Glioblastoma disrupts cortical network activity at multiple spatial and temporal scales.

The emergence of glioblastoma in cortical tissue initiates early and persistent neural hyperexcitability with signs ranging from mild cognitive impairment to convulsive seizures. The influence of peritumoral synaptic density, expansion dynamics, and spatial contours of excess glutamate upon higher order neuronal network modularity is unknown. We combined cellular and widefield imaging of calcium and glutamate fluorescent reporters in two glioblastoma mouse models with distinct synaptic microenvironments and infiltration profiles. Functional metrics of neural ensembles are dysregulated during tumor invasion depending on the stage of malignant progression and tumor cell proximity. Neural activity is differentially modulated during periods of accelerated and inhibited tumor expansion. Abnormal glutamate accumulation precedes and outpaces the spatial extent of baseline neuronal calcium signaling, indicating these processes are uncoupled in tumor cortex. Distinctive excitability homeostasis patterns and functional connectivity of local and remote neuronal populations support the promise of precision genetic diagnosis and management of this devastating brain disease.

Developing a pathway to clinical trials for CACNA1A-related epilepsies: A patient organization perspective.

CACNA1A-related disorders are rare neurodevelopmental disorders linked to variants in the CACNA1A gene. This gene encodes the α1 subunit of the P/Q-type calcium channel Cav2.1, which is globally expressed in the brain and crucial for fast synaptic neurotransmission. The broad spectrum of CACNA1A-related neurological disorders includes developmental and epileptic encephalopathies, familial hemiplegic migraine type 1, episodic ataxia type 2, spinocerebellar ataxia type 6, together with unclassified presentations with developmental delay, ataxia, intellectual disability, autism spectrum disorder, and language impairment. The severity of each disorder is also highly variable. The spectrum of CACNA1A-related seizures is broad across both loss-of-function and gain-of-function variants and includes absence seizures, focal seizures with altered consciousness, generalized tonic-clonic seizures, tonic seizures, status epilepticus, and infantile spasms. Furthermore, over half of CACNA1A-related epilepsies are refractory to current therapies. To date, almost 1700 CACNA1A variants have been reported in ClinVar, with over 400 listed as Pathogenic or Likely Pathogenic, but with limited-to-no clinical or functional data. Robust genotype-phenotype studies and impacts of variants on protein structure and function have also yet to be established. As a result, there are few definitive treatment options for CACNA1A-related epilepsies. The CACNA1A Foundation has set out to change the landscape of available and effective treatments and improve the quality of life for those living with CACNA1A-related disorders, including epilepsy. Established in March 2020, the Foundation has built a robust preclinical toolbox that includes patient-derived induced pluripotent stem cells and novel disease models, launched clinical trial readiness initiatives, and organized a global CACNA1A Research Network. This Research Network is currently composed of over 60 scientists and clinicians committed to collaborating to accelerate the path to CACNA1A-specific treatments and one day, a cure.

Designing a plan to find treatments for epilepsies linked to the CACNA1A gene and test them in clinical trials for FDA approval CACNA1A-related disorders are rare conditions that affect brain development and are caused by changes in the CACNA1A gene. This gene provides instructions for making a protein called Cav2.1, which plays a crucial role in fast communication between nerve cells. The disorders can lead to various neurological problems such as seizures, epilepsy, developmental delays, intellectual disability, and autism. The severity of these disorders varies, and individuals may experience a broad range of seizures. More than 1700 different genetic changes in the CACNA1A gene have been identified, with over 400 considered likely to cause the disorders. However, there is limited information on the clinical and molecular aspects of these changes. Despite the significant impact on individuals' lives, there are currently no definitive treatments for CACNA1A-related epilepsies. To address this gap, the CACNA1A Foundation was established in March 2020. The Foundation aims to improve the lives of individuals with CACNA1A-related disorders, including epilepsy. It has developed a comprehensive set of tools, including patient-derived cells and new disease models, to advance research. Additionally, the Foundation has initiated initiatives to prepare for clinical trials and has formed a global CACNA1A Research Network with over 60 scientists and clinicians collaborating to develop specific treatments and, ultimately, find a cure.

Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy.

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

Abnormal patterns of sleep and waking behaviors are accompanied by neocortical oscillation disturbances in an Ank3 mouse model of epilepsy-bipolar disorder comorbidity.

ANK3 is a leading bipolar disorder (BD) candidate gene in humans and provides a unique opportunity for studying epilepsy-BD comorbidity. Previous studies showed that deletion of Ank3-1b, a BD-associated variant of Ank3 in mice leads to increased firing threshold and diminished action potential dynamic range of parvalbumin (PV) interneurons and absence epilepsy, thus providing a biological mechanism linking epilepsy and BD. To explore the behavioral overlap of these disorders, we characterized behavioral patterns of Ank3-1b KO mice during overnight home-cage activity and examined network activity during these behaviors using paired video and EEG recordings. Since PV interneurons contribute to the generation of high-frequency gamma oscillations, we anticipated changes in the power of neocortical EEG signals in the gamma frequency range (> 25 Hz) during behavioral states related to human BD symptoms, including abnormal sleep, hyperactivity, and repetitive behaviors. Ank3-1b KO mice exhibited an overall increase in slow gamma (~25-45 Hz) power compared to controls, and slow gamma power correlated with seizure phenotype severity across behaviors. During sleep, increased slow gamma power correlated with decreased time spent in the rapid eye movement (REM) stage of sleep. Seizures were more common during REM sleep compared to non-REM (NREM) sleep. We also found that Ank3-1b KO mice were hyperactive and exhibited a repetitive behavior phenotype that co-occurred with increased slow gamma power. Our results identify a novel EEG biomarker associating Ank3 genetic variation with BD and epilepsy and suggest modulation of gamma oscillations as a potential therapeutic target.

Targeted suppression of mTORC2 reduces seizures across models of epilepsy.

Epilepsy is a neurological disorder that poses a major threat to public health. Hyperactivation of mTOR complex 1 (mTORC1) is believed to lead to abnormal network rhythmicity associated with epilepsy, and its inhibition is proposed to provide some therapeutic benefit. However, mTOR complex 2 (mTORC2) is also activated in the epileptic brain, and little is known about its role in seizures. Here we discover that genetic deletion of mTORC2 from forebrain neurons is protective against kainic acid-induced behavioral and EEG seizures. Furthermore, inhibition of mTORC2 with a specific antisense oligonucleotide robustly suppresses seizures in several pharmacological and genetic mouse models of epilepsy. Finally, we identify a target of mTORC2, Nav1.2, which has been implicated in epilepsy and neuronal excitability. Our findings, which are generalizable to several models of human seizures, raise the possibility that inhibition of mTORC2 may serve as a broader therapeutic strategy against epilepsy.

A hyperthermic seizure unleashes a surge of spreading depolarizations in Scn1a-deficient mice.

Spreading depolarization (SD) is a massive wave of cellular depolarization that slowly migrates across the brain gray matter. Cortical SD is frequently generated following brain injury, while less is understood about its potential contribution to genetic disorders of hyperexcitability, such as SCN1A-deficient epilepsy, in which febrile seizure often contributes to disease initiation. Here we report that spontaneous SD waves are predominant EEG abnormalities in the Scn1a-deficient mouse (Scn1a+/R1407X) and undergo sustained intensification following a single hyperthermic seizure. Chronic DC-band EEG recording detected spontaneous SDs, seizures, and seizure-SD complexes in Scn1a+/R1407X mice but not WT littermates. The SD events were infrequent, while a single hyperthermia-induced seizure robustly increased SD frequency over 4-fold during the initial postictal week. This prolonged neurological aftermath could be suppressed by memantine administration. Video, electromyogram, and EEG spectral analysis revealed distinct neurobehavioral patterns; individual seizures were associated with increased motor activities, while SDs were generally associated with immobility. We also identified a stereotypic SD prodrome, detectable over a minute before the onset of the DC potential shift, characterized by increased motor activity and bilateral EEG frequency changes. Our study suggests that cortical SD is a pathological manifestation in SCN1A-deficient epileptic encephalopathy.

Remote neuronal activity drives glioma progression through SEMA4F.

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

Remote neuronal activity drives glioma infiltration via Sema4f.

The tumor microenvironment (TME) plays an essential role in malignancy and neurons have emerged as a key component of the TME that promotes tumorigenesis across a host of cancers. Recent studies on glioblastoma (GBM) highlight bi-directional signaling between tumors and neurons that propagates a vicious cycle of proliferation, synaptic integration, and brain hyperactivity; however, the identity of neuronal subtypes and tumor subpopulations driving this phenomenon are incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumors promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity dependent infiltrating population present at the leading edge of mouse and human tumors that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified Sema4F as a key regulator of tumorigenesis and activity-dependent infiltration. Furthermore, Sema4F promotes the activity-dependent infiltrating population and propagates bi-directional signaling with neurons by remodeling tumor adjacent synapses towards brain network hyperactivity. Collectively, our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, while revealing new mechanisms of tumor infiltration that are regulated by neuronal activity.

WWOX P47T partial loss-of-function mutation induces epilepsy, progressive neuroinflammation, and cerebellar degeneration in mice phenocopying human SCAR12.

WWOX gene loss-of-function (LoF) has been associated with neuropathologies resulting in developmental, epileptic, and ataxic phenotypes of varying severity based on the level of WWOX dysfunction. WWOX gene biallelic germline variant p.Pro47Thr (P47T) has been causally associated with a new form of autosomal recessive cerebellar ataxia with epilepsy and intellectual disability (SCAR12, MIM:614322). This mutation affecting the WW1 protein binding domain of WWOX, impairs its interaction with canonical proline-proline-X-tyrosine motifs in partner proteins. We generated a mutant knock-in mouse model of Wwox P47T mutation that phenocopies human SCAR12. WwoxP47T/P47T mice displayed epilepsy, profound social behavior and cognition deficits, and poor motor coordination, and unlike KO models that survive only for 1 month, live beyond 1 year of age. These deficits progressed with age and mice became practically immobile, suggesting severe cerebellar dysfunction. WwoxP47T/P47T mice brains revealed signs of progressive neuroinflammation with elevated astro-microgliosis that increased with age. Cerebellar cortex displayed significantly reduced molecular and granular layer thickness and a strikingly reduced number of Purkinje cells with degenerated dendrites. Transcriptome profiling from various brain regions of WW domain LoF mice highlighted widespread changes in neuronal and glial pathways, enrichment of bioprocesses related to neuroinflammation, and severe cerebellar dysfunction. Our results show significant pathobiological effects and potential mechanisms through which WWOX partial LoF leads to epilepsy, cerebellar neurodegeneration, neuroinflammation, and ataxia. Additionally, the mouse model described here will be a useful tool to understand the role of WWOX in common neurodegenerative conditions in which this gene has been identified as a novel risk factor.

Glioma epileptiform activity and progression are driven by IGSF3-mediated potassium dysregulation.

Seizures are a frequent pathophysiological feature of malignant glioma. Recent studies implicate peritumoral synaptic dysregulation as a driver of brain hyperactivity and tumor progression; however, the molecular mechanisms that govern these phenomena remain elusive. Using scRNA-seq and intraoperative patient ECoG recordings, we show that tumors from seizure patients are enriched for gene signatures regulating synapse formation. Employing a human-to-mouse in vivo functionalization pipeline to screen these genes, we identify IGSF3 as a mediator of glioma progression and dysregulated neural circuitry that manifests as spreading depolarization (SD). Mechanistically, we discover that IGSF3 interacts with Kir4.1 to suppress potassium buffering and found that seizure patients exhibit reduced expression of potassium handlers in proliferating tumor cells. In vivo imaging reveals that dysregulated synaptic activity emanates from the tumor-neuron interface, which we confirm in patients. Our studies reveal that tumor progression and seizures are enabled by ion dyshomeostasis and identify SD as a driver of disease.

The Role of Hyperexcitability in Gliomagenesis.

Glioblastoma is the most common malignant primary brain tumor. Recent studies have demonstrated that excitatory or activity-dependent signaling-both synaptic and non-synaptic-contribute to the progression of glioblastoma. Glutamatergic receptors may be stimulated via neuron-tumor synapses or release of glutamate by the tumor itself. Ion currents generated by these receptors directly alter the structure of membrane adhesion molecules and cytoskeletal proteins to promote migratory behavior. Additionally, the hyperexcitable milieu surrounding glioma increases the rate at which tumor cells proliferate and drive recurrent disease. Inhibition of excitatory signaling has shown to effectively reduce its pro-migratory and -proliferative effects.

Loss of functional System x-c uncouples aberrant postnatal neurogenesis from epileptogenesis in the hippocampus of Kcna1-KO mice.

Mutations in Kv1.1 (Kcna1) voltage-gated potassium channels in humans and mice generate network hyperexcitability, enhancing aberrant postnatal neurogenesis in the dentate subgranular zone, resulting in epilepsy and hippocampal hypertrophy. While Kcna1 loss stimulates proliferation of progenitor cell subpopulations, the identity of extrinsic molecular triggers linking network hyperexcitability to aberrant postnatal neurogenesis remains incomplete. System x-c (Sxc) is an inducible glutamate/cysteine antiporter that regulates extracellular glutamate. Here, we find that the functional unit of Sxc, xCT (Slc7a11), is upregulated in regions of Kcna1 knockout (KO) hippocampus, suggesting a contribution to both hyperplasia and epilepsy. However, Slc7a11 KO suppressed and rescued hippocampal enlargement without altering seizure severity in Kcna1-Slc7a11-KO mice. Microglial activation, but not astrocytosis, was also reduced. Our study identifies Sxc-mediated glutamate homeostasis as an essential non-synaptic trigger coupling aberrant postnatal neurogenesis and neuroimmune crosstalk, revealing that neurogenesis and epileptogenesis in the dentate gyrus are not mutually contingent events.

Emx1-Cre Is Expressed in Peripheral Autonomic Ganglia That Regulate Central Cardiorespiratory Functions.

The Emx1-IRES-Cre transgenic mouse is commonly used to direct genetic recombination in forebrain excitatory neurons. However, the original study reported that Emx1-Cre is also expressed embryonically in peripheral autonomic ganglia, which could potentially affect the interpretation of targeted circuitry contributing to systemic phenotypes. Here, we report that Emx1-Cre is expressed in the afferent vagus nerve system involved in autonomic cardiorespiratory regulatory pathways. Our imaging studies revealed expression of Emx1-Cre driven tdtomato fluorescence in the afferent vagus nerve innervating the dorsal medulla of brainstem, cell bodies in the nodose ganglion, and their potential target structures at the carotid bifurcation such as the carotid sinus and the superior cervical ganglion (SCG). Photostimulation of the afferent terminals in the nucleus tractus solitarius (NTS) in vitro using Emx1-Cre driven ChR2 reliably evoked EPSCs in the postsynaptic neurons with electrophysiological characteristics consistent with the vagus afferent nerves. In addition, optogenetic stimulation targeting the Emx1-Cre expressing structures identified in this study, such as vagus nerve, carotid bifurcation, and the dorsal medulla surface transiently depressed cardiorespiratory rate in urethane anesthetized mice in vivo Together, our study demonstrates that Emx1-IRES-Cre is expressed in the key peripheral autonomic nerve system and can modulate cardiorespiratory function independently of forebrain expression. These results raise caution when interpreting systemic phenotypes of Emx1-IRES-Cre conditional recombinant mice, and also suggest the utility of this line to investigate modulators of the afferent vagal system.

Kctd7 deficiency induces myoclonic seizures associated with Purkinje cell death and microvascular defects.

Mutations in the potassium channel tetramerization domain-containing 7 (KCTD7) gene are associated with a severe neurodegenerative phenotype characterized by childhood onset of progressive and intractable myoclonic seizures accompanied by developmental regression. KCTD7-driven disease is part of a large family of progressive myoclonic epilepsy syndromes displaying a broad spectrum of clinical severity. Animal models of KCTD7-related disease are lacking, and little is known regarding how KCTD7 protein defects lead to epilepsy and cognitive dysfunction. We characterized Kctd7 expression patterns in the mouse brain during development and show that it is selectively enriched in specific regions as the brain matures. We further demonstrate that Kctd7-deficient mice develop seizures and locomotor defects with features similar to those observed in human KCTD7-associated diseases. We also show that Kctd7 is required for Purkinje cell survival in the cerebellum and that selective degeneration of these neurons is accompanied by defects in cerebellar microvascular organization and patterning. Taken together, these results define a new model for KCTD7-associated epilepsy and identify Kctd7 as a modulator of neuron survival and excitability linked to microvascular alterations in vulnerable regions.

X-linked serotonin 2C receptor is associated with a non-canonical pathway for sudden unexpected death in epilepsy.

Sudden Unexpected Death in Epilepsy is a leading cause of epilepsy-related mortality, and the analysis of mouse Sudden Unexpected Death in Epilepsy models is steadily revealing a spectrum of inherited risk phenotypes based on distinct genetic mechanisms. Serotonin (5-HT) signalling enhances post-ictal cardiorespiratory drive and, when elevated in the brain, reduces death following evoked audiogenic brainstem seizures in inbred mouse models. However, no gene in this pathway has yet been linked to a spontaneous epilepsy phenotype, the defining criterion of Sudden Unexpected Death in Epilepsy. Most monogenic models of Sudden Unexpected Death in Epilepsy invoke a failure of inhibitory synaptic drive as a critical pathogenic step. Accordingly, the G protein-coupled, membrane serotonin receptor 5-HT2C inhibits forebrain and brainstem networks by exciting GABAergic interneurons, and deletion of this gene lowers the threshold for lethal evoked audiogenic seizures. Here, we characterize epileptogenesis throughout the lifespan of mice lacking X-linked, 5-HT2C receptors (loxTB Htr2c). We find that loss of Htr2c generates a complex, adult-onset spontaneous epileptic phenotype with a novel progressive hyperexcitability pattern of absences, non-convulsive, and convulsive behavioural seizures culminating in late onset sudden mortality predominantly in male mice. RNAscope localized Htr2c mRNA in subsets of Gad2+ GABAergic neurons in forebrain and brainstem regions. To evaluate the contribution of 5-HT2C receptor-mediated inhibitory drive, we selectively spared their deletion in GAD2+ GABAergic neurons of pan-deleted loxTB Htr2c mice, yet unexpectedly found no amelioration of survival or epileptic phenotype, indicating that expression of 5-HT2C receptors in GAD2+ inhibitory neurons was not sufficient to prevent hyperexcitability and lethal seizures. Analysis of human Sudden Unexpected Death in Epilepsy and epilepsy genetic databases identified an enrichment of HTR2C non-synonymous variants in Sudden Unexpected Death in Epilepsy cases. Interestingly, while early lethality is not reflected in the mouse model, we also identified variants mainly among male Sudden Infant Death Syndrome patients. Our findings validate HTR2C as a novel, sex-linked candidate gene modifying Sudden Unexpected Death in Epilepsy risk, and demonstrate that the complex epilepsy phenotype does not arise solely from 5-HT2C-mediated synaptic disinhibition. These results strengthen the evidence for the serotonin hypothesis of Sudden Unexpected Death in Epilepsy risk in humans, and advance current efforts to develop gene-guided interventions to mitigate premature mortality in epilepsy.

Proceedings of the Sleep and Epilepsy Workshop: Section 3 Mortality: Sleep, Night, and SUDEP.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with refractory epilepsy. Likely pathophysiological mechanisms include seizure-induced cardiac and respiratory dysregulation. A frequently identified feature in SUDEP cases is that they occur at night. This raises the question of a role for sleep state in regulating of SUDEP. An association with sleep has been identified in a number of studies with patients and in animal models. The focus of this section of the Sleep and Epilepsy Workshop was on identifying and understanding the role for sleep and time of day in the pathophysiology of SUDEP.

Kcnq2/Kv7.2 controls the threshold and bi-hemispheric symmetry of cortical spreading depolarization.

Spreading depolarization is a slowly propagating wave of massive cellular depolarization associated with acute brain injury and migraine aura. Genetic studies link depolarizing molecular defects in Ca2+ flux, Na+ current in interneurons, and glial Na+-K+ ATPase with spreading depolarization susceptibility, emphasizing the important roles of synaptic activity and extracellular ionic homeostasis in determining spreading depolarization threshold. In contrast, although gene mutations in voltage-gated potassium ion channels that shape intrinsic membrane excitability are frequently associated with epilepsy susceptibility, it is not known whether epileptogenic mutations that regulate membrane repolarization also modify spreading depolarization threshold and propagation. Here we report that the Kcnq2/Kv7.2 potassium channel subunit, frequently mutated in developmental epilepsy, is a spreading depolarization modulatory gene with significant control over the seizure-spreading depolarization transition threshold, bi-hemispheric cortical expression, and diurnal temporal susceptibility. Chronic DC-band cortical EEG recording from behaving conditional Kcnq2 deletion mice (Emx1cre/+::Kcnq2flox/flox) revealed spontaneous cortical seizures and spreading depolarization. In contrast to the related potassium channel deficient model, Kv1.1-KO mice, spontaneous cortical spreading depolarizations in Kcnq2 cKO mice are tightly coupled to the terminal phase of seizures, arise bilaterally, and are observed predominantly during the dark phase. Administration of the non-selective Kv7.2 inhibitor XE991 to Kv1.1-KO mice partly reproduced the Kcnq2 cKO-like spreading depolarization phenotype (tight seizure coupling and bilateral symmetry) in these mice, indicating that Kv7.2 currents can directly and actively modulate spreading depolarization properties. In vitro brain slice studies confirmed that Kcnq2/Kv7.2 depletion or pharmacological inhibition intrinsically lowers the cortical spreading depolarization threshold, whereas pharmacological Kv7.2 activators elevate the threshold to multiple depolarizing and hypometabolic spreading depolarization triggers. Together these results identify Kcnq2/Kv7.2 as a distinctive spreading depolarization regulatory gene, and point to spreading depolarization as a potentially significant pathophysiological component of KCNQ2-linked epileptic encephalopathy syndromes. Our results also implicate KCNQ2/Kv7.2 channel activation as a potential adjunctive therapeutic target to inhibit spreading depolarization incidence.

Early 17ß-estradiol treatment reduces seizures but not abnormal behaviour in mice with expanded polyalanine tracts in the Aristaless related homeobox gene (ARX).

Children with severe intellectual disability have an increased prevalence of refractory seizures. Steroid treatment may improve seizure outcomes, but the mechanism remains unknown. Here we demonstrate that short term, daily delivery of an exogenous steroid 17ß-estradiol (40 ng/g) in early postnatal life significantly reduced the number and severity of seizures, but did not improve behavioural deficits, in mice modelling mutations in the Aristaless-related homeobox gene (ARX), expanding the first (PA1) or second (PA2) polyalanine tract. Frequency of observed seizures on handling (n = 14/treatment/genotype) were significantly reduced in PA1 (32% reduction) and more modestly reduced in PA2 mice (14% reduction) with steroid treatment compared to vehicle. Spontaneous seizures were assessed (n = 7/treatment/genotype) at 7 weeks of age coinciding with a peak of seizure activity in untreated mice. PA1 mice treated with steroids no longer present with the most severe category of prolonged myoclonic seizures. Treated PA2 mice had an earlier onset of seizures coupled with a subsequent reduction in seizures later in postnatal life, with a complete absence of any seizures during the analysis at 7 weeks of age. Despite the reduction in seizures, 17ß-estradiol treated mice showed no improvement in behavioural or cognitive outcomes in adulthood. For the first time we show that these deficits due to mutations in Arx are already present before seizure onset and do not worsen with seizures. ARX is a transcription factor and Arx PA mutant mice have deregulated transcriptome profiles in the developing embryonic brain. At postnatal day 10, treatment completion, RNAseq identified 129 genes significantly deregulated (Log2FC > ± 0.5, P-value<0.05) in the frontal cortex of mutant compared to wild-type mice. This list reflects genes deregulated in disease and was particularly enriched for known genes in neurodevelopmental disorders and those involved in signalling and developmental pathways. 17ß-estradiol treatment of mutant mice significantly deregulated 295 genes, with only 23 deregulated genes overlapping between vehicle and steroid treated mutant mice. We conclude that 17ß-estradiol treatment recruits processes and pathways to reduce the frequency and severity of seizures in the Arx PA mutant mice but does not precisely correct the deregulated transcriptome nor improve mortality or behavioural and cognitive deficits.

Night Watch on the Titanic: Detecting Early Signs of Epileptogenesis in Alzheimer Disease.

Aberrant cortical network excitability is an inextricable feature of Alzheimer disease (AD) that can negatively impact memory and accelerate cognitive decline. Surface electroencephalogram spikes and intracranial recordings of nocturnal silent seizures in human AD, coupled with the abnormal neural synchrony that precedes development of behavioral seizures in mouse AD models, build the case for epileptogenesis as an early therapeutic target for AD. Since most individuals with AD do not develop overt seizures, leveraging functional biomarkers of epilepsy risk to stratify a heterogeneous AD patient population for treatment is research priority for successful clinical trial design. Who will benefit from antiseizure interventions, which one, and when should it begin?

ß spectrin-dependent and domain specific mechanisms for Na+ channel clustering.

Previously, we showed that a hierarchy of spectrin cytoskeletal proteins maintains nodal Na+ channels (Liu et al., 2020). Here, using mice lacking ß1, ß4, or ß1/ß4 spectrins, we show this hierarchy does not function at axon initial segments (AIS). Although ß1 spectrin, together with AnkyrinR (AnkR), compensates for loss of nodal ß4 spectrin, it cannot compensate at AIS. We show AnkR lacks the domain necessary for AIS localization. Whereas loss of ß4 spectrin causes motor impairment and disrupts AIS, loss of ß1 spectrin has no discernable effect on central nervous system structure or function. However, mice lacking both neuronal ß1 and ß4 spectrin show exacerbated nervous system dysfunction compared to mice lacking ß1 or ß4 spectrin alone, including profound disruption of AIS Na+ channel clustering, progressive loss of nodal Na+ channels, and seizures. These results further define the important role of AIS and nodal spectrins for nervous system function.