Epilepsy Research

ImportanceEpilepsy is one of the most common neurological disorders globally. A significant proportion of patients fail to achieve effective seizure control with medication and ultimately develop drug-resistant epilepsy, particularly mesial temporal lobe epilepsy (MTLE). While surgical resection and laser interstitial thermal therapy (LITT) are effective treatments for drug-resistant MTLE, these procedures may be associated with severe adverse events. In contrast, allogeneic induced pluripotent stem cell (iPSC)-based therapy is expected to offer a novel, potentially safer therapeutic approach with fewer side effects for patients with drug-resistant MTLE. ObjectiveTo evaluate the safety and preliminary efficacy of a single intracranial injection of ALC05 (iPSC-derived GABAergic interneurons) in patients with unilateral MTLE, and to assess the therapeutic effects of different dosage levels. Design, Setting, and ParticipantsThis single-center, randomized, double-blind, Phase 1 clinical trial will enroll 12 subjects with unilateral MTLE. All subjects will be randomly assigned to either the low-dose or high-dose group in a 1:1 ratio. To minimize risks at each dose level, the first subject in each dose group will be monitored for safety for at least 3 months following ALC05 injection and must demonstrate acceptable safety and tolerability before the remaining subjects are enrolled. The primary outcome will be the incidence and severity of adverse events (AEs) and serious adverse events (SAEs). Secondary outcomes include cell engraftment and survival, responder rate, and seizure frequency. The follow-up period for this study is 1 year. After completing the follow-up period within this study, subjects will enter a 15-year long-term safety follow-up. DiscussionMTLE remains a significant challenge in neurology. The results of this study will provide critical data regarding the feasibility and preliminary efficacy of ALC05 in treating MTLE and may offer a transformative therapeutic option for this condition.

Objective: Electrical brain stimulations (EBS) are central to epileptic network identification and functional mapping during stereo-electroencephalography (SEEG), yet stimulation frequencies remain empirical, and standardized across patients and brain regions, producing false negatives and false positives, and potentially compromising surgical outcome. We investigated theta-range EBS (7 Hz) in the temporal lobe, a prominent physiological frequency band in this region, and compared it with conventional 1-Hz and 50-Hz protocols. Methods: We analyzed 1,408 temporal EBS in 25 patients with drug-resistant epilepsy. Epileptic responses (afterdischarges, seizures) and clinical signs were assessed across the epileptic network and temporal structures (amygdala, hippocampus, neocortex, parahippocampal gyrus, white matter), and analyzed according to stimulation parameters (frequency, intensity, duration, total charge). Results: At matched intensity and duration, 7-Hz EBS were associated with a higher occurrence of afterdischarges and clinical signs than 1-Hz EBS in several temporal structures (e.g., parahippocampal epileptogenic zone: p=0.014). Effects on usual seizure induction were less consistent. Comparisons with 50 Hz showed no systematic significant differences, with responses observed at one or both frequencies depending on structure and outcome. When controlling for total charge, frequency-related differences were attenuated. Some effects were sporadically observed at both intermediate frequency and charge quantity. No adverse events occured. Significance: Theta-range stimulation modulates electrophysiological and clinical responses during SEEG mapping and may provide complementary information to conventional frequencies. These findings support exploring a broader range of stimulation frequencies, rather than relying solely on standard protocols.

Objective: The postictal state is a major yet underrecognised component of epilepsy burden. We aimed to develop a structured patient-reported instrument to quantify postictal recovery, characterise its multidimensional burden and identify demographic, clinical, psychiatric and treatment-related factors associated with postictal severity and duration. Methods: We conducted a prospective, single-centre observational cohort study (Timone Hospital, Marseille, February 2025 - March 2026). Consecutive patients aged >=15 years admitted for scalp or stereo-EEG video-monitoring were included. Patients completed the Postictal Recovery Scale (PRS), an 11-domain questionnaire assessing fatigue, mood, sensory, motor, language, orientation, time perception and postictal amnesia. Items were rated from 0 (severe impairment) to 3 (no symptoms), yielding a total score of 0-33. Internal consistency was assessed using Cronbach alpha. Associations between PRS scores, subjective postictal duration and covariates were analysed using group comparisons, correlations and regression models. Results: Of 107 enrolled patients, 96 were included. PRS showed good internal consistency (Cronbach alpha; = 0.79). 96% of patients reported experiencing postictal symptoms, with fatigue (80%) and postictal amnesia (79%) being the most frequent and severe manifestations. Recovery exceeded one hour in 21% of patients. Greater postictal impairment was associated with higher interictal anxiety (Spearman {rho} = -0.32, p = 0.0018) and depressive symptoms (Spearman {rho} = -0.40, p = 0.0001), whereas demographic, epilepsy-related and treatment variables showed no significant associations. Altered postictal time perception was reported by 40% of patients and was associated with disorientation, but not psychiatric symptoms. Subjective postictal duration was longer than subjective ictal duration (Wilcoxon test, p < 0.0001). Significance: The postictal state is a frequent and multidimensional patient-reported experience. Greater postictal severity, particularly concerning anxiety and depression, is associated with interictal psychiatric comorbidity, while altered temporal experience emerges as a distinct dimension of postictal dysfunction. These findings support integrating postictal measures into clinical practice and trials.

Ferroptosis is a form of non-apoptotic cell death in which iron catalyzes the formation of reactive oxygen species, leading to lipid peroxidation. Experimentally, this process has recently been associated with seizures based on the increased levels of specific markers (4-hydroxynonenal and malondialdehyde) in the brain and plasma. Clinically, iron deposits have been identified in resected tissue from patients with refractory temporal lobe epilepsy. Quantitative susceptibility mapping (QSM) offers an opportunity to detect these accumulations in vivo. In this study, we investigated how pilocarpine-induced status epilepticus contributes to the generation of iron deposits in diverse cerebral regions and whether QSM can detect these deposits longitudinally. We scanned 14 animals (n=10 experimental; n=4 control) at five different time points (pre-status epilepticus induction and 1, 7, 14, 21 days post-induction) using QSM. We identified iron deposits in the caudate putamen, hippocampus, thalamus, and primary somatosensory cortex of experimental animals, which is consistent with histological findings. The initial size of the hippocampal iron deposits significantly increased over the following weeks. None of these effects was observed in the control animals. The presence of cerebral iron depositions in an animal model of pilocarpine-induced status epilepticus suggests that ferroptosis may be involved in the onset, development, and progression of spontaneous recurrent seizures. Furthermore, non-invasive, longitudinal in vivo mapping of brain iron deposits could be a potential imaging marker in neurological disorders such as epilepsy. Future experiments will be required to determine the origin of the iron and avoid its progressive accumulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/712677v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@14abf67org.highwire.dtl.DTLVardef@5c08fborg.highwire.dtl.DTLVardef@51c40forg.highwire.dtl.DTLVardef@1eb5f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundIdiopathic epilepsy (IE) is the most common chronic nervous system disorder of dogs, and its cause is poorly understood. Emerging evidence suggests that microbiome alterations can occur with IE via the microbiota-gut-brain axis. Therefore, we analyzed the fecal microbiomes of 98 dogs (49 IE, 49 control) in a pairwise case-control observational study using 16S rRNA gene sequencing. ResultsAlthough the microbial community was mostly similar between groups, IE was associated with a modest but significant shift in Weighted-Unifrac distance (P = 0.042). We used six differential abundance (DA) methods to identify differentially abundant amplicon sequencing variants (ASVs) between IE and control groups. Notably, one Collinsella ASV was found to be significantly more abundant in IE dogs by all six methods. The gut microbial compositions varied drastically across households (accounting for about 69% of the total variation), but did not have significant differences between sex, age, or breed. Phenobarbital administration in IE dogs had a significant effect on seizure control, and was not associated with changes in the microbiome. ConclusionOur findings suggest a relationship between gut microbiomes and IE. However, the specific mechanism needs to be further investigated.

ObjectiveBrain infection is an underrecognized global cause of epilepsy due to the ensuing neuroinflammation and neurological damage. Immune system response, including underlying neuroinflammation, is dynamically shaped by the intestinal microbiome. In experimental rodent epilepsy models, seizure burden and antiseizure medication (ASM) activity can be dramatically influenced by gut dysbiosis, including in the Theilers murine encephalomyelitis virus (TMEV) infection model of acute symptomatic seizures and long-term epilepsy. We previously demonstrated that experimentally induced gut dysbiosis via repeated antibiotic administration alters seizure burden and carbamazepine (CBZ) anticonvulsant activity in this model (1). However, whether dysbiosis and CBZ differentially shape neuropathological damage and neuroinflammation following TMEV infection was not reported. MethodsHere, we extended our earlier study to quantify the extent to which antibiotic-induced gut dysbiosis and repeated CBZ administration during TMEV infection altered the severity of acute neuropathology. Hippocampal tissue was analyzed 7 days post-infection using quantitative immunofluorescence to assess neuronal death, microglial and astroglial reactivity, and neuronal proliferation across CA1, CA3, and dentate gyrus (DG) subregions. ResultsDysbiosis markedly exacerbated hippocampal neurodegeneration and gliosis, accompanied by increased glial proliferation, whereas CBZ administration reversed these effects in a hippocampal region-dependent manner. Collectively, these findings demonstrate that the gut microbiome primes hippocampal neuroimmune responses to viral infection-induced acute seizures and modifies associated neuropathology in a hippocampal region-specific manner. SignificanceThis work identifies the gut-brain axis as a critical determinant of neuroinflammatory damage after infection-induced symptomatic seizures, highlighting the gut microbiome as a potential therapeutic target to alleviate the worldwide epilepsy burden. HighlightsO_LIExperimentally-evoked gut dysbiosis exacerbates hippocampal neurodegeneration after brain viral infection. C_LIO_LIExperimentally-evoked gut dysbiosis increases microgliosis and glial proliferation after brain viral infection. C_LIO_LICarbamazepine reversed dysbiosis-induced neuroinflammation and neurodegeneration. C_LIO_LIExperimentally-evoked gut dysbiosis differentially modulates glial response in the dentate gyrus. C_LI

Background: Statistical Analysis Plans (SAPs) are essential for trial transparency and credibility but are resource-intensive to produce. While Large Language Models (LLMs) have shown promise in drafting protocols, their ability to generate high-quality, protocol-compliant SAPs remains untested against current content guidance. This study developed and validated an LLM-based pipeline for drafting SAPs from clinical trial protocols. Methods: We developed a structured, section-by-section prompting pipeline aligned with standard SAP guidance. We applied this pipeline to nine clinical trial protocols using three leading LLMs: OpenAI GPT-5, Anthropic Claude Sonnet 4, and Google Gemini 2.5 Pro. The resulting 27 SAPs were evaluated against a 46-item quality checklist derived from the published SAP guidelines. Items were double-scored by independent trial statisticians on a 0 to 3 scale for accuracy. We compared performance across LLMs and between item types (descriptive vs. statistical reasoning) using mixed-effects logistic regression. Results: Across 9 trials, the models produced SAP drafts with high overall accuracy (77% to 78%), with no difference in performance between the three LLMs (p=0.79) but varied by content type (p < 0.001). All models performed well on descriptive items (e.g., administrative details, trial design), with lower accuracy for items requiring statistical reasoning (e.g., modelling strategies, sensitivity analyses). Accuracy for statistical items ranged from 67% to 72%, whereas descriptive items achieved 81% to 83% accuracy. Qualitatively, models were prone to specific failure modes in complex sections, such as omitting necessary details for secondary outcome models or hallucinating sensitivity analyses. Discussion: Current LLMs can effectively draft portions of SAPs, offering the potential for substantial time savings in trial documentation. However, a human-in-the-loop approach remains mandatory; while models demonstrate strong capability in producing descriptive content, their independent application to complex statistical methodology design still requires further methodological development and training. Future work should explore advanced prompt engineering, such as retrieval-augmented generation or agentic workflows, to improve reasoning capabilities.

Background: The detection of subtle epileptogenic lesions such as focal cortical dysplasias (FCDs) is a clinical challenge in the management of drug-resistant focal epilepsy (DRFE). Ultra-high field (UHF) MRI offers increased signal-to-noise ratios and spatial resolution compared to 3Tesla (T) MRI and may improve diagnostic yield. Here, we present a 9.4T MRI cohort study of patients with DRFE. Methods: We recruited n=21 DRFE patients (with 3T-MRI findings: 2 positive, 3 equivocal, 16 negative) undergoing presurgical workup, and n=20 healthy controls for 9.4T MRI (0.8 mm isotropic MP2RAGE, slabs of 0.375 x 0.375 x 0.8 mm T2*-weighted GRE) and 3T MRI (MP2RAGE, FLAIR) acquisitions. Visual review for possible epileptogenic lesions was performed by clinical experts. For histopathologically confirmed FCD lesions, we extracted surface-based quantitative features (cortical thickness, qT1, FLAIR, T2*, and QSM values) across cortical depths and distances from the lesion centre and performed high-resolution cortical profiling of 9.4T T2* values. Results: No new epileptogenic lesions were visually identified at 9.4T in 3T MRI negative patients. In the two patients with histopathologically confirmed lesions, the FCD IIb lesions were visible with distinct qualitative and quantitative features at both field strengths. One of these FCD IIb showed a focal cortical T2* reduction at 9.4T that could here be quantified via automated cortical profiling, consistent with the previously described "black line sign". Conclusion: 9.4T MRI findings in epileptogenic lesions underlying DRFE are consistent with those on 3T MRI. While additional lesions were not identified in patients with negative 3T MRI, higher resolution T2*-weighted sequences can reveal a feature not seen at 3T: Cortical profiling of FCDs highlights the black line sign and can possibly help refine surgical or ablation targeting for some FCDs. Further optimization of UHF protocols and analysis methods on larger cohorts may reveal clinically applicable diagnostic benefits.

In the management of atrial fibrillation, the most frequently prescribed oral anticoagulant is apixaban, given at a fixed dose of 5mg BID. Apixaban is predominantly metabolized by cytochrome P4503A4 (CYP3A4) and is also a substrate for the drug efflux transporter P-glycoprotein (P-gp). In nearly 300,000 Medicare patients with AF receiving apixaban, we previously showed that concomitant therapy with drugs that inhibit both CYP3A4 and P-gp, specifically amiodarone or diltiazem, significantly increased serious bleeding that caused hospitalization and/or death. We hypothesized that this adverse effect was mediated by an increase in apixaban plasma concentrations caused by concomitant therapy that reduced drug elimination. Utilizing left-over samples obtained from clinically indicated blood draws that would typically be discarded, the Vanderbilt University Medical Center biobank BioVU contains >353,000 samples linked to de-identified electronic medical records (EMRs), with both DNA and plasma harvested. Of 35 samples drawn from patients taking apixaban 5mg BID, 5 were identified to be drawn from patients concomitantly taking drugs inhibiting both CYP3A4 and P-gp. Using a chromogenic anti-Xa assay, we found that plasma concentrations of apixaban were significantly higher (347{+/-}64 ng/mL; mean{+/-}SEM) for patients receiving concomitant CYP3A4/P-gp-inhibiting drugs compared to those not treated with these drugs (166{+/-}67 ng/mL; P=0.025, Mann Whitney). There were no differences between the 2 patient groups with respect to age, weight, or serum creatinine. The results of this pilot study provide preliminary data to support our hypothesis, and they demonstrate the practicality of obtaining pharmacokinetic data from a large cohort of plasma samples linked to deidentified EMRs. This approach could be used to define the role of apixaban levels in high-risk clinical scenarios and to better understand the relationship between drug levels and bleeding risk.

Background: Clinical evidence on the contemporary management and functional outcomes of patients with Wernicke encephalopathy remains limited. This study aimed to clarify the nationwide patterns of thiamine administration and functional outcomes at discharge. Methods: Using the Japanese nationwide inpatient Diagnosis Procedure Combination database, we identified patients hospitalized with Wernicke encephalopathy between July 2010 and March 2024. Initial intravenous thiamine doses were categorized as low ([&le;]300 mg/day), medium (301-900 mg/day), or high (>900 mg/day). Outcomes included in-hospital mortality and functional status (Barthel Index) at discharge. Results: We identified 7856 patients with Wernicke encephalopathy. Over the 13-year study period, the proportion of patients receiving initial high-dose thiamine increased markedly from 5.4% to 49.0%, while the frequency of low-dose therapy decreased from 83.0% to 37.9%. Despite prompt intervention [median time to initial administration: 0 days (interquartile range, 0 to 0 days)], 56.1% of patients were discharged with impaired activities of daily living (Barthel Index <90), and the in-hospital mortality rate was 3.8%. Conclusions: High-dose thiamine treatment is increasingly implemented for Wernicke encephalopathy in Japan. Although in-hospital mortality was relatively low, the high prevalence of functional impairment at discharge, despite early treatment initiation, indicates substantial burden of Wernicke encephalopathy. Given the limited clinical evidence, further research investigating the optimal thiamine dose and develop effective primary prevention strategies for Wernicke encephalopathy is needed.

Background and Purpose: 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography (static PET) has mixed specificity and sensitivity in targeting epileptic zones in the noninvasive stage of epilepsy surgery evaluations. We compared the signal quality of static PET compared to a method of interictal dynamic PET (iD-PET). Materials and Methods: We calculated the signal quality of static PET and iD-PET obtained from a cohort of patients with focal epilepsy. We developed a Bayesian regional estimated signal quality (BRESQ) technique to objectively compare signal-to-noise ratios (SNRs) by region of interest (ROI) within subjects. Results: Adjusted for ROI size and neighboring regions, iDPET was superior to sPET with probability >95% in 8/36 regions; >90% in 21/36 regions; >80% in 29/36 regions. The top five regions with the largest adjusted SNR differences (greatest magnitude of iDPET superiority) were the Temporal Mesial (Left and Right), Occipital Lateral (Left and Right), and the Left Frontal Inferior Base. Conclusions: We found that iDPET yielded a superior SNR in most ROI. BRESQ offers a scalable and generalizable method to quantify signal quality between brain mapping modalities.

Schizophrenia (SCZ) and type 2 diabetes mellitus (T2DM) are common comorbid disorders that severely impair patient prognosis and quality of life. This study aimed to explore the association between the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and MTHFR promoter methylation in patients with comorbid SCZ and T2DM. A total of 120 participants were enrolled from Liaocheng Fourth Peoples Hospital between January 2025 and June 2025, comprising 30 subjects in each of the four groups: SCZ group, T2DM group, SCZ-T2DM comorbid (SCZ+T2DM) group, and healthy control (CTL) group. Corresponding primers were designed for genetic analysis, and methylation-specific PCR (MSP) was performed to detect the methylation level of the MTHFR promoter. Genotype distribution of the MTHFR C677T polymorphism was consistent with Hardy-Weinberg equilibrium (HWE) (p>0.05). The C677T polymorphism was significantly associated with an elevated risk of SCZ and T2DM comorbidity (p<0.05). Notably, the methylation rate of the MTHFR promoter in the SCZ+T2DM group (95.00%) was not significantly higher than that in the CTL group (90.00%) (p>0.05). In conclusion, the MTHFR gene may serve as a susceptibility gene for SCZ-T2DM comorbidity, whereas MTHFR promoter methylation is not associated with the pathogenesis of this comorbid condition. These results indicate that genetic variation in MTHFR, rather than promoter methylation, contributes critically to the comorbidity of SCZ and T2DM in the Han Chinese population. Our findings may provide novel molecular insights into their shared pathophysiology and inform future clinical strategies for patients with this complex phenotype.

Epilepsy is a chronic neurological disorder requiring multi-faceted management, including seizure detection, syndrome diagnosis, prognostication, antiseizure medication recommendation, epileptogenic zone localization, and surgical outcome prediction. Although numerous deep learning approaches have been developed for individual tasks, these models are typically siloed and modality-specific (e.g., EEG for seizure detection, MRI for localization), failing to reflect the multidisciplinary nature of real-world epilepsy care, where epileptologists, neuroradiologists, neurosurgeons, neuropsychologists and neuropsychiatrists jointly interpret heterogeneous evidence to guide decisions. In this work, we propose a clinical guideline-grounded hybrid multi-agent framework for holistic epilepsy management. Heterogeneous patient data is processed through modality-specific discriminative and generative models, where textual interpretations from generative agents are combined with structured predictions from discriminative models as auxiliary guidance. This aggregated evidence is passed to a central orchestrating agent grounded in international epilepsy guidelines, which evaluates multi-modal findings within structured clinical pathways and performs iterative cross-agent coordination for evidence-informed decision-making. We evaluate our framework across two datasets spanning six epilepsy management tasks and also introduce a publicly available multi-modal, multi-task epilepsy benchmark. Results demonstrate that integrating discriminative evidence with guideline-grounded generative coordination yields more reliable and comprehensive decisions compared to conventional LLM-based and task-specific baselines. Our dataset and code is available at URL.

BackgroundHyperglycemia after intracerebral hemorrhage (ICH) may be associated with worse outcomes. In this study, we evaluated the association of early post-ICH glucose trajectories and clinical outcomes. MethodsWe performed a secondary analysis of the ATACH-2 trial dataset. Hyperglycemia was defined as a blood glucose of [&ge;]140 mg/dl. Glucose levels at 0h, 24h, 48h, and 72h were analyzed using a linear mixed effects model, with fixed effects for time and random intercept/slopes. Patient-specific estimates were used to predict glucose values at 0h and 72h, informed by all four timepoints, to classify patients into the following glycemic trajectory groups: (1) early hyperglycemia, (2) late hyperglycemia, (3) persistent hyperglycemia, and (4) persistent normoglycemia. Outcomes were compared using univariate analysis and log-rank test survival analysis. Good outcomes were defined as a modified Rankin Score of 0 to 2. The association between glycemic trajectories and functional outcomes was tested using logistic regression models adjusted for patient demographics and clinical variables. ResultsOf 1000 patients (median age 62 [IQR 52-71]; 38% female) in the study, 81 (8.1%) had early hyperglycemia, 59 (5.9%) late hyperglycemia, 225 (22.5%) persistent hyperglycemia, and 635 (63.5%) persistent normoglycemia. On univariate analysis, 45.8% of patients with persistent normoglycemia had favorable 90-day functional outcomes compared to 30.9% in early, 30.5% in late, and 32.0% in persistent hyperglycemia patients (p<0.001). The late hyperglycemia patients had the highest rate of hematoma expansion (35.3%, p=0.029) and the lowest Kaplan Meier-estimated survival (86%, p=0.015). In adjusted multivariable regression models, early hyperglycemia was significantly associated with a poor functional outcome (OR 2.27, 95% CI 1.10-4.68, p=0.026). ConclusionEarly hyperglycemia was associated with worse functional outcomes, while late and persistent hyperglycemia were associated with worse survival rates. These findings suggest that glycemic trajectories may affect or predict prognosis. This highlights the importance of continuous glucose monitoring and glycemic control strategies after ICH.

Importance: NGLY1 (N-Glycanase 1) Deficiency is an ultra-rare autosomal recessive disorder affecting ~165 patients worldwide, characterized by developmental delay, hyperkinetic movement disorders, and shortened life expectancy. Despite its severe neurological manifestations, comprehensive neuroimaging characterization has been limited to case reports and small descriptive studies. Objective: To investigate alterations in brain morphology in patients with NGLY1 Deficiency and determine whether these metrics associate with clinical phenotypes. Design, Setting, and Participants: This case series analyzed real-world MRI scans performed on 11 patients with NGLY1 Deficiency between 1999-2023 at sites across the globe. Ages ranged from 2 to 19 years at scan time (5 female, 6 male). Exposure: Molecular diagnosis of NGLY1 Deficiency. Main Outcomes and Measures: Cortical and subcortical morphology, including subcortical volume, and cortical thickness, surface area, volume, and curvature, were measured with 3-dimensional T1-weighted magnetic resonance imaging (MRI) scans. Z-scores were calculated using normative models from CentileBrain for patients >3 years old or custom models for patients <3 years old. Clinical phenotypes were matched to Human Phenotype Ontology codes. Results: 16 scans from 11 patients met quality criteria for analysis. Both age groups (under and over 3 years old) showed significantly reduced subcortical volumes, particularly in bilateral thalamus and putamen. Younger patients demonstrated widespread reductions in cortical surface area, volume, and curvature, indicating altered gyrification patterns. Older patients showed thinner dorsal and thicker ventral cortical regions with limited surface area reductions. Thalamic volume reduction in older patients correlated with gait disturbance, dysphagia, and EEG abnormalities, with additional cortical associations with sleep and hearing abnormalities. Seizure presence in younger patients correlated with altered cortical thickness, surface area, and curvature patterns. Conclusions and Relevance: NGLY1 Deficiency is associated with pervasive alterations in brain development affecting both subcortical and cortical morphology. Age-dependent patterns of cortical alterations indicate disrupted neurodevelopmental trajectories that may reflect impaired neuronal migration and/or altered synaptic pruning. Correlations with clinical variables suggest that these measures may serve as useful biomarkers for tracking disease progression and/or treatment efficacy. These findings provide a comprehensive neuroimaging characterization of NGLY1 Deficiency and establish a foundation for understanding brain structure-function relationships in this ultra-rare disorder.

BackgroundHow does neuronal activity change as an animal transitions from being awake to a state of general anesthesia? Previous studies used C. elegans to investigate awake and anesthetized states, emergence from anesthesia, and to establish metrics characterizing how system-wide neuronal dynamics differ under these conditions. This study employs a new technique to image pan-neuronal activity in C. elegans continuously during induction of anesthesia with isoflurane. MethodsC. elegans worms expressing pan-neuronal nuclear RFP and cytosolic GCaMP6s were imaged with light sheet microscopy to measure single cell activity in the majority of neurons in the animals head during induction via isoflurane exposure. Stable concentrations of isoflurane were maintained throughout the experiment by measured flow vaporization of isoflurane into a specially designed gas enclosure compatible with the imaging system. Building on our previous work investigating emergence from anesthesia, we analyzed ensemble neuronal activity, spectrograms of frequency over time, and metrics of information flow between neurons. ResultsInduction of isoflurane anesthesia caused a progressive reduction in neuronal activity over the course of 40 minutes. Spectrograms indicated a loss of bulk signal power across all frequencies, notably in low frequencies too. State Decoupling and Internal Predictability were among the most useful metrics for discriminating the anesthetized state, demonstrating induction kinetics that are the inverse of emergence. However, each animal does not arrive at the anesthetized state at the same time; response times are highly individualized. ConclusionsInformation metrics of neurodynamic activity demonstrate that isoflurane induction results in a gradual increase in neuronal disconnection and disorganization. Thus, at the level of individual neuron connectivity and system dynamics, the induction of anesthesia in C. elegans nematodes is in essence the reverse of emergence. Induction however occurs more rapidly and shows marked variability between individuals. Future genetic studies will show which molecular targets define sensitivity to volatile anesthetics like isoflurane. Summary StatementIsoflurane-induced unconsciousness is a common phenomenon across species. Does the induction of anesthesia arise by distinct state transitions, or through gradual changes in system dynamics when activity is measured at the level of individual neurons?

Purpose: DNM1-related disorder is a rare developmental and epileptic encephalopathy. The current understanding of the clinical spectrum is based on sparse patient descriptions. Here, we compile the largest DNM1 cohort to date, to characterize the genotypic and phenotypic landscape of the disorder. Methods: Phenotypic data was manually curated from 95 individuals from multiple sources and harmonized using the Human Phenotype Ontology framework. Results: Disease-causing variants in DNM1 cluster in mutational hotspots within the gene, which achieve Strong and Moderate evidence for pathogenicity based on ACMG guidelines. The overall DNM1 phenotype was homogeneous compared to other genetic epilepsy conditions: SCN2A, SCN8A, STXBP1, and SYNGAP1. The p.R237W (n=15) variant was associated with bilateral tonic-clonic seizures, infantile spasms, and dystonia. The p.I398_R399insCR (n=14) variant was associated with severe hypotonia, profound global delay, and cortical visual impairment. Five individuals with homozygous loss-of-function variants were clinically similar to dominant-negative DNM1-related disorder, but microcephaly and brain MRI abnormalities were more common in this group. Conclusion: A harmonized cohort of individuals with DNM1-related disorder was analyzed to define mutational hotspots and reveal novel genotype-phenotype correlations. Due to the homogeneous phenotype, disease mechanism, and high proportion of recurrent variants, DNM1 represents an attractive target for targeted therapy development.

Polyhydramnios, Megalencephaly, and Symptomatic Epilepsy syndrome (PMSE/STRADA-related disorder) is a rare neurodevelopmental disorder characterized by megalencephaly (ME), early-onset drug-resistant epilepsy, neurocognitive impairment, and high early mortality, often due to status epilepticus. PMSE is caused by a multi-exon deletion in STRADA, encoding STRADA, which regulates the mechanistic target of rapamycin (mTOR) pathway. GABAergic inhibitory interneurons (INs) critically modulate the excitatory:inhibitory balance in cortical and hippocampal networks, and IN deficits contribute to epileptogenesis in several epileptic encephalopathies. However, no studies have investigated INs in PMSE. We used a multimodal approach to study INs in a Strada-/- mouse model engineered with the same causative 5-exon deletion identified in human PMSE. We demonstrate that Strada/STRADA loss causes a reduction of INs in the somatosensory cortex and a corresponding increase in the striatum, representative of remnant ganglionic eminence progenitor origin, in Strada-/- mice and a single PMSE brain tissue specimen. RNA sequencing comparing wildtype to Strada-/- cortex and striatum corroborated these findings, revealing increased IN-related gene expression (e.g., Dlx2) in the striatum and decreased IN-related gene expression (e.g., Pvalb) in the developing cortex. Cytoskeletal (e.g., Tpp3, Kank4, Map1a) and mTOR-associated genes (e.g., Rictor, Cryab) are differentially expressed in the developing cortex, mature striatum, and mature cortex of Strada-/- mice. Functional validation confirmed enlarged INs in mouse and human Strada/STRADA-deficient brain and enhanced S6 phosphorylation in Strada-/- striatum. Together, these findings suggest STRADA/Strada loss contributes to failed IN migration -- the first such report in a developmental, mTOR-associated megalencephaly syndrome -- highlighting INs as a therapeutic target for seizure prevention in PMSE. Key PointsO_LI- Reduced numbers of cortical inhibitory interneurons were observed in the cerebral cortex of Strada-/- mice, with striatal interneuron aggregation C_LIO_LI- Reduced numbers of cortical inhibitory interneurons, with an aggregation in striatum, were observed in human PMSE brain, supporting the observations in Strada-/- mouse C_LIO_LI- Transcriptomic analysis in Strada-/- mice reveals evidence of early developmental interneuron and cytoskeletal dysfunction C_LIO_LI- We introduce a loss of cortical interneurons as a salient feature of PMSE developmental pathogenesis, potentially contributing to a loss of inhibitory modulation C_LIO_LI- This is the first study proposing interneuron migration impairment in the developmental pathogenesis of an mTOR-associated megalencephaly syndrome C_LI

PCDH19-Cluster Epilepsy (PCDH19-CE) is a rare neurological disorder caused by mutations in the PCDH19 (Protocadherin-19) gene and is characterized by early-onset seizures and cognitive impairment. In contrast to most X-linked disorders, PCDH19 mutations predominantly affect heterozygous females, while hemizygous males are largely spared. Although advances have been made to understand the pathological mechanism underlying PCDH19-CE, key downstream targets and compensatory pathways are yet to be elucidated. Using CRISPR/Cas9 technology, we generated both a mouse model of PCDH19-CE and a human embryonic stem cell (ESC) model. Transcriptomic analysis identified genes that were differentially expressed in the brains of heterozygous (Pcdh19WT/mut) female mice compared with wildtype (WT) and homozygous (Pcdh19mut/mut) female mice. Pathway analysis of these differentially expressed genes (DEGs) revealed enrichment in pathways involved in neuronal development, ion channel activity, synaptic development and neuronal signalling. Neurons differentiated from human ESCs carrying a PCDH19 mutation exhibited similar gene expression patterns, with heterozygous neurons displaying a distinct expression pattern compared to both WT and homozygous mutant neurons. In contrast to the molecular phenotype, neurons derived from homozygous mutant cells showed highly elongated neurites while neurons from heterozygous cells showed intermediate neurite elongation. This suggests that neurite morphology correlates directly with levels of WT PCDH19. Overall, our findings indicate that heterozygous PCDH19 mutations are associated with defects in the expression of genes involved in developmental, signalling, and neuronal pathways in both mouse and human disease models, while certain morphological phenotypes appear to depend on the levels of WT PCDH19.

Background Phonemic awareness deficits are a core feature of Specific Learning Disorder-Reading (SLD-R). How task- and language-specific factors influence these deficits in alphasyllabary languages may help clarify the cognitive mechanisms underlying reading impairment in SLD-R. Methods Thirty children with a DSM-5 diagnosis of SLD-R (mean age 11.4 years) and 29 age-matched typically developing children were given phoneme blending (words and pseudowords) and segmentation tasks in Malayalam. The effects of age and consonant clusters on task performance were evaluated. Results Children with SLD-R performed significantly worse than controls across most phonemic awareness tasks, with the largest deficits observed in pseudoword blending and word blending, and smaller deficits in segmentation. No significant difference was observed for initial phoneme deletion. In typically developing children, age showed strong positive correlations with phonemic performance across most tasks, whereas the SLD-R group showed weak or absent correlations, except in word blending and initial phoneme deletion. Consonant clusters significantly affected performance in both groups, with SLD-R showing more severe deficits. Conclusions Phonemic awareness deficits observed in SLD-R in alphasyllabary languages like Malayalam are more prominent in tasks where lexical support is absent, like pseudoword blending. These deficits vary across task types and linguistic complexity. Phonemic awareness improves with age in typically developing children, while improvement is uneven in children with SLD-R. The findings suggest that phonemic awareness deficits are a core feature of SLD-R across languages, but their manifestation is shaped by orthographic and linguistic characteristics of the writing system.