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.

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.

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.

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.

Pathogenesis of peritumoral hyperexcitability in an immunocompetent CRISPR-based glioblastoma model.

Seizures often herald the clinical appearance of gliomas or appear at later stages. Dissecting their precise evolution and cellular pathogenesis in brain malignancies could inform the development of staged therapies for these highly pharmaco-resistant epilepsies. Studies in immunodeficient xenograft models have identified local interneuron loss and excess glial glutamate release as chief contributors to network disinhibition, but how hyperexcitability in the peritumoral microenvironment evolves in an immunocompetent brain is unclear. We generated gliomas in WT mice via in utero deletion of key tumor suppressor genes and serially monitored cortical epileptogenesis during tumor infiltration with in vivo electrophysiology and GCAMP7 calcium imaging, revealing a reproducible progression from hyperexcitability to convulsive seizures. Long before seizures, coincident with loss of inhibitory cells and their protective scaffolding, gain of glial glutamate antiporter xCT expression, and reactive astrocytosis, we detected local Iba1+ microglial inflammation that intensified and later extended far beyond tumor boundaries. Hitherto unrecognized episodes of cortical spreading depolarization that arose frequently from the peritumoral region may provide a mechanism for transient neurological deficits. Early blockade of glial xCT activity inhibited later seizures, and genomic reduction of host brain excitability by deleting MapT suppressed molecular markers of epileptogenesis and seizures. Our studies confirmed xenograft tumor-driven pathobiology and revealed early and late components of tumor-related epileptogenesis in a genetically tractable, immunocompetent mouse model of glioma, allowing the complex dissection of tumor versus host pathogenic seizure mechanisms.

Arx expansion mutation perturbs cortical development by augmenting apoptosis without activating innate immunity in a mouse model of X-linked infantile spasms syndrome.

X-linked infantile spasms syndrome (ISSX) is a clinically devastating developmental epileptic encephalopathy with life-long impact. Arx(GCG)10+7 , a mouse model of the most common triplet-repeat expansion mutation of ARX, exhibits neonatal spasms, electrographic phenotypes and abnormal migration of GABAergic interneuron subtypes. Neonatal presymptomatic treatment with 17ß-estradiol (E2) in Arx(GCG)10+7 reduces spasms and modifies progression of epilepsy. Cortical pathology during this period, a crucial point for clinical intervention in ISSX, has largely been unexplored, and the pathogenic cellular defects that are targeted by early interventions are unknown. In the first postnatal week, we identified a transient wave of elevated apoptosis in Arx(GCG)10+7 mouse cortex that is non-Arx cell autonomous, since mutant Arx-immunoreactive (Arx+) cells are not preferentially impacted by cell death. NeuN+ (also known as Rbfox3) survival was also not impacted, suggesting a vulnerable subpopulation in the immature Arx(GCG)10+7 cortex. Inflammatory processes during this period might explain this transient elevation in apoptosis; however, transcriptomic and immunohistochemical profiling of several markers of inflammation revealed no innate immune activation in Arx(GCG)10+7 cortex. Neither neonatal E2 hormone therapy, nor ACTH(1-24), the frontline clinical therapy for ISSX, diminished the augmented apoptosis in Arx(GCG)10+7 , but both rescued neocortical Arx+ cell density. Since early E2 treatment effectively prevents seizures in this model, enhanced apoptosis does not solely account for the seizure phenotype, but may contribute to other aberrant brain function in ISSX. However, since both hormone therapies, E2 and ACTH(1-24), elevate the density of cortical Arx+-interneurons, their early therapeutic role in other neurological disorders hallmarked by interneuronopathy should be explored.This article has an associated First Person interview with the first author of the paper.

PIK3CA variants selectively initiate brain hyperactivity during gliomagenesis.

Glioblastoma is a universally lethal form of brain cancer that exhibits an array of pathophysiological phenotypes, many of which are mediated by interactions with the neuronal microenvironment1,2. Recent studies have shown that increases in neuronal activity have an important role in the proliferation and progression of glioblastoma3,4. Whether there is reciprocal crosstalk between glioblastoma and neurons remains poorly defined, as the mechanisms that underlie how these tumours remodel the neuronal milieu towards increased activity are unknown. Here, using a native mouse model of glioblastoma, we develop a high-throughput in vivo screening platform and discover several driver variants of PIK3CA. We show that tumours driven by these variants have divergent molecular properties that manifest in selective initiation of brain hyperexcitability and remodelling of the synaptic constituency. Furthermore, secreted members of the glypican (GPC) family are selectively expressed in these tumours, and GPC3 drives gliomagenesis and hyperexcitability. Together, our studies illustrate the importance of functionally interrogating diverse tumour phenotypes driven by individual, yet related, variants and reveal how glioblastoma alters the neuronal microenvironment.

Adult loss of Cacna1a in mice recapitulates childhood absence epilepsy by distinct thalamic bursting mechanisms.

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.

Therapeutic inhibition of mTORC2 rescues the behavioral and neurophysiological abnormalities associated with Pten-deficiency.

Dysregulation of the mammalian target of rapamycin (mTOR) signaling, which is mediated by two structurally and functionally distinct complexes, mTORC1 and mTORC2, has been implicated in several neurological disorders1-3. Individuals carrying loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene, a negative regulator of mTOR signaling, are prone to developing macrocephaly, autism spectrum disorder (ASD), seizures and intellectual disability2,4,5. It is generally believed that the neurological symptoms associated with loss of PTEN and other mTORopathies (for example, mutations in the tuberous sclerosis genes TSC1 or TSC2) are due to hyperactivation of mTORC1-mediated protein synthesis1,2,4,6,7. Using molecular genetics, we unexpectedly found that genetic deletion of mTORC2 (but not mTORC1) activity prolonged lifespan, suppressed seizures, rescued ASD-like behaviors and long-term memory, and normalized metabolic changes in the brain of mice lacking Pten. In a more therapeutically oriented approach, we found that administration of an antisense oligonucleotide (ASO) targeting mTORC2's defining component Rictor specifically inhibits mTORC2 activity and reverses the behavioral and neurophysiological abnormalities in adolescent Pten-deficient mice. Collectively, our findings indicate that mTORC2 is the major driver underlying the neuropathophysiology associated with Pten-deficiency, and its therapeutic reduction could represent a promising and broadly effective translational therapy for neurological disorders where mTOR signaling is dysregulated.