WONOEP appraisal: Biomarkers and treatment strategies beyond the synapse

Abstract

Epilepsy is a heterogeneous neurological disorder affecting more than 70 million people worldwide, posing significant challenges for clinicians due to its complex etiology, diverse manifestations, variable treatment responses, and the inability to predict seizures or disease onset reliably. Despite advances in antiseizure medications, approximately 30% of patients remain treatment‐resistant, highlighting the urgent need for therapies with antiepileptogenic or disease‐modifying effects. To optimize and individualize strategies to predict and treat seizures and epilepsy, efforts to identify biomarkers of epilepsy risk, epileptogenesis, seizures, and therapy response are ongoing. This article reports key presentations and discussions from the 2023 Workshop on Neurobiology of Epilepsy (WONOEP XVII) in Kilkea, Ireland on novel epilepsy biomarkers and treatment strategies beyond the synapse and does not constitute a comprehensive review of biomarkers or treatment strategies. Much of the focus in epilepsy research has centered on identifying primarily neuronal processes or components. The 2023 WONOEP presentations discussed advances in plasma biomarkers for posttraumatic seizures and outcomes, perivascular spaces in posttraumatic epilepsy and poststroke epilepsy, imaging biomarkers of astrogliosis, and plasma microRNA biomarkers of intellectual disability and autism in tuberous sclerosis complex. Furthermore, research on immuno‐ and anti‐inflammatory therapies and blood–brain barrier in drug‐resistant focal epilepsies and infantile epileptic spasms syndrome was presented, as well as on the effects of antiseizure and cardioprotective medications on cardiac injury in temporal lobe epilepsy. The review also emphasizes the need for further interdisciplinary collaboration to accelerate the translation of these findings into clinical practice, ultimately improving outcomes and quality of life for people with epilepsy.

Key points.

• Plasma proteins, circulating miRNAs, extracellular vesicles, and neuroimaging are emerging epilepsy biomarkers.

• Novel therapeutic approaches for epilepsies target glia, immune pathways, and the blood–brain barrier.

• Integrating multiomics, digital health, and systems biology may speed biomarker discovery and personalized medicine.

• Emerging biomarkers for neuropsychiatric and systemic comorbidities highlight new opportunities for precision medicine in epilepsy.

1. INTRODUCTION

Biomarkers are invaluable in enhancing the diagnosis and treatment of epilepsy. As a heterogeneous condition with diverse underlying causes and clinical manifestations, epilepsy has long posed both diagnostic and therapeutic challenges. Biomarkers, which encompass a wide range of biological indicators, from genetic markers to neuroimaging findings and neuroelectric signals, offer a promising avenue for revolutionizing our approach to this complex disorder (as reviewed in Ahmad et al. ¹ ). Furthermore, new work is now expanding on circulating biomarkers beyond the neuronal components, including S100 calcium‐binding protein B (S100B) ² and ubiquitin C‐terminal hydrolase 1 (UCHL1). ³ , ⁴

These biomarkers not only hold the potential to provide early and accurate diagnostic information but also play a crucial role in tailoring personalized treatment strategies. By identifying specific biomarkers associated with epilepsy subtypes, drug responses, or disease progression, clinicians can make more informed and personalized decisions regarding medication selection and dosage adjustments. ⁵ Furthermore, the ongoing research into biomarkers may unlock novel insights into the pathophysiological mechanisms underlying epilepsy, paving the way for the development of targeted therapies and ultimately improving the quality of life for individuals living with this condition.

In addition to its intrinsic complexity, epilepsy often brings with it a host of underexplored comorbidities, such as depression ⁶ , ⁷ and cardiovascular issues, ⁸ , ⁹ , ¹⁰ which significantly impact the overall well‐being of affected individuals. Certain conditions may also share underlying pathobiological mechanisms. These comorbidities, although sometimes overshadowed by the focus on seizure control, are of great importance in the comprehensive management of epilepsy. Depression, for instance, can be both a consequence of the chronic burden of living with seizures and a contributor to their frequency and severity. ⁶ Similarly, epilepsy has been associated with an increased risk of cardiovascular problems, including hypertension and arrhythmias, which can further complicate the clinical picture. ¹¹ , ¹² In this context, biomarkers for comorbidities like depression and cardiovascular issues become essential. These biomarkers offer the potential to identify at‐risk individuals early in their disease trajectory, enabling timely intervention and tailored treatment strategies. For depression, biomarkers might include specific neurochemical markers in the brain or blood, which could help predict the development of depressive symptoms in epilepsy patients. ¹³ , ¹⁴ Likewise, in the realm of cardiovascular issues, biomarkers may encompass genetic indicators, inflammation markers, or specific cardiac parameters that identify those at heightened risk. ¹⁵ , ¹⁶ By implementing such biomarker‐driven approaches, clinicians can not only enhance the overall quality of care but also better address the intertwined complexities of epilepsy and its comorbidities, ultimately improving the patients' holistic well‐being and overall health outcomes. In essence, the exploration of biomarkers in these underexplored comorbidities represents a crucial step toward a more comprehensive and personalized management of epilepsy, aligning with the broader goal of optimizing patient care. Furthermore, the treatment for comorbidities in the epilepsy population is rarely discussed. The pharmacodynamic properties of certain antiseizure medications (ASMs) can affect the progression of common comorbidities in epilepsy. For instance, hypertension, dyslipidemia, and ischemic stroke ⁴ are the most common comorbidities in hospitalized elderly patients with epilepsy. ¹⁷ , ¹⁸ , ¹⁹ Therefore, the indirect effect of ASMs on the cardiovascular system and potential drug–drug interaction should be taken into consideration in certain populations.

The Workshop on Neurobiology of Epilepsy (WONOEP XVII), organized in 2023 by the Neurobiology Commission of the International League Against Epilepsy (ILAE), focused on the extracellular space in epilepsy. One of the main areas for discussion was biomarkers and treatment strategies beyond the synapse. Here, we summarize the presentations and relevant discussions centered around novel biomarker research and treatment strategies, while emphasizing the importance of considering comorbidities. The scope of this article reflects selected topics presented and discussed at WONOEP XVII and is not intended to serve as a comprehensive review of all biomarkers or therapeutic approaches in epilepsy. Moreover, it underscores the significance of not only focusing on seizure control but also comprehensively addressing comorbid conditions like depression, cardiovascular issues, and other neuropsychiatric comorbidities, recognizing their intricate interplay with epilepsy and the potential for biomarkers to enhance our understanding and management of these often‐overlooked aspects of the condition.

2. BIOMARKERS FOR EPILEPSY DIAGNOSIS AND PROGNOSIS

Biomarkers play a crucial role in advancing the diagnosis and prognosis of epilepsy. These biological indicators, which can be detected through various methods such as electroencephalography (EEG), neuroimaging, or molecular assays, provide valuable insights into the underlying mechanisms of epilepsy.

2.1. EEG biomarkers

EEG biomarkers are fundamental tools in the diagnosis, monitoring, and understanding of epilepsy. Prognostic models using EEG measures may be helpful in predicting ASM efficacy. ²⁰ EEG measures the weak electrical signals of the brain detectable through electrodes placed on the scalp, capturing patterns that reflect neuronal firing. ²¹ In epilepsy, abnormal electrical discharges, particularly during seizures, provide insight into the condition. ²² A key EEG biomarker in epilepsy is the interictal/sporadic epileptiform discharge (IED), ²³ which represents abnormal brain activity occurring in seizure‐free intervals. IEDs, such as spikes, sharp waves, and spike–wave complexes, may suggest epileptic brain activity and may serve as indicators of an underlying seizure disorder even when seizures themselves are not recorded during EEG monitoring (Table S1). ²⁴ For example, in focal epilepsy, IEDs may arise from specific regions of the brain and may help localize the epileptic focus, providing crucial information for surgical planning in drug‐resistant cases. ²⁵ In generalized epilepsies, IEDs can appear as generalized spike–wave discharges or polyspikes, helping to distinguish generalized epilepsy syndromes from focal epilepsies. ²⁶ High‐frequency oscillations (HFOs) can be classified into ripples (80–200 Hz) and fast ripples (200–500 Hz) based on their spectral characteristics. Certain HFOs, including oscillatory events in the gamma range (30–100 Hz), have been linked to epileptogenic cortex and may reflect neuronal ensembles capable of generating seizures. ²⁷ , ²⁸ , ²⁹ , ³⁰ However, most robust evidence supporting HFOs as biomarkers comes from intracranial EEG studies, where the proximity to cortical sources allows reliable detection. By contrast, scalp recordings of HFOs face ongoing technical challenges, such as reduced signal amplitude, interference from physiological artifacts, and limitations in electrode spatial resolution. Consequently, although HFOs detected during invasive monitoring have shown utility in identifying surgical targets in drug‐resistant epilepsy (DRE), their role in noninvasive clinical practice remains an active area of research rather than established routine. These challenges underscore the need for complementary approaches that extend beyond conventional EEG, including advanced signal‐analysis methods, multimodal neuroimaging, and circulating biomarkers. When considered together, IEDs, HFOs, and changes in frequency‐band dynamics represent some of the most promising EEG‐based signatures under active investigation to support diagnosis and treatment planning, and ongoing research aims to improve their reliability and clinical applicability.

2.2. Novel neuroimaging techniques to advance biomarker discovery in epilepsy

2.2.1. Neuroimaging the perivascular space to highlight individuals at risk of developing epilepsy

Neuroimaging biomarkers can play an important role in the diagnosis and prognosis of epilepsy, complementing traditional methods such as EEG. ³¹ Advanced neuroimaging techniques like magnetic resonance imaging (MRI) provide detailed structural and functional information that aids in the localization and characterization of epileptic foci. By combining neuroimaging with EEG findings, clinicians can more accurately identify the regions of the brain responsible for seizures, particularly in cases where EEG alone might not offer sufficient clarity.

In the context of epilepsy resulting from traumatic brain injury (TBI), neuroimaging biomarkers offer critical insights into posttraumatic epilepsy (PTE) and associated cognitive impairments. TBI is a leading cause of emergency visits and hospitalizations, with a significant proportion of moderate to severe cases developing epilepsy over time. ³² Advanced imaging techniques, particularly MRI, have identified enlarged perivascular spaces (ePVSs) as potential biomarkers for distinguishing patients who develop PTE from those who do not. MRI studies have revealed differences in ePVSs between chronic TBI patients with and without PTE, suggesting a link between these structural changes and the development of epilepsy. ³³ Furthermore, in cases of poststroke epilepsy (PSE), asymmetry in perivascular spaces observed on high‐resolution MRI has been identified as a predictor of PSE, offering a novel approach to identifying high‐risk patients and guiding individualized treatment strategies. ³⁴ , ³⁵ The etiological relationship between these acquired epilepsies and perivascular spaces is unclear. ³⁶ However, ePVSs are often taken as a marker of glymphatic system dysfunction, which could affect the clearance of metabolic solutes and waste products. Given that posttraumatic and poststroke seizures are associated with increased levels of glutamate ³⁷ and inflammatory markers, ³⁸ , ³⁹ it may be that the impaired clearance of these extracellular products contributes to a lowered seizure threshold.

2.2.2. Visualizing the molecular processes of astrogliosis to advance biomarker discovery in PTE

Beyond structural biomarkers, functional imaging with positron emission tomography (PET) has also shown promise in epilepsy prognosis. This noninvasive imaging technique enables the visualization of molecular processes in the human body by utilizing radiotracers that bind to specific molecular targets. To date, several PET radiotracers have been developed for molecular imaging of the brain, particularly to investigate neuroinflammation in brain diseases, in both preclinical and clinical research. ⁴⁰ , ⁴¹ , ⁴² , ⁴³ , ⁴⁴ , ⁴⁵ , ⁴⁶ However, many of these tracers face limitations when examining neuroinflammation, such as reduced binding due to genetic polymorphisms in target proteins, nonspecific binding, and limited clinical utility due to the short half‐lives of the radioisotopes. ⁴¹ , ⁴² , ⁴³ , ⁴⁴ , ⁴⁵ Consequently, the development of novel PET radiotracers for neuroinflammation has become a key focus in the field. For example, innovative tracers like [¹⁸F]‐fluorodeprenyl‐D2 ([¹⁸F]‐FDED), an alternative to [¹¹C]‐L‐deprenyl, both of which target monoamine oxidase‐B (MAO‐B) upregulated in activated astrocytes, offer a longer half‐life, making [¹⁸F]‐FDED more suitable for widespread clinical use. ⁴⁰ , ⁴¹ , ⁴⁷

Complementary to PET‐based approaches, magnetic resonance spectroscopy (MRS) and chemical exchange saturation transfer MRS (CEST‐MRS) enable noninvasive measurement of brain metabolites and neurotransmitter concentrations, including glutamate and γ‐aminobutyric acid (GABA). GluCEST imaging has shown promise for lateralizing temporal lobe epilepsy foci based on glutamate level changes, and GABA‐MRS can quantify inhibitory neurotransmitter deficits, together offering a window into the excitatory–inhibitory imbalance implicated in epileptogenesis. ⁴⁸ Furthermore, recent advances in PET radiotracer development have expanded the toolkit for imaging neuroinflammation beyond translocator protein (TSPO)‐based ligands and MAO‐B tracers. Morgan et al. ⁴⁹ demonstrated that the P2X7 receptor radiotracer ¹⁸F‐JNJ‐64413739 showed increased brain uptake correlating with status epilepticus (SE) severity in mice. Critically, the radioligand was also applied to ex vivo human brain tissue from patients with drug‐resistant temporal lobe epilepsy, where autoradiographic binding was characterized across cortical regions, ⁵⁰ supporting P2X7R‐based PET as a promising translational approach for imaging neuroinflammation in epilepsy.

Unpublished data presented at WONOEP demonstrated that the implementation of [¹⁸F]‐FDED PET in preclinical TBI showed promising results of the novel tracer, with rapid uptake of the tracer into the brain and increased tracer uptake in post‐TBI rats compared to sham animals, indicating increased astrogliosis. Although these findings provide valuable insights into early astrogliosis in response to acute injury, further studies are underway to map astrogliosis long‐term, as well as relate astrogliosis to seizure burden and treatment response, thereby validating the utility of PET imaging as a biomarker of brain astrogliosis.

In summary, imaging biomarkers represent emerging tools for characterizing brain alterations that contribute to epileptogenesis following TBI or stroke. Although promising, their prognostic use remains under active investigation and has not yet become routine in clinical decision‐making. MRI‐ and PET‐based measures can reveal complementary aspects of network disruption, neuroinflammation, glial activation, and metabolic imbalance, thereby improving our understanding of mechanisms that may predispose individuals to develop epilepsy. A comparative overview of MRI and PET biomarkers is provided in Table 1.

TABLE 1.

Comparison of MRI‐ and PET‐based imaging biomarkers in epilepsy.

Feature	MRI	PET
Biological target	Structural abnormalities (e.g., cortical malformations, hippocampal sclerosis); microstructure (DTI); neurotransmitter and metabolite concentrations (MRS, CEST‐MRS; e.g., glutamate, GABA, NAA, glutathione); functional connectivity (fMRI)	Neurotransmitter systems (e.g., GABA, glutamate, dopamine); glucose metabolism (FDG‐PET); neuroinflammation via TSPO ligands, MAO‐B tracers (e.g., [18F]‐FDED), and P2X7 receptor radiotracers (e.g., [18F]‐JNJ‐64413739)
Strengths	High spatial resolution; widely available; noninvasive; no ionizing radiation; standard of care for presurgical evaluation; MRS/CEST‐MRS enables noninvasive quantification of excitatory–inhibitory balance	Provides molecular specificity; sensitive to early pathophysiological changes; enables in vivo visualization of receptor binding and inflammatory processes; allows quantitative measurement of metabolic and molecular processes
Limitations	Primarily indirect measures of neuronal activity; findings can be nonspecific; deep or subtle abnormalities may remain undetected; MRS has limited spatial resolution and requires specialized pulse sequences (e.g., MEGA‐PRESS for GABA)	Costly; involves exposure to ionizing radiation; limited availability; lower spatial resolution than MRI; requires complementary imaging for anatomical localization; challenging to quantify; novel tracers (TSPO, MAO‐B, P2X7R) face issues including genetic polymorphisms affecting binding and limited clinical validation
Translational readiness	High: structural MRI, fMRI, DTI routinely used in clinical practice; MRS established in research settings with growing clinical adoption	Moderate: FDG‐PET established in surgical workup; novel tracers (e.g., TSPO, MAO‐B, P2X7R) still in preclinical/early clinical phases
Key references	31, 33, 34, 35, 36, 48	41, 44, 45, 47, 49, 50

Abbreviations: [¹⁸F]‐FDED, [¹⁸F]‐fluorodeprenyl‐D2; CEST, chemical exchange saturation transfer; DTI, diffusion tensor imaging; FDG‐PET, fluorodeoxyglucose positron emission tomography; fMRI, functional MRI; GABA, γ‐aminobutyric acid; MAO‐B, monoamine oxidase‐B; MEGA‐PRESS, Meshcher–Garwood point resolved spectroscopy; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; NAA, N‐acetylaspartate; PET, positron emission tomography; TSPO, translocator protein.

2.3. Circulating biomarkers

In addition to brain imaging biomarkers, circulating molecular biomarkers are emerging as promising tools for assessing injury severity and neuroinflammatory responses following TBI. However, their potential for predicting seizure development or treatment response remains under investigation and has not yet been validated in clinical practice. ⁵ Recent research has explored innovative molecular biomarkers that can serve as early indicators of seizure risk and treatment response (Table S2).

In a preclinical study using the lateral fluid percussion injury (LFPI) model in adult rats, a panel of plasma protein biomarkers was identified, which revealed predictive markers for early seizure occurrence and exposure to ASM, such as levetiracetam. ⁵¹ The panel used by the investigators was comprised of inflammatory, cellular injury, and neurodegeneration markers and was assayed using a reverse‐phase protein microarray. This was used to identify changes in the expression of these markers in the plasma at different timepoints after LFPI, while the rats received prophylactic levetiracetam treatment, as done in the clinical setting, or vehicle. Rats were tracked with video‐EEG to monitor for ongoing seizures. Using machine learning, subsets of plasma biomarkers were identified that differentiated rats treated with levetiracetam (HMGB1, phosphorylated tau protein [pTAU‐Thr²³¹], UCHL1), early posttraumatic seizures (pTAU‐Thr²³¹, UCHL1 in vehicle‐treated rats; interferon gamma in levetiracetam‐treated rats), and prior craniotomy surgery in sham or LFPI rats (pTAU‐Thr²³¹, S100B). The study highlighted that many clinically relevant factors may affect the expression of biomarkers and predictive models need to consider such variables to improve accuracy and remove confounders. ⁵²

Beyond posttraumatic contexts, circulating cytokines and chemokines have emerged as clinically actionable biomarkers in other severe epilepsy presentations. In a landmark study, Hanin et al. ⁵³ profiled serum and cerebrospinal fluid (CSF) cytokines in patients with new onset refractory SE (NORSE), including its subtype febrile infection‐related epilepsy syndrome (FIRES). Elevated levels of proinflammatory mediators including IL‐6, TNF‐α, IL‐8, CCL2, and CXCL10 were identified in both serum and CSF, with specific cytokine signatures predicting functional outcomes and treatment response. Notably, cytokine levels decreased following immunotherapy initiation, supporting their use not only as prognostic biomarkers but also as pharmacodynamic markers to monitor treatment efficacy. ⁵³ This body of work represents one of the most advanced examples of circulating biomarker‐guided therapy in epilepsy to date, and underscores the potential for inflammatory profiling to inform precision treatment strategies in severe epileptic encephalopathies.

Another promising development in the field of molecular biomarkers is the study of microRNAs (miRNAs). These small, noncoding RNA molecules regulate gene expression and have been implicated in a wide range of neurological disorders, including epilepsy. In the context of epilepsy, miRNAs are being explored as both diagnostic and prognostic biomarkers due to their ability to reflect changes in brain activity and inflammation. ⁵⁴ , ⁵⁵ Importantly, cellular damage or stress causes the release of miRNAs from cells, including passive (lytic) release and via controlled pathways within extracellular vesicles. A recent preclinical study demonstrated that a portion of circulating miRNAs originate from neurons and glia in mice after chemically induced SE. ⁵⁶ This suggests brain‐originated miRNAs reach the circulation, whereupon they can be measured, supporting a pathophysiological basis for these molecules as mechanistic biomarkers. For example, blood levels of miRNAs such as miR‐134 and miR‐146a have been found to be dysregulated in patients with epilepsy and animal models of the disease, suggesting their potential as biomarkers for seizure activity and disease progression. ⁵⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶² Elevated levels of miR‐134 have been associated with seizure onset in experimental models, and miR‐146a is believed to play a role in neuroinflammation, a key process in epileptogenesis. ⁶³ Limitations have emerged, however, in the diagnostic value of plasma miRNAs for forecasting epilepsy after TBI. Two recent studies reported that although several brain‐enriched miRNAs could differentiate rats with TBI from controls, none had predictive value for the later development of epilepsy. ⁶⁴ , ⁶⁵ Moreover, despite their potential, molecular biomarkers, including miRNAs, face significant challenges in clinical translation. A major issue is the lack of robustness and reproducibility across studies. Biomarker research in epilepsy is often complicated by the variability in patient populations, the heterogeneity of epileptic syndromes, and inconsistent methodologies for identifying and validating biomarkers. This variability hinders the reproducibility of findings and makes it difficult to establish standardized molecular biomarkers that can be applied reliably in clinical practice. Additionally, the multifactorial nature of epilepsy, shaped by genetic, environmental, and individual factors, adds another layer of complexity to biomarker discovery and validation. To overcome these challenges, ongoing rigorous standards for biomarker validation are required. ⁵⁴ , ⁵⁵ Promisingly, these have been incorporated in recent studies on plasma miRNAs, ⁶⁴ , ⁶⁵ and continuing to apply standardized protocols for molecular biomarker research, conducting multicenter validation studies, and ensuring adherence to strict quality control measures are essential to improve the reliability and clinical relevance of these biomarkers. Ultimately, reproducibility across laboratories and large‐scale multicenter validation remain among the greatest hurdles to overcome before miRNAs and other molecular biomarkers can be confidently implemented in clinical practice. Without such coordinated efforts, the field risks generating fragmented findings that fail to translate into tangible diagnostic tools.

3. BIOMARKERS FOR COMORBIDITIES IN EPILEPSY

In addition to seizures, epilepsy is associated with numerous comorbidities, including neuropsychiatric and cognitive impairments, peptic ulcers, arthritis, respiratory and cardiovascular complications, sleep disorders, ⁶⁶ , ⁶⁷ and in extreme cases, death. ⁶⁸ These comorbidities can have a multifactorial relationship with epilepsy, where they may either contribute to the development of epilepsy, arise as a consequence of epilepsy, share a mechanistic basis or common risk factors, or exist in a bidirectional relationship with epilepsy. ⁶⁸ Approximately half of all adults with active epilepsy have at least one comorbid medical disorder, and the prevalence of comorbidities among individuals with epilepsy is up to eight times higher than in the general population. ⁶⁸ As summarized in Table S3, there is a growing body of research focused on identifying biomarkers associated with epilepsy‐related comorbidities.

The presence of comorbidities has significant implications for the management of epilepsy, influencing both treatment decisions and prognosis. For example, in elderly epilepsy patients, it is common to use ASMs alongside treatments for cardiovascular conditions. However, the effects of epilepsy and its treatments on the cardiovascular system remain underexplored in clinical studies. Clinical studies have shown that generalized convulsive seizures can cause cardiac stress in approximately 25% of patients, even in the absence of overt cardiac symptoms. ¹¹ , ⁶⁹ Using serum biomarkers like cardiac troponins and copeptin, researchers have observed elevated levels of these markers in patients postseizure, indicating potential cardiac stress. ⁶⁹ In post‐SE animal models, ¹² elevated troponin levels have been observed at different timepoints and have been linked to the frequency of seizures, suggesting that epilepsy can lead to stressed cardiomyopathy (unpublished work, Z.L). In patients with convulsive SE, increased cardiac injury markers are associated with worse clinical outcomes and higher mortality risk, ⁷⁰ making cardiac biomarkers valuable tools for short‐term prognosis and risk assessment in epilepsy patients. ⁷¹ , ⁷² Previous animal studies have demonstrated that the short‐term administration of beta‐blockers either prior to the induction of SE ⁷³ or immediately after SE ⁷⁴ has been linked to a decrease in cardiac troponin I levels, altered cardiac electrophysiological abnormalities, and reduced life‐threatening arrhythmias. This supports the hypothesis that recurrent epileptic seizures result in sympathetic activation and catecholamine surges, leading to stress‐related cardiomyopathy. ⁷⁵

Besides cardiac biomarkers, serum‐based biomarkers may also be useful to aid standard clinical testing in early risk assessment of comorbidities in epilepsy. ⁶⁵ , ⁷⁶ , ⁷⁷ For instance, miRNAs, which regulate gene expression and are involved in various diseases as mentioned previously, have shown promise as biomarkers for comorbidities in epilepsy. In young children with tuberous sclerosis complex (TSC; <4 months of age), several miRNAs have been identified as early indicators of neurodevelopmental comorbidities such as autism spectrum disorder and intellectual disability. ⁷⁸ By detecting these miRNAs early, clinicians can better predict the development of these conditions and guide interventions to improve social and behavioral outcomes for affected children.

Therefore, expanding epilepsy research to focus on identifying biomarkers for comorbidities will enhance both diagnosis and prognosis. These biomarkers help with determining more comprehensive care, allowing clinicians to predict and manage not only seizures but also the wide range of comorbid conditions that can complicate the lives of individuals with epilepsy. These emerging markers provide an opportunity for holistic patient care, where comorbidity management is guided by biomarker‐informed precision medicine. Incorporating such biomarker strategies into clinical workflows could ultimately shift the paradigm from reactive treatment to proactive prevention, improving both seizure control and long‐term health outcomes for people with epilepsy.

4. TREATMENT STRATEGIES BEYOND THE SYNAPSE

The current treatment landscape for epilepsy primarily focuses on balancing excitatory and inhibitory neurotransmission. Most available ASMs aim to manage the symptoms by modulating the neural pathways involved in seizure generation. These mechanisms include the modulation of voltage‐gated ion channels, ⁷⁹ enhancing GABA‐mediated inhibition, inhibiting ionotropic glutamate receptor‐mediated excitations, and directly modulating the synaptic release machinery. ⁸⁰ , ⁸¹ However, although these treatments can help alleviate the symptoms of epilepsy, they fall short in targeting the mechanisms driving epileptogenesis and do not change the molecular or neuronal network disturbances responsible for the enduring hyperexcitability in established epilepsy. Despite advances in pharmacotherapy, approximately one third of patients continue to suffer from DRE, characterized by persistent seizures even with appropriate medication trials. ⁸² Moreover, individuals with DRE are more prone to epilepsy‐related comorbidities, such as cognitive impairments, mood disorders, and cardiovascular issues, further complicating their management. ⁸³

Beyond these examples, a growing body of preclinical literature has identified therapeutic targets that operate outside conventional synaptic mechanisms (Figure 1). The mTOR pathway, dysregulated in tuberous sclerosis complex and other epilepsies, represents one of the most extensively studied nonsynaptic targets; rapamycin and its analogue everolimus have demonstrated antiepileptogenic effects across multiple models, and everolimus is now clinically approved as adjunctive therapy for seizures in TSC. ⁸⁴ Blood–brain barrier disruption and the consequent extravasation of serum albumin into the brain parenchyma activate astrocytic TGF‐β/ALK5 signaling, driving neuroinflammation, impaired glutamate homeostasis, and ultimately epileptogenesis. Losartan, an angiotensin II receptor blocker, has been shown to prevent acquired epilepsy via suppression of this pathway in preclinical models, ⁸⁵ and retrospective clinical data suggest reduced epilepsy incidence among hypertensive patients receiving angiotensin receptor blockers. ⁸⁶ Targeting the IL‐1β biosynthesis pathway via caspase‐1 inhibition with VX‐765, in combination with IL‐1 receptor antagonism (anakinra), has shown antiepileptogenic effects in post‐SE models. ⁸⁴ The S1P receptor modulator fingolimod has demonstrated disease‐modifying effects in models of both acquired and genetic epilepsy through anti‐inflammatory mechanisms including reduction of TNF‐α, IL‐1β, and microglial activation. ⁸⁷ Additionally, activation of the Nrf2 pathway by sulforaphane, alone or in combination with N‐acetylcysteine, has shown promise in reducing oxidative stress‐mediated epileptogenesis. ⁸⁴ Collectively, these examples illustrate the breadth of nonsynaptic therapeutic strategies under preclinical investigation (Table S4), although important translational gaps remain, including limited dose–response data, uncertain brain target engagement, and the need for treatment protocols that can be realistically applied in clinical settings. ⁸⁴ , ⁸⁸

FIGURE 1.

Pathophysiological mechanisms and therapeutic targets beyond the synapse in epilepsy. Schematic overview is shown of interconnected nonsynaptic mechanisms contributing to epileptogenesis and seizure persistence. In addition to classical antiseizure medications (ASMs) targeting synaptic components (center; e.g., voltage‐gated Na⁺ channels, T‐type Ca²⁺ channels, SV2A, γ‐aminobutyric acid type A [GABA_A], and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid [AMPA]/kainate receptors), multiple extrasynaptic processes are implicated. These include (1) neuroinflammation and microglial activation, (2) astrogliosis and astrocytic dysfunction, (3) blood–brain barrier disruption, (4) mTOR pathway dysregulation, (5) oxidative stress and mitochondrial dysfunction, (6) peripheral/systemic contributions, (7) glymphatic system impairment, and (8) epigenetic modifications. Each mechanism is associated with potential biomarkers and therapeutic targets, highlighting opportunities for disease‐modifying strategies. Arrows indicate crosstalk between pathways, emphasizing their interdependence in epilepsy pathophysiology. An artificial intelligence‐assisted tool (Perplexity) was used to support the design and visual layout of this figure. The authors reviewed, edited, and validated all content and take full responsibility for its scientific accuracy. [¹⁸F]‐FDED, [¹⁸F]‐fluorodeprenyl‐D2; ACTH, adrenocorticotropic hormone; ePVS, enlarged perivascular spaces; HPA, hypothalamic–pituitary–adrenal; MAO‐B, monoamine oxidase‐B; MRI, magnetic resonance imaging; ROS, reactive oxygen species.

Given the limitations of ASMs, there is a growing need to develop treatment strategies that go beyond symptomatic control and address the broader pathophysiology of epilepsy. Prolonged use of ASMs, particularly in DRE, may be associated with increased risks of adverse effects and potential interactions with medications used to treat comorbid conditions, highlighting the importance of careful monitoring. ⁸⁹ These issues highlight the necessity for therapies that not only target seizure reduction but also modify the progression of epilepsy and its associated comorbidities. Although emerging preclinical evidence suggests that epilepsy prevention (i.e., antiepileptogenesis) ⁷⁹ , ⁹⁰ and disease modification ⁸¹ , ⁹¹ are possible, and clinical trials are ongoing, ⁹² it is too early to know to what extent these findings will translate to the clinic. To fast‐track the development and repurposing of truly disease‐modifying treatments, a deep understanding of the pathophysiological mechanisms underlying DRE and its resistance to ASMs is essential.

The rapidly advancing knowledge about patient populations with genetic causes of epilepsy is leading to identification of disease pathways and targeted treatments. ⁵ , ⁸¹ , ⁹³ Emerging precision approaches include a multitude of modalities, including new chemical entities, new biological entities, antisense oligonucleotides, and gene therapies. ⁸⁰ The epilepsy field is undergoing significant changes, and refocusing on understanding drivers of intrinsic disease severity will benefit the development of treatments with novel mechanisms of action. ⁹⁴ Why do some patients become seizure‐free on ASM, whereas others do not? If we consider intrinsic epilepsy severity, determined by an interplay of genetic and acquired factors, homeostatic responses, and the epileptic perturbation itself, compounding into drug resistance, these different factors become levers to potentially intercept drug resistance. An interesting avenue is to consider DRE as the outcome of failing homeostatic mechanisms. ⁹⁵ , ⁹⁶ Homeostasis fails to maintain brain stability within boundaries that respond to standard symptomatic treatment. Patient‐centric research, including human genetics and the use of brain samples from patients to investigate the pathophysiological mechanism beyond the synapse, will be critical to identify these biological processes that are driving the varied severity of epilepsy in patients.

4.1. Systems biology approaches to identify and develop novel treatment strategies

In recent years, integrating bioinformatics and digital frameworks has played an important role in searching for new therapeutic targets in epilepsy. Advances in computational biology allow researchers to analyze large datasets from genomic, transcriptomic, and proteomic studies to identify novel molecular pathways involved in seizure generation and drug resistance. ⁴⁸ , ⁶⁸ , ⁷¹ , ⁷⁹ , ⁸⁰ Bioinformatics tools enable the mapping of complex biological networks, revealing potential targets that may have been previously overlooked. ⁵ , ⁸¹ , ⁹⁷ A recent example is the use of correlated gene networks to identify upstream regulators in disease conditions based on the analysis of hundreds of individual brain samples from patients with different types of structural epilepsy. ⁹⁸ This analysis confirmed established hallmarks of epilepsy biology and uncovered a detailed molecular disease map for future approaches. These digital approaches can accelerate the drug discovery process by simulating target interactions and predicting their effects on the pathways underlying epilepsy. Although this approach allows the generation and refinement of novel hypotheses, the need for laboratory experiments has not been circumvented to date. Challenges in drug discovery for epilepsy include deep understanding of disease biology, a lack of high‐throughput screening cascades encompassing disease complexity, and the demonstration of clinical benefit of targeting these novel biological pathways. There is a need for more efficient approaches to validate artificial intelligence (AI)‐derived hypotheses coming from big datasets. Linking observations at different scales from gene to disease pathophysiology and clinical benefit will be essential to bridge the translational gap. ⁵² Integrating omics and systems biology frameworks into epilepsy research offers exciting new opportunities for identifying nonsynaptic targets, immune‐modulatory pathways, and innovative strategies that could circumvent traditional pharmacological challenges. ⁵

Machine learning algorithms and AI are increasingly being utilized to predict patient responses to existing ASMs and to propose tailored treatments based on individual genetic and clinical profiles. ⁵² , ⁹⁹ These technologies provide valuable insights into the heterogeneity of DRE, allowing for a more personalized approach to treatment. The integration of these digital frameworks into epilepsy research opens new possibilities for identifying nonsynaptic targets, immune‐modulatory pathways, and other novel approaches that could bypass traditional pharmacological challenges.

Furthermore, in exploring new treatment options for DRE, one key consideration is how therapies interact with the brain. Candidate therapeutics for epilepsies are traditionally designed so that they can effectively cross the blood–brain barrier (BBB) and target and modify pathogenic mechanisms of epilepsies. However, emerging research suggests that in some instances, direct brain access may not be necessary for achieving therapeutic effects (unpublished work, S.L.M, A.S.G), and the therapeutic effects of certain treatments for neurological disorders may be indirectly achieved through modification of peripheral or systemic pathogenic processes that eventually affect brain function. In a preclinical model of infantile epileptic spasms syndrome (IESS), systemic administration of the anti‐inflammatory agent anakinra (interleukin 1 receptor antagonist), demonstrated disease‐modifying effects, such as improvements in motor skills and cognitive function (unpublished work, S.L.M, A.S.G). Although anakinra typically exhibits limited access to the central nervous system under physiological conditions, clinical and experimental evidence indicates that its central penetration can increase when the BBB is compromised, allowing greater therapeutic engagement with neuroinflammatory pathways. ¹⁰⁰ Thus, both peripheral immune modulation and context‐dependent central effects may contribute to its mechanisms of action. Clinically, anakinra has been successfully used in patients with FIRES or NORSE, supporting further investigation of IL‐1 signaling as a therapeutic target in severe epileptic encephalopathies. ⁹ , ¹⁰¹ In addition, the therapeutic effects of the peptide‐hormone adrenocorticotropic hormone (ACTH) in the treatment of IESS ¹⁰² , ¹⁰³ , ¹⁰⁴ are thought to at least partially be an indirect effect of glucocorticoids released from the adrenal glands. However, adrenalectomy did not prevent the ACTH‐mediated regulation of epileptic spasms, suggesting that additional pathways may be implicated. ¹⁰⁵ Increasing evidence that ACTH may modify peripheral inflammatory responses associated with IESS is emerging. ¹⁰⁶ , ¹⁰⁷

Taken together, treatment strategies beyond the synapse are critical for addressing the limitations of current ASM treatment, especially in DRE. By focusing on novel therapeutic targets, such as immunomodulation, neuroinflammation, and brain–body interactions, researchers and clinicians can develop more comprehensive therapies to manage epilepsy, offering new options to patients who remain unresponsive to standard treatments.

5. CONCLUSIONS AND FUTURE DIRECTIONS

In conclusion, biomarkers have emerged as pivotal tools in transforming the diagnosis, prognosis, and treatment of epilepsy. Their ability to provide early insights into the complex mechanisms of epilepsy, as well as its associated comorbidities, holds immense potential for improving patient outcomes. By identifying molecular, imaging, and serum‐based biomarkers, clinicians can enhance the precision of epilepsy diagnosis, predict treatment responses, and offer personalized care tailored to the specific needs of each patient. In particular, biomarkers such as IEDs, miRNAs, and imaging markers like ePVSs are being investigated for their potential relevance in epilepsy detection and understanding its progression. These tools are invaluable not only for seizure management but also for assessing broader comorbidities, including cardiovascular, sleep, and cognitive impairments, that significantly affect quality of life.

Despite improvements in ASM diversity and tolerability, a significant proportion of patients with DRE continue to experience uncontrolled seizures. For these individuals, biomarkers offer a promising avenue for discovering new therapeutic targets and more effectively managing the condition. The use of biomarkers in epilepsy research enables the identification of novel molecular pathways, provides insights into immune‐related mechanisms, and helps in predicting comorbidities such as cardiac stress and psychiatric conditions.

Furthermore, the integration of bioinformatics and digital frameworks accelerates the discovery of nonsynaptic targets, bringing precision medicine closer to reality for epilepsy patients.

As this is a meeting report, the findings highlighted here represent those discussed at WONOEP XVII rather than a complete survey of the field. Although treatment strategies remain essential in reducing seizure frequency, the field is moving toward a more comprehensive approach, where biomarkers guide personalized interventions that go beyond symptomatic control to address the underlying causes of epilepsy. Innovative treatments such as immunotherapies demonstrate the potential to modify disease progression, especially in drug‐resistant cases. ⁵³ , ⁸¹ , ⁹¹ , ¹⁰⁸ However, the true breakthrough lies in the combination of advanced biomarkers and new therapies, which together promise a future where epilepsy is more accurately diagnosed, better managed, and ultimately, more curable. The next step will require multicenter validation of candidate biomarkers, integration of multiomics and AI‐driven analyses, and clinical trials testing disease‐modifying strategies guided by biomarkers. These coordinated efforts will be critical to ensure that discoveries in biomarker research translate into reliable, reproducible, and clinically actionable tools that reshape epilepsy care.

AUTHOR CONTRIBUTIONS

Mirte Scheper and Zining Liu contributed equally to this work. They led the draft of the manuscript. All authors contributed to and edited the manuscript. Pablo M. Casillas‐Espinosa and David C. Henshall provided critical review, substantive editing, and supervision of the manuscript. All authors approved the final version of the manuscript.

FUNDING INFORMATION

A.S.G. received research funding from NINDS NS127524, the US Department of Defense (W81XWH‐22‐1‐0210, W81XWH‐22‐1‐0510, EP220067, HT9425‐24‐1‐0134), the Isabelle Rapin and Harold Oaklander Child Neurology Research Fund in the Isabelle Rapin Child Neurology Division, and the Abbe Goldstein/Joshua Lurie and Laurie Marsh/Dan Levitz families. She has received research funding from the CURE Epilepsy Infantile Spasms Initiative. S.L.M. received research funding from NINDS NS127524, the US Department of Defense (W81XWH‐22‐1‐0210, W81XWH‐22‐1‐0510, EP220067, HT9425‐24‐1‐0134), and the Abbe Goldstein/Joshua Lurie and Laurie Marsh/Dan Levitz families. He has received research funding from the CURE Epilepsy Infantile Spasms Initiative. D.C.H. received funding from Science Foundation Ireland Research Centre and was cofunded under the European Regional Development Fund and by FutureNeuro industry partners (16/RC/3948, 21/RC/10294_P2), the Higher Education Authority (SeeDeepER), Wellcome Trust (222 648/Z/21/Z), European Union Horizon 2020 (964712), and UCB. P.M.C.‐E. is supported by the National Health and Medical Research Council (APP1087172 and APP2013629), the Monash Future Leader Fellowship (FLPF24‐0237761657), the Veski FAIR Fellowship, the Medical Research Future Fund stem cell therapy missions grant (MRF1201781), and the Department of Defense USA Epilepsy Research Program (DoD ERP IDA, grant #EP200022; DoD ERPA RPA, grant #EP220067). G.H. received funding from the South‐Eastern Norway Regional Health Authority (grant #2021006).

CONFLICT OF INTEREST STATEMENT

S.D. is an employee of UCB Pharma. A.S.G. was editor‐in‐chief of Epilepsia Open until August 2024, is associate editor for Neurobiology of Disease, and has received royalties from Elsevier and Wolters Kluwer for publications. She is consulting physician for Synergy Medical Solutions. She has no conflicts of interest in regard to this study. S.L.M. is on the editorial board of Brain and Development, Pediatric Neurology, and Physiological Research. He receives compensation from MedLink for his work as associate editor and royalties from two books he coedited. S.L.M. holds a US patent for the multiple hit rat model (#7863499); no financial profits or conflicts have ensued as a result of this patent. He has no conflicts of interest in regard to this study. G.H. has served as a lecturer for Eisai and UCB and has received a travel grant from Teva, unrelated to this study. None of the other authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Supporting information

TABLE S1. Electroencephalography‐based biomarkers under investigation in epilepsy.

TABLE S2. (A) Circulating protein biomarkers investigated in epilepsy. (B) Circulating miRNA biomarkers investigated in epilepsy.

TABLE S3. Biomarkers of comorbidities in epilepsy.

TABLE S4. Emerging treatment strategies beyond the synapse in epilepsy.

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.