Epilepsy and sudden unexpected death in epilepsy in a mouse model of human -linked developmental and epileptic encephalopathy.

Voltage-gated sodium channel β1 subunits are essential proteins that regulate excitability. They modulate sodium and potassium currents, function as cell adhesion molecules and regulate gene transcription following regulated intramembrane proteolysis. Biallelic pathogenic variants in , encoding β1, are linked to developmental and epileptic encephalopathy 52, with clinical features overlapping Dravet syndrome.

Genome-wide CRISPRi Screen in Human iNeurons to Identify Novel Focal Cortical Dysplasia Genes.

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed an CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels.

Deriving early single-rosette brain organoids from human pluripotent stem cells.

Brain organoid methods are complicated by multiple rosette structures and morphological variability. We have developed a human brain organoid technique that generates self-organizing, single-rosette cortical organoids (SOSR-COs) with reproducible size and structure at early timepoints. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex.

Protocol for selecting single human pluripotent stem cells using a modified micropipetter.

Single-cell clonal selection is a critical procedure for generating a homogeneous population of human pluripotent stem cells. Here, we present a protocol that repurposes the STRIPPER Micropipetter, normally used for in vitro fertilization, to pick single stem cells. We describe steps for tool and reagent preparation, single-cell picking, and colony passaging.

Sudden Unexpected Death in Epilepsy and Respiratory Defects in a Mouse Model of DEPDC5-Related Epilepsy.

Objectives: DEPDC5 is a common causative gene in familial focal epilepsy with or without malformations of cortical development. Its pathogenic variants also confer a significantly higher risk for sudden unexpected death in epilepsy (SUDEP), providing opportunities to investigate the pathophysiology intersecting neurodevelopment, epilepsy, and cardiorespiratory function. There is an urgent need to gain a mechanistic understanding of DEPDC5-related epilepsy and SUDEP, identify biomarkers for patients at high risk, and develop preventive interventions.

Microglial depletion after brain injury prolongs inflammation and impairs brain repair, adult neurogenesis and pro-regenerative signaling.

The adult zebrafish brain, unlike mammals, has a remarkable regenerative capacity. Although inflammation in part hinders regeneration in mammals, it is necessary for zebrafish brain repair. Microglia are resident brain immune cells that regulate the inflammatory response.

A Shared Pathogenic Mechanism for Valproic Acid and Knockout in a Brain Organoid Model of Neural Tube Defects.

Neural tube defects (NTDs), including anencephaly and spina bifida, are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or severe lifelong complications (spina bifida). Despite hundreds of genetic mouse models of neural tube defect phenotypes, the genetics of human NTDs are poorly understood.

Predictors of referral for long-term EEG monitoring for Medicare beneficiaries with drug-resistant epilepsy.

Objective: For people with drug-resistant epilepsy, the use of epilepsy surgery is low despite favorable odds of seizure freedom. To better understand surgery utilization, we explored factors associated with inpatient long-term EEG monitoring (LTM), the first step of the presurgical pathway.

Methods: Using 2001-2018 Medicare files, we identified patients with incident drug-resistant epilepsy using validated criteria of ≥2 distinct antiseizure medication (ASM) prescriptions and ≥1 drug-resistant epilepsy encounter among patients with ≥2 years pre- and ≥1 year post-diagnosis Medicare enrollment.

Surge of neurophysiological coupling and connectivity of gamma oscillations in the dying human brain.

The brain is assumed to be hypoactive during cardiac arrest. However, animal models of cardiac and respiratory arrest demonstrate a surge of gamma oscillations and functional connectivity. To investigate whether these preclinical findings translate to humans, we analyzed electroencephalogram and electrocardiogram signals in four comatose dying patients before and after the withdrawal of ventilatory support.

A Shared Pathogenic Mechanism for Valproic Acid and SHROOM3 Knockout in a Brain Organoid Model of Neural Tube Defects.

Neural tube defects (NTDs) including anencephaly and spina bifida are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or life-long severe complications (spina bifida). Despite hundreds of genetic mouse models having neural tube defect phenotypes, the genetics of human NTDs are poorly understood.

Loss of POGZ alters neural differentiation of human embryonic stem cells.

POGZ is a pogo transposable element derived protein with multiple zinc finger domains. Many de novo loss-of-function (LoF) variants of the POGZ gene are associated with autism and other neurodevelopmental disorders. However, the role of POGZ in human cortical development remains poorly understood.

Identification of neural oscillations and epileptiform changes in human brain organoids.

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations.

Definitions of Drug-Resistant Epilepsy for Administrative Claims Data Research.

Background And Objective: To assess the accuracy of definitions of drug-resistant epilepsy applied to administrative claims data.

Methods: We randomly sampled 450 patients from a tertiary health system with ≥1 epilepsy/convulsion encounter, ≥2 distinct antiseizure medications (ASMs) from 2014 to 2020, and ≥2 years of electronic medical records (EMR) data. We established a drug-resistant epilepsy diagnosis at a specific visit by reviewing EMR data and using a rubric based on the 2010 International League Against Epilepsy definition.

STRADA-mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation.

Genetic diseases involving overactivation of the mechanistic target of rapamycin (mTOR) pathway, so-called "mTORopathies," often manifest with malformations of cortical development (MCDs), epilepsy, and cognitive impairment. How mTOR pathway hyperactivation results in abnormal human cortical development is poorly understood. To study the effect of mTOR hyperactivity on early stages of cortical development, we focused on Pretzel Syndrome (polyhydramnios, megalencephaly, symptomatic epilepsy; PMSE syndrome), a rare mTORopathy caused by homozygous germline mutations in the STRADA gene.

Multimodal Analysis of STRADA Function in Brain Development.

mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9-13) in the pseudokinase STE20-related kinase adaptor alpha (; ), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited mouse N2a cells, a germline mouse knockout (KO-/-) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the founder mutation.

A native function for RAN translation and CGG repeats in regulating fragile X protein synthesis.

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5'-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production.

Altered Synaptic Drive onto Birthdated Dentate Granule Cells in Experimental Temporal Lobe Epilepsy.

Dysregulated adult hippocampal neurogenesis occurs in many temporal lobe epilepsy (TLE) models. Most dentate granule cells (DGCs) generated in response to an epileptic insult develop features that promote increased excitability, including ectopic location, persistent hilar basal dendrites (HBDs), and mossy fiber sprouting. However, some appear to integrate normally and even exhibit reduced excitability compared to other DGCs.

Modeling genetic epilepsies in a dish.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, provide a powerful platform for mechanistic studies of disorders of neurodevelopment and neural networks. hPSC models of autism, epilepsy, and other neurological disorders are also advancing the path toward designing and testing precision therapies. The field is evolving rapidly with the addition of genome-editing approaches, expanding protocols for the two-dimensional (2D) differentiation of different neuronal subtypes, and three-dimensional (3D) human brain organoid cultures.

deletion in adult mice results in seizures and SUDEP.

Pathogenic loss-of-function variants in are linked to Dravet syndrome (DS). Previous work suggested that neuronal pathfinding defects underlie epileptogenesis and SUDEP in the null mouse model of DS. We tested this hypothesis by inducing deletion in adult mice that had developed normally.

Prominent role of forebrain excitatory neurons in SCN8A encephalopathy.

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities.

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts.

For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes.