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. Two of the four patients exhibited a rapid and marked surge of gamma power, surge of cross-frequency coupling of gamma waves with slower oscillations, and increased interhemispheric functional and directed connectivity in gamma bands. High-frequency oscillations paralleled the activation of beta/gamma cross-frequency coupling within the somatosensory cortices. Importantly, both patients displayed surges of functional and directed connectivity at multiple frequency bands within the posterior cortical "hot zone," a region postulated to be critical for conscious processing. This gamma activity was stimulated by global hypoxia and surged further as cardiac conditions deteriorated in the dying patients. These data demonstrate that the surge of gamma power and connectivity observed in animal models of cardiac arrest can be observed in select patients during the process of dying.

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. METHODS: Depdc5 was specifically deleted in excitatory or inhibitory neurons in the mouse brain to determine neuronal subtypes that drive epileptogenesis and SUDEP. Electroencephalogram (EEG), cardiac, and respiratory recordings were performed to determine cardiorespiratory phenotypes associated with SUDEP. Baseline respiratory function and the response to hypoxia challenge were also studied in these mice. RESULTS: Depdc5 deletion in excitatory neurons in cortical layer 5 and dentate gyrus caused frequent generalized tonic-clonic seizures and SUDEP in young adult mice, but Depdc5 deletion in cortical interneurons did not. EEG suppression immediately following ictal offset was observed in fatal and non-fatal seizures, but low amplitude rhythmic theta frequency activity was lost only in fatal seizures. In addition, these mice developed baseline respiratory dysfunction prior to SUDEP, during which ictal apnea occurred long before terminal cardiac asystole. INTERPRETATION: Depdc5 deletion in excitatory neurons is sufficient to cause DEPDC5-related epilepsy and SUDEP. Ictal apnea and respiratory dysregulation play critical roles in SUDEP. Our study also provides a novel mouse model to investigate the underlying mechanisms of DEPDC5-related epilepsy and SUDEP. ANN NEUROL 2023;94:812-824.

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. To explore the microglial role in repair, we used liposomal clodronate or colony stimulating factor-1 receptor (csf1r) inhibitor to suppress microglia after brain injury, and also examined regeneration in two genetic mutant lines that lack microglia. We found that microglial ablation impaired telencephalic regeneration after injury. Microglial suppression attenuated cell proliferation at the intermediate progenitor cell amplification stage of neurogenesis. Notably, the loss of microglia impaired phospho-Stat3 (signal transducer and activator of transcription 3) and ß-Catenin signaling after injury. Furthermore, the ectopic activation of Stat3 and ß-Catenin rescued neurogenesis defects caused by microglial loss. Microglial suppression also prolonged the post-injury inflammatory phase characterized by neutrophil accumulation, likely hindering the resolution of inflammation. These findings reveal specific roles of microglia and inflammatory signaling during zebrafish telencephalic regeneration that should advance strategies to improve mammalian brain repair.

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. Here we generated multiple POGZ LoF lines in H9 human embryonic stem cells (hESCs) using CRISPR/CAS9 genome editing. These lines were then differentiated into neural structures, similar to those found in early to mid-fetal human brain, a critical developmental stage for studying disease mechanisms of neurodevelopmental disorders. We found that the loss of POGZ reduced neural stem cell proliferation in excitatory cortex-patterned neural rosettes, structures analogous to the cortical ventricular zone in human fetal brain. As a result, fewer intermediate progenitor cells and early born neurons were generated. In addition, neuronal migration from the apical center to the basal surface of neural rosettes was perturbed due to the loss of POGZ. Furthermore, cortical-like excitatory neurons derived from multiple POGZ homozygous knockout lines exhibited a more simplified dendritic architecture compared to wild type lines. Our findings demonstrate how POGZ regulates early neurodevelopment in the context of human cells, and provide further understanding of the cellular pathogenesis of neurodevelopmental disorders associated with POGZ variants.

Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome.

OBJECTIVE: SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Nav 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment. This study was carried out to determine whether reducing the abundance of the Scn8a transcript with an antisense oligonucleotide (ASO) would delay seizure onset and prolong survival in a mouse model of SCN8A encephalopathy. METHODS: ASO treatment was tested in a conditional mouse model with Cre-dependent expression of the pathogenic patient SCN8A mutation p.Arg1872Trp (R1872W). This model exhibits early onset of seizures, rapid progression, and 100% penetrance. An Scn1a +/- haploinsufficient mouse model of Dravet syndrome was also treated. ASO was administered by intracerebroventricular injection at postnatal day 2, followed in some cases by stereotactic injection at postnatal day 30. RESULTS: We observed a dose-dependent increase in length of survival from 15 to 65 days in the Scn8a-R1872W/+ mice treated with ASO. Electroencephalographic recordings were normal prior to seizure onset. Weight gain and activity in an open field were unaffected, but treated mice were less active in a wheel running assay. A single treatment with Scn8a ASO extended survival of Dravet syndrome mice from 3 weeks to >5 months. INTERPRETATION: Reduction of Scn8a transcript by 25 to 50% delayed seizure onset and lethality in mouse models of SCN8A encephalopathy and Dravet syndrome. Reduction of SCN8A transcript is a promising approach to treatment of intractable childhood epilepsies. Ann Neurol 2020;87:339-346.

Variant-specific changes in persistent or resurgent sodium current in SCN8A-related epilepsy patient-derived neurons.

Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872>L (Patient 1); p.V1592>L (Patient 2); and p.N1759>S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

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. However, the application of these techniques to study complex neurological disorders, including the epilepsies, remains a challenge. Here, we review previous work using both 2D and 3D hPSC models of genetic epilepsies, as well as recent advances in the field. We also describe new strategies for applying these technologies to disease modeling of genetic epilepsies, and discuss current challenges and future directions.

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. To examine the relationship between DGC birthdate, morphology, and network integration in a model of TLE, we retrovirally birthdated either early-born [EB; postnatal day (P)7] or adult-born (AB; P60) DGCs. Male rats underwent pilocarpine-induced status epilepticus (SE) or sham treatment at P56. Three to six months after SE or sham treatment, we used whole-cell patch-clamp and fluorescence microscopy to record spontaneous excitatory and inhibitory currents from birthdated DGCs. We found that both AB and EB populations of DGCs recorded from epileptic rats received increased excitatory input compared with age-matched controls. Interestingly, when AB populations were separated into normally integrated (normotopic) and aberrant (ectopic or HBD-containing) subpopulations, only the aberrant populations exhibited a relative increase in excitatory input (amplitude, frequency, and charge transfer). The ratio of excitatory-to-inhibitory input was most dramatically upregulated for ectopically localized DGCs. These data provide definitive physiological evidence that aberrant integration of post-SE, AB DGCs contributes to increased synaptic drive and support the idea that ectopic DGCs serve as putative hub cells to promote seizures.SIGNIFICANCE STATEMENT Adult dentate granule cell (DGC) neurogenesis is altered in rodent models of temporal lobe epilepsy (TLE). Some of the new neurons show abnormal morphology and integration, but whether adult-generated DGCs contribute to the development of epilepsy is controversial. We examined the synaptic inputs of age-defined populations of DGCs using electrophysiological recordings and fluorescent retroviral reporter birthdating. DGCs generated neonatally were compared with those generated in adulthood, and adult-born (AB) neurons with normal versus aberrant morphology or integration were examined. We found that AB, ectopically located DGCs exhibit the most pro-excitatory physiological changes, implicating this population in seizure generation or progression.

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. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.

Somatic Depdc5 deletion recapitulates electroclinical features of human focal cortical dysplasia type IIA.

Epileptogenic mechanisms in focal cortical dysplasia (FCD) remain elusive, as no animal models faithfully recapitulate FCD seizures, which have distinct electrographic features and a wide range of semiologies. Given that DEPDC5 plays significant roles in focal epilepsies with FCD, we used in utero electroporation with clustered regularly interspaced short palindromic repeats gene deletion to create focal somatic Depdc5 deletion in the rat embryonic brain. Animals developed spontaneous seizures with focal pathological and electroclinical features highly clinically relevant to FCD IIA, paving the way toward understanding its pathogenesis and developing mechanistic-based therapies. Ann Neurol 2018;83:140-146.

Fibroblast growth factor 2 regulates activity and gene expression of human post-mitotic excitatory neurons.

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature, post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline, the vast majority of cells are post-mitotic, and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f, we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1, ETV4, SPRY4, and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3, SYT2, and GALR1. Since several of these genes have been implicated in MDD previously, these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD.

Rabies tracing of birthdated dentate granule cells in rat temporal lobe epilepsy.

OBJECTIVE: To understand how monosynaptic inputs onto adult-born dentate granule cells (DGCs) are altered in experimental mesial temporal lobe epilepsy (mTLE) and whether their integration differs from early-born DGCs that are mature at the time of epileptogenesis. METHODS: A dual-virus tracing strategy combining retroviral birthdating with rabies virus-mediated putative retrograde trans-synaptic tracing was used to identify and compare presynaptic inputs onto adult-born and early-born DGCs in the rat pilocarpine model of mTLE. RESULTS: Our results demonstrate that hilar ectopic DGCs preferentially synapse onto adult-born DGCs after pilocarpine-induced status epilepticus (SE), whereas normotopic DGCs synapse onto both adult-born and early-born DGCs. We also find that parvalbumin- and somatostatin- interneuron inputs are greatly diminished onto early-born DGCs after SE. However, somatostatin- interneuron inputs onto adult-born DGCs are maintained, likely due to preferential sprouting. Intriguingly, CA3 pyramidal cell backprojections that specifically target adult-born DGCs arise in the epileptic brain, whereas axons of interneurons and pyramidal cells in CA1 appear to sprout across the hippocampal fissure to preferentially synapse onto early-born DGCs. INTERPRETATION: These data support the presence of substantial hippocampal circuit remodeling after an epileptogenic insult that generates prominent excitatory monosynaptic inputs, both local recurrent and widespread feedback loops, onto DGCs. Both adult-born and early-born DGCs are targets of new inputs from other DGCs as well as from CA3 and CA1 pyramidal cells after pilocarpine treatment, changes that likely contribute to epileptogenesis in experimental mTLE. Ann Neurol 2017;81:790-803.

Convulsive seizures and SUDEP in a mouse model of SCN8A epileptic encephalopathy.

De novo mutations of the voltage-gated sodium channel gene SCN8A have recently been recognized as a cause of epileptic encephalopathy, which is characterized by refractory seizures with developmental delay and cognitive disability. We previously described the heterozygous SCN8A missense mutation p.Asn1768Asp in a child with epileptic encephalopathy that included seizures, ataxia, and sudden unexpected death in epilepsy (SUDEP). The mutation results in increased persistent sodium current and hyperactivity of transfected neurons. We have characterized a knock-in mouse model expressing this dominant gain-of-function mutation to investigate the pathology of the altered channel in vivo. The mutant channel protein is stable in vivo. Heterozygous Scn8a(N1768D/+) mice exhibit seizures and SUDEP, confirming the causality of the de novo mutation in the proband. Using video/EEG analysis, we detect ictal discharges that coincide with convulsive seizures and myoclonic jerks. Prior to seizure onset, heterozygous mutants are not defective in motor learning or fear conditioning, but do exhibit mild impairment of motor coordination and social discrimination. Homozygous mutant mice exhibit earlier seizure onset than heterozygotes and more rapid progression to death. Analysis of the intermediate phenotype of functionally hemizygous Scn8a(N1768D/-) mice indicates that severity is increased by a double dose of mutant protein and reduced by the presence of wild-type protein. Scn8a(N1768D) mutant mice provide a model of epileptic encephalopathy that will be valuable for studying the in vivo effects of hyperactive Nav1.6 and the response to therapeutic interventions.

Concise Review: Exciting Cells: Modeling Genetic Epilepsies with Patient-Derived Induced Pluripotent Stem Cells.

Human induced pluripotent stem cell (iPSC) models of epilepsy are becoming a revolutionary platform for mechanistic studies and drug discovery. The skyrocketing pace of epilepsy gene discovery is vastly outstripping the development of in vivo animal models. Currently, antiepileptic drug prescribing to patients with specific genetic epilepsies is based on small-scale clinical trials and empiricism; however, rapid production of patient-derived iPSC models will allow for precision therapy. We review iPSC-based studies that have already afforded novel discoveries in diseases with epileptic phenotypes, as well as challenges to using iPSC-based neurological disease models. We also discuss iPSC-derived cardiomyocyte studies of arrhythmia-inducing ion channelopathies that exemplify novel drug discovery and use of multielectrode array technology that can be translated to epilepsy research. Beyond initial studies of Rett, Timothy, Phelan-McDermid, and Dravet syndromes, the stage is set for groundbreaking iPSC-based mechanistic and therapeutic discoveries in genetic epilepsies with the potential to impact patient treatment and quality of life.

SCN8A encephalopathy: Research progress and prospects.

On April 21, 2015, the first SCN8A Encephalopathy Research Group convened in Washington, DC, to assess current research into clinical and pathogenic features of the disorder and prepare an agenda for future research collaborations. The group comprised clinical and basic scientists and representatives of patient advocacy groups. SCN8A encephalopathy is a rare disorder caused by de novo missense mutations of the sodium channel gene SCN8A, which encodes the neuronal sodium channel Nav 1.6. Since the initial description in 2012, approximately 140 affected individuals have been reported in publications or by SCN8A family groups. As a result, an understanding of the severe impact of SCN8A mutations is beginning to emerge. Defining a genetic epilepsy syndrome goes beyond identification of molecular etiology. Topics discussed at this meeting included (1) comparison between mutations of SCN8A and the SCN1A mutations in Dravet syndrome, (2) biophysical properties of the Nav 1.6 channel, (3) electrophysiologic effects of patient mutations on channel properties, (4) cell and animal models of SCN8A encephalopathy, (5) drug screening strategies, (6) the phenotypic spectrum of SCN8A encephalopathy, and (7) efforts to develop a bioregistry. A panel discussion of gaps in bioregistry, biobanking, and clinical outcomes data was followed by a planning session for improved integration of clinical and basic science research. Although SCN8A encephalopathy was identified only recently, there has been rapid progress in functional analysis and phenotypic classification. The focus is now shifting from identification of the underlying molecular cause to the development of strategies for drug screening and prioritized patient care.

Abnormal neuronal patterning occurs during early postnatal brain development of Scn1b-null mice and precedes hyperexcitability.

Voltage-gated Na(+) channel (VGSC) ß1 subunits, encoded by SCN1B, are multifunctional channel modulators and cell adhesion molecules (CAMs). Mutations in SCN1B are associated with the genetic epilepsy with febrile seizures plus (GEFS+) spectrum disorders in humans, and Scn1b-null mice display severe spontaneous seizures and ataxia from postnatal day (P)10. The goal of this study was to determine changes in neuronal pathfinding during early postnatal brain development of Scn1b-null mice to test the hypothesis that these CAM-mediated roles of Scn1b may contribute to the development of hyperexcitability. c-Fos, a protein induced in response to seizure activity, was up-regulated in the Scn1b-null brain at P16 but not at P5. Consistent with this, epileptiform activity was observed in hippocampal and cortical slices prepared from the P16 but not from the P5-P7 Scn1b-null brain. On the basis of these results, we investigated neuronal pathfinding at P5. We observed disrupted fasciculation of parallel fibers in the P5 null cerebellum. Further, P5 null mice showed reduced neuron density in the dentate gyrus granule cell layer, increased proliferation of granule cell precursors in the hilus, and defective axonal extension and misorientation of somata and processes of inhibitory neurons in the dentate gyrus and CA1. Thus, Scn1b is critical for neuronal proliferation, migration, and pathfinding during the critical postnatal period of brain development. We propose that defective neuronal proliferation, migration, and pathfinding in response to Scn1b deletion may contribute to the development of hyperexcitability.

Using Patient-Derived Induced Pluripotent Stem Cells to Model and Treat Epilepsies.

Human induced pluripotent stem cells (iPSCs) are transforming the fields of disease modeling and precision therapy. For the treatment of neurological disorders, iPSCs introduce the possibility for targeted cell-based therapies by deriving patient-specific neural tissue in vitro that may ultimately be used for transplantation. We review iPSC technologies and their applications that have already advanced our understanding of neurological disorders, focusing on the epilepsies. We also discuss the application of powerful new tools such as genome editing and multi-well, multi-electrode array recording platforms to iPSC disease modeling and therapy development for the epilepsies. Despite some limitations, the field of iPSCs is evolving rapidly and is quickly becoming vital for understanding mechanisms of genetic epilepsies and for future patient-specific therapeutic applications.

Excitotoxic brain injury in adult zebrafish stimulates neurogenesis and long-distance neuronal integration.

Zebrafish maintain a greater capacity than mammals for central nervous system repair after injury. Understanding differences in regenerative responses between different vertebrate species may shed light on mechanisms to improve repair in humans. Quinolinic acid is an excitotoxin that has been used to induce brain injury in rodents for modeling Huntington's disease and stroke. When injected into the adult rodent striatum, this toxin stimulates subventricular zone neurogenesis and neuroblast migration to injury. However, most new neurons fail to survive and lesion repair is minimal. We used quinolinic acid to lesion the adult zebrafish telencephalon to study reparative processes. We also used conditional transgenic lineage mapping of adult radial glial stem cells to explore survival and integration of neurons generated after injury. Telencephalic lesioning with quinolinic acid, and to a lesser extent vehicle injection, produced cell death, microglial infiltration, increased cell proliferation, and enhanced neurogenesis in the injured hemisphere. Lesion repair was more complete with quinolinic acid injection than after vehicle injection. Fate mapping of her4-expressing radial glia showed injury-induced expansion of radial glial stem cells that gave rise to neurons which migrated to injury, survived at least 8 weeks and formed long-distance projections that crossed the anterior commissure and synapsed in the contralateral hemisphere. These findings suggest that quinolinic acid lesioning of the zebrafish brain stimulates adult neural stem cells to produce robust regeneration with long-distance integration of new neurons. This model should prove useful for elucidating reparative mechanisms that can be applied to restorative therapies for mammalian brain injury.

Gain-of-function mutation in Gnao1: a murine model of epileptiform encephalopathy (EIEE17)?

G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gαo, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1 (+/G184S)) that prevents Go turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41-70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.

OBJECTIVE: Neuronal channelopathies cause brain disorders, including epilepsy, migraine, and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders remain uncertain. One particularly devastating channelopathy is Dravet syndrome (DS), a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Na(v) 1.1. Heterologous expression of mutant channels suggests loss of function, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Mouse model studies suggest that decreased Na(v) 1.1 function in interneurons causes disinhibition. We aim to determine how mutant SCN1A affects human neurons using the induced pluripotent stem cell (iPSC) method to generate patient-specific neurons. METHODS: Here we derive forebrain-like pyramidal- and bipolar-shaped neurons from 2 DS subjects and 3 human controls by iPSC reprogramming of fibroblasts. DS and control iPSC-derived neurons are compared using whole-cell patch clamp recordings. Sodium current density and intrinsic neuronal excitability are examined. RESULTS: Neural progenitors from DS and human control iPSCs display a forebrain identity and differentiate into bipolar- and pyramidal-shaped neurons. DS patient-derived neurons show increased sodium currents in both bipolar- and pyramidal-shaped neurons. Consistent with increased sodium currents, both types of patient-derived neurons show spontaneous bursting and other evidence of hyperexcitability. Sodium channel transcripts are not elevated, consistent with a post-translational mechanism. INTERPRETATION: These data demonstrate that epilepsy patient-specific iPSC-derived neurons are useful for modeling epileptic-like hyperactivity. Our findings reveal a previously unrecognized cell-autonomous epilepsy mechanism potentially underlying DS, and offer a platform for screening new antiepileptic therapies.