived cortical control tissue (n = 27, age range: 0–44 years), as well as by western blot analysis of contactin-3 (n = 7 vs n = 7, age range: 0–3 years for both TSC and controls) and immunohistochemistry (n = 5 TSC vs n = 4 controls). The expression of contactin-3 was further analyzed in fetal and postnatal control tissue by western blotting and in-situ hybridization, as well as in the SH-SY5Y neuroblastoma cell line differentiation model in vitro. Results: CNTN3 gene expression was lower in cortical tubers from patients across a wide range of ages (fold change = − 0.5, p < 0.001) as compared to controls. Contactin-3 protein expression was lower in the age range of 0–3 years old (fold change = − 3.8, p < 0.001) as compared to the age-matched controls. In control brain tissue, contactin-3 gene and protein expression could be detected during fetal development, peaked around birth and during infancy and declined in the adult brain. CNTN3 expression was induced in the differentiated SH-SY5Y neuroblastoma cells in vitro (fold change = 6.2, p < 0.01). Conclusions: Our data show a lower expression of contactin-3 in cortical tubers of TSC patients during early postnatal period as compared to controls, which may affect normal brain development and might contribute to neuropsychiatric co-morbidities observed in patients with TSC.