Faculty Bibliography

Despite the approval of ~20 additional antiseizure medications (ASMs) since the 1980s, one-third of epilepsy patients experience seizures despite therapy. Drug-resistant epilepsy (DRE) is associated with cognitive and psychiatric comorbidities, socioeconomic impairment, injuries, and a 9.3-13.4 times higher mortality rate than in seizure-free patients. Improved seizure control can reduce morbidity and mortality. Two new ASMs were launched in the United States in 2020: cenobamate for focal epilepsy in adults and fenfluramine for Dravet syndrome (DS). They offer markedly improved efficacy. Cenobamate achieved 21% seizure freedom with the highest dose and decreased tonic-clonic seizures by 93% during maintenance treatment in a randomized clinical trial (RCT). In long-term, open-label studies, 10%-36% of patients were seizure-free for a median duration of ~30-45 months. Fenfluramine treatment in DS reduced convulsive seizure frequency by 56% over placebo at the highest dose, with 8% of patients free of convulsive seizures, and 25% with only one convulsive seizure over 14 weeks. These results were sustained for up to 3 years in open-label extension studies. Mortality was reduced 5-fold. These results are superior to all other approved ASMs, placing these two drugs among the most effective antiseizure therapies. The adverse event profiles resemble those of other ASMs. Despite greater efficacy and similar toxicity, these medications are infrequently used. Two years after US market entry, < 5% of either adults with focal DRE or patients with DS were treated with either cenobamate or fenfluramine. We believe this is a failure of our medical system, resulting from limited knowledge about these drugs stemming partly from the separation of academia from industry; restrictions to access created by health care payors, hospitals, and regulatory agencies; and insufficient post-launch information about the efficacy and safety of these ASMs.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

Introduction: Previous case-control studies of sudden unexpected death in epilepsy (SUDEP) patients failed to identify ECG features (peri-ictal heart rate, heart rate variability, corrected QT interval, postictal heart rate recovery, and cardiac rhythm) predictive of SUDEP risk. This implied a need to derive novel metrics to assess SUDEP risk from ECG. Methods: We applied Single Spectrum Analysis and Independent Component Analysis (SSA-ICA) to remove artifact from ECG recordings. Then cross-frequency phase-phase coupling (PPC) was applied to a 20-s mid-seizure window and a contour of −3 dB coupling strength was determined. The contour centroid polar coordinates, amplitude (alpha) and angle (theta), were calculated. Association of alpha and theta with SUDEP was assessed and a logistic classifier for alpha was constructed. Results: Alpha was higher in SUDEP patients, compared to non-SUDEP patients (p < 0.001). Theta showed no significant difference between patient populations. The receiver operating characteristic (ROC) of a logistic classifier for alpha resulted in an area under the ROC curve (AUC) of 94% and correctly classified two test SUDEP patients. Discussion: This study develops a novel metric alpha, which highlights non-linear interactions between two rhythms in the ECG, and is predictive of SUDEP risk.

BACKGROUND AND OBJECTIVES/OBJECTIVE:Mood, anxiety disorders, and suicidality are more frequent in people with epilepsy than in the general population. Yet, their prevalence and the types of mood and anxiety disorders associated with suicidality at the time of the epilepsy diagnosis are not established. We sought to answer these questions in patients with newly diagnosed focal epilepsy and to assess their association with suicidal ideation and attempts. METHODS:statistics, and logistic regression analyses. RESULTS:A total of 151 (43.5%) patients had a psychiatric diagnosis; 134 (38.6%) met the criteria for a mood and/or anxiety disorder, and 75 (21.6%) reported suicidal ideation with or without attempts. Mood (23.6%) and anxiety (27.4%) disorders had comparable prevalence rates, whereas both disorders occurred together in 43 patients (12.4%). Major depressive disorders (MDDs) had a slightly higher prevalence than bipolar disorders (BPDs) (9.5% vs 6.9%, respectively). Explanatory variables of suicidality included MDD, BPD, panic disorders, and agoraphobia, with BPD and panic disorders being the strongest variables, particularly for active suicidal ideation and suicidal attempts. DISCUSSION/CONCLUSIONS:In patients with newly diagnosed focal epilepsy, the prevalence of mood, anxiety disorders, and suicidality is higher than in the general population and comparable to those of patients with established epilepsy. Their recognition at the time of the initial epilepsy evaluation is of the essence.

OBJECTIVE:High-fat and low-carbohydrate diets can reduce seizure frequency in some treatment-resistant epilepsy patients, including the more flexible modified Atkins diet (MAD), which is more palatable, mimicking fasting and inducing high ketone body levels. Low-carbohydrate diets may shift brain energy production, particularly impacting neuron- and astrocyte-linked metabolism. METHODS:We evaluated the effect of short-term MAD on molecular mechanisms in adult epilepsy patients from surgical brain tissue and plasma compared to control participants consuming a nonmodified higher carbohydrate diet (n = 6 MAD, mean age = 43.7 years, range = 21-53, diet for average 10 days; n = 10 control, mean age = 41.9 years, range = 28-64). RESULTS: = .48). Brain proteomics and RNAseq identified few differences, including 2.75-fold increased hippocampal MT-ND3 and trends (p < .01, false discovery rate > 5%) in hippocampal nicotinamide adenine dinucleotide (NADH)-related signaling pathways (activated oxidative phosphorylation and inhibited sirtuin signaling). SIGNIFICANCE/CONCLUSIONS:Short-term MAD was associated with metabolic differences in plasma and resected epilepsy brain tissue when compared to control participants, in combination with trending expression changes observed in hippocampal NADH-related signaling pathways. Future studies should evaluate how brain molecular mechanisms are altered with long-term MAD in a larger cohort of epilepsy patients, with correlations to seizure frequency, epilepsy syndrome, and other clinical variables. [Clinicaltrials.gov NCT02565966.].

Cannabidiol (CBD), a non-euphoric component of cannabis, reduces seizures in multiple forms of pediatric epilepsies, but the mechanism(s) of anti-seizure action remain unclear. In one leading model, CBD acts at glutamatergic axon terminals, blocking the pro-excitatory actions of an endogenous membrane phospholipid, lysophosphatidylinositol (LPI), at the G-protein-coupled receptor GPR55. However, the impact of LPI-GPR55 signaling at inhibitory synapses and in epileptogenesis remains underexplored. We found that LPI transiently increased hippocampal CA3-CA1 excitatory presynaptic release probability and evoked synaptic strength in WT mice, while attenuating inhibitory postsynaptic strength by decreasing GABA_ARγ₂ and gephyrin puncta. LPI effects at excitatory and inhibitory synapses were eliminated by CBD pre-treatment and absent after GPR55 deletion. Acute pentylenetrazole-induced seizures elevated GPR55 and LPI levels, and chronic lithium-pilocarpine-induced epileptogenesis potentiated LPI's pro-excitatory effects. We propose that CBD exerts potential anti-seizure effects by blocking LPI's synaptic effects and dampening hyperexcitability.

Cross-frequency coupling (CFC) between theta and high frequency oscillations (HFOs) is predominant during active wakefulness, REM sleep and behavioral and learning tasks in rodent hippocampus. Evidence suggests that these state-dependent CFCs are linked to spatial navigation and memory consolidation processes. CFC studies currently include only the cortical and subcortical structures. To our knowledge, the study of nucleus tractus solitarius (NTS)-cortical structure CFC is still lacking. Here we investigate CFC in simultaneous local field potential recordings from hippocampal CA1 and the NTS during behavioral states in freely moving rats. We found a significant increase in theta (6-8 Hz)-HFO (120-160 Hz) coupling both within the hippocampus and between NTS theta and hippocampal HFOs during REM sleep. Also, the hippocampal HFOs were modulated by different but consistent phases of hippocampal and NTS theta oscillations. These findings support the idea that phase-amplitude coupling is both state- and frequency-specific and CFC analysis may serve as a tool to help understand the selective functions of neuronal network interactions in state-dependent information processing. Importantly, the increased NTS theta-hippocampal HFO coupling during REM sleep may represent the functional connectivity between these two structures which reflects the function of the hippocampus in visceral learning with the sensory information provided by the NTS. This gives a possible insight into an association between the sensory activity and REM-sleep dependent memory consolidation.

OBJECTIVE:In vitro data prompted U.S Food and Drug Administration warnings that lamotrigine, a common sodium channel modulating anti-seizure medication (NaM-ASM), could increase the risk of sudden death in patients with structural or ischaemic cardiac disease, however, its implications for Sudden Unexpected Death in Epilepsy (SUDEP) are unclear. METHODS:This retrospective, nested case-control study identified 101 sudden unexpected death in epilepsy (SUDEP) cases and 199 living epilepsy controls from Epilepsy Monitoring Units (EMUs) in Australia and the USA. Differences in proportions of lamotrigine and NaM-ASM use were compared between cases and controls at the time of admission, and survival analyses from the time of admission up to 16 years were conducted. Multivariable logistic regression and survival analyses compared each ASM subgroup adjusting for SUDEP risk factors. RESULTS:Proportions of cases and controls prescribed lamotrigine (P = 0.166), one NaM-ASM (P = 0.80), or ≥2NaM-ASMs (P = 0.447) at EMU admission were not significantly different. Patients taking lamotrigine (adjusted hazard ratio [aHR] = 0.56; P = 0.054), one NaM-ASM (aHR = 0.8; P = 0.588) or ≥2 NaM-ASMs (aHR = 0.49; P = 0.139) at EMU admission were not at increased SUDEP risk up to 16 years following admission. Active tonic-clonic seizures at EMU admission associated with >2-fold SUDEP risk, irrespective of lamotrigine (aHR = 2.24; P = 0.031) or NaM-ASM use (aHR = 2.25; P = 0.029). Sensitivity analyses accounting for incomplete ASM data at follow-up suggest undetected changes to ASM use are unlikely to alter our results. SIGNIFICANCE/CONCLUSIONS:This study provides additional evidence that lamotrigine and other NaM-ASMs are unlikely to be associated with an increased long-term risk of SUDEP, up to 16 years post-EMU admission.

Malformations of cortical development (MCD) are neurological conditions involving focal disruptions of cortical architecture and cellular organization that arise during embryogenesis, largely from somatic mosaic mutations, and cause intractable epilepsy. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat condition-related epilepsy. Here we report a genetic landscape from 283 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation including in utero electroporation of mice and single-nucleus RNA sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associated with distinct pathophysiological and clinical phenotypes. The unique single-cell level spatiotemporal expression patterns of mutated genes in control and patient brains indicate critical roles in excitatory neurogenic pools during brain development and in promoting neuronal hyperexcitability after birth.

Epilepsy has a high prevalence and can severely impair quality of life and increase the risk of premature death. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in drug-resistant epilepsy and most often results from respiratory and cardiac impairments due to brainstem dysfunction. Epileptic activity can spread widely, influencing neuronal activity in regions outside the epileptic network. The brainstem controls cardiorespiratory activity and arousal and reciprocally connects to cortical, diencephalic, and spinal cord areas. Epileptic activity can propagate trans-synaptically or via spreading depression (SD) to alter brainstem functions and cause cardiorespiratory dysfunction. The mechanisms by which seizures propagate to or otherwise impair brainstem function and trigger the cascading effects that cause SUDEP are poorly understood. We review insights from mouse models combined with new techniques to understand the pathophysiology of epilepsy and SUDEP. These techniques include in vivo, ex vivo, invasive and non-invasive methods in anesthetized and awake mice. Optogenetics combined with electrophysiological and optical manipulation and recording methods offer unique opportunities to study neuronal mechanisms under normal conditions, during and after non-fatal seizures, and in SUDEP. These combined approaches can advance our understanding of brainstem pathophysiology associated with seizures and SUDEP and may suggest strategies to prevent SUDEP.