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Sleep is thought to be restorative to brain energy homeostasis, but it is not clear how this is achieved. We show here that Drosophila glia exhibit a daily cycle of glial mitochondrial oxidation and lipid accumulation that is dependent on prior wake and requires the Drosophila APOE orthologs NLaz and GLaz, which mediate neuron-glia lipid transfer. In turn, a full night of sleep is required for glial lipid clearance, mitochondrial oxidative recovery and maximal neuronal mitophagy. Knockdown of neuronal NLaz causes oxidative stress to accumulate in neurons, and the neuronal mitochondrial integrity protein, Drp1, is required for daily glial lipid accumulation. These data suggest that neurons avoid accumulation of oxidative mitochondrial damage during wake by using mitophagy and passing damage to glia in the form of lipids. We propose that a mitochondrial lipid metabolic cycle between neurons and glia reflects a fundamental function of sleep relevant for brain energy homeostasis., (© 2024. The Author(s).)

Background and Objectives: Heterozygous variants in RAR-related orphan receptor B ( RORB ) have recently been associated with susceptibility to idiopathic generalized epilepsy. However, few reports have been published so far describing pathogenic variants of this gene in patients with epilepsy and intellectual disability (ID). In this study, we aimed to delineate the epilepsy phenotype associated with RORB pathogenic variants and to provide arguments in favor of the pathogenicity of variants., Methods: Through an international collaboration, we analyzed seizure characteristics, EEG data, and genotypes of a cohort of patients with heterozygous variants in RORB . To gain insight into disease mechanisms, we performed ex vivo cortical electroporation in mouse embryos of 5 selected variants, 2 truncating and 3 missense, and evaluated on expression and quantified changes in axonal morphology., Results: We identified 35 patients (17 male, median age 10 years, range 2.5-23 years) carrying 32 different heterozygous variants in RORB , including 28 single-nucleotide variants or small insertions/deletions (12 missense, 12 frameshift or nonsense, 2 splice-site variants, and 2 in-frame deletions), and 4 microdeletions; de novo in 18 patients and inherited in 10. Seizures were reported in 31/35 (89%) patients, with a median age at onset of 3 years (range 4 months-12 years). Absence seizures occurred in 25 patients with epilepsy (81%). Nineteen patients experienced a single seizure type: absences, myoclonic absences, or absences with eyelid myoclonia and focal seizures. Nine patients had absence seizures combined with other generalized seizure types. One patient had presented with absences associated with photosensitive occipital seizures. Three other patients had generalized tonic-clonic seizures without absences. ID of variable degree was observed in 85% of the patients. Expression studies in cultured neurons showed shorter axons for the 5 tested variants, both truncating and missense variants, supporting an impaired protein function., Discussion: In most patients, the phenotype of the RORB -related disorder associates absence seizures with mild-to-moderate ID. In silico and in vitro evaluation of the variants in our cohort, including axonal morphogenetic experiments in cultured neurons, supports their pathogenicity, showing a hypomorphic effect.

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) as a result of a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied with a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in 39 pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, c.1197-8G>A (GenBank: NM_001288739.1), which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of enlarged synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.Arg399Trp and p.Gly401Asp, had a similar DEE phenotype. In contrast, one individual with a missense variant affecting exon 10b, p.Pro405Leu, which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants as well as the possibility of splice site variants acting through a dominant-negative mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Pathogenic variants in SCN1B, the gene encoding voltage-gated sodium channel b1/b1B subunits are associated with a spectrum of epileptic disorders. This study describes a child with early myoclonic encephalopathy and a compound heterozygous variant in the SCN1B gene (p.Arg85Cys and c.3G>C/p.Met1), along with the child’s clinical response to anti-seizure medications (ASMs) and the ketogenic diet. We reviewed the current clinical literature pertinent to SCN1B-related epilepsy. Methods: We described the evaluation and management of a patient with SCN1B-related developmental and epileptic encephalopathy (DEE). We used the Medline and Pubmed databases to review the various neurological manifestations associated with SCN1B genetic variants, and summarize the functional studies performed on SCN1B variants. Results: We identified 20 families and six individuals (including the index case described herein) reported to have SCN1B-related epilepsy. Individuals with monoallelic pathogenic variants in SCN1B often present with genetic epilepsy with febrile seizures plus (GEFS+), while those with biallelic pathogenic variants may present with developmental and epileptic encephalopathy (DEE). Individuals with DEE present with seizures of various semiologies (commonly myoclonic seizures) and status epilepticus at early infancy and are treated with various antiseizure medications. In our index case, adjunctive fenfluramine was started at 8 months of age at 0.2 mg/kg/day with gradual incremental increases to the final dose of 0.7 mg/kg/day over 5 weeks. Fenfluramine was effective in the treatment of seizures, resulting in a 50% reduction in myoclonic seizures, status epilepticus, and generalized tonic-clonic seizures, as well as a 70−90% reduction in focal seizures, with no significant adverse effects. Following the initiation of fenfluramine at eight months of age, there was also a 50% reduction in the rate of hospitalizations. Conclusions: SCN1B pathogenic variants cause epilepsy and neurodevelopmental impairment with variable expressivity and incomplete penetrance. The severity of disease is associated with the zygosity of the pathogenic variants. Biallelic variants in SCN1B can result in early myoclonic encephalopathy, and adjunctive treatment with fenfluramine may be an effective treatment for SCN1B-related DEE. Further research on the efficacy and safety of using newer ASMs, such as fenfluramine in patients under the age of 2 years is needed.

Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Over the past decade, pathogenic variants in all members of the ASXL family of genes, ASXL1, ASXL2, and ASXL3, have been found to lead to clinically distinct but overlapping syndromes. Bohring-Opitz syndrome (BOPS) was first described as a clinical syndrome and later found to be associated with pathogenic variants in ASXL1. This syndrome is characterized by developmental delay, microcephaly, characteristic facies, hypotonia, and feeding difficulties. Subsequently, pathogenic variants in ASXL2 were found to lead to Shashi-Pena syndrome (SHAPNS) and in ASXL3 to lead to Bainbridge-Ropers syndrome (BRPS). While SHAPNS and BRPS share many core features with BOPS, there also seem to be emerging clear differences. Here, we present five cases of BOPS, one case of SHAPNS, and four cases of BRPS. By adding our cohort to the limited number of previously published patients, we review the overlapping features of ASXL-related diseases that bind them together, while focusing on the characteristics that make each neurodevelopmental syndrome unique. This will assist in diagnosis of these overlapping conditions and allow clinicians to more comprehensively counsel affected families., (© 2021 Wiley Periodicals LLC.)

The blood-brain barrier (BBB) is an evolutionarily conserved, structural, and functional separation between circulating blood and the central nervous system (CNS). By controlling permeability into and out of the nervous system, the BBB has a critical role in the precise regulation of neural processes. Here, we review recent studies demonstrating that permeability at the BBB is dynamically controlled by circadian rhythms and sleep. An endogenous circadian rhythm in the BBB controls transporter function, regulating permeability across the BBB. In addition, sleep promotes the clearance of metabolites along the BBB, as well as endocytosis across the BBB. Finally, we highlight the implications of this regulation for diseases, including epilepsy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

CREBBP loss-of function variants cause Rubinstein-Taybi syndrome (RTS). There have been two separate reports of patients with missense variants in exon 30 or 31 of CREBBP in individuals lacking the characteristic facial and limb dysmorphism associated with RTS. Frequent features in this condition include variable intellectual disability, short stature, autistic behavior, microcephaly, feeding problems, epilepsy, recurrent upper airway infections, and mild hearing impairment. We report three further patients with de novo exon 31 CREBBP missense variants. The first individual has a c.5357G>A p. (Arg1786His) variant affecting the same codon as one of the previously described patients. Both these patients could be recognized by clinicians as mild RTS. Our second patient has a c.5602C>T p.(Arg1868Trp) variant that has been described in five other individuals who all share a strikingly similar phenotype. The third individual has a novel c.5354G>A p.(Cys1785Try) variant. Our reports expand the clinical spectrum to include ventriculomegaly, absent corpus callosum, staphyloma, cochlear malformations, and exomphalos. These additional cases also help to establish genotype-phenotype correlations in this disorder. After the first and last authors of the previous two reports, we propose to call this disorder "Menke-Hennekam syndrome" to establish it as a clinical entity distinct from RTS and to provide a satisfactory name for adoption by parents and professionals, thus facilitating appropriate clinical management and research., (© 2019 Wiley Periodicals, Inc.)

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder usually caused by mutations in methyl-CpG-binding protein 2 (MeCP2). RTT is typified by apparently normal development until 6-18 mo of age, when motor and communicative skills regress and hand stereotypies, autonomic symptoms, and seizures present. Restoration of MeCP2 function selectively to astrocytes reversed several deficits in a murine model of RTT, but the mechanism of this rescue is unknown. Astrocytes carry out many essential functions required for normal brain functioning, including extracellular K + buffering. Kir4.1, an inwardly rectifying K + channel, is largely responsible for the channel-mediated K + regulation by astrocytes. Loss-of-function mutations in Kir4.1 in human patients result in a severe neurodevelopmental disorder termed EAST or SESAME syndrome. Here, we evaluated astrocytic Kir4.1 expression in a murine model of Rett syndrome. We demonstrate by chromatin immunoprecipitation analysis that Kir4.1 is a direct molecular target of MeCP2. Astrocytes from Mecp2 -deficient mice express significantly less Kir4.1 mRNA and protein, which translates into a >50% deficiency in Ba 2+ -sensitive Kir4.1-mediated currents, and impaired extracellular potassium dynamics. By examining astrocytes in isolation, we demonstrate that loss of Kir4.1 is cell autonomous. Assessment through postnatal development revealed that Kir4.1 expression in Mecp2 -deficient animals never reaches adult, wild-type levels, consistent with a neurodevelopmental disorder. These are the first data implicating a direct MeCP2 molecular target in astrocytes and provide novel mechanistic insight explaining a potential mechanism by which astrocytic dysfunction may contribute to RTT.

Kir4.1 is an inwardly rectifying K(+) channel expressed exclusively in glial cells in the central nervous system. In glia, Kir4.1 is implicated in several functions including extracellular K(+) homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and facilitation of glutamate uptake. Knockout of Kir4.1 in rodent models leads to severe neurological deficits, including ataxia, seizures, sensorineural deafness, and early postnatal death. Accumulating evidence indicates that Kir4.1 plays an integral role in the central nervous system, prompting many laboratories to study the potential role that Kir4.1 plays in human disease. In this article, we review the growing evidence implicating Kir4.1 in a wide array of neurological disease. Recent literature suggests Kir4.1 dysfunction facilitates neuronal hyperexcitability and may contribute to epilepsy. Genetic screens demonstrate that mutations of KCNJ10, the gene encoding Kir4.1, causes SeSAME/EAST syndrome, which is characterized by early onset seizures, compromised verbal and motor skills, profound cognitive deficits, and salt-wasting. KCNJ10 has also been linked to developmental disorders including autism. Cerebral trauma, ischemia, and inflammation are all associated with decreased astrocytic Kir4.1 current amplitude and astrocytic dysfunction. Additionally, neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis demonstrate loss of Kir4.1. This is particularly exciting in the context of Huntington disease, another neurodegenerative disorder in which restoration of Kir4.1 ameliorated motor deficits, decreased medium spiny neuron hyperexcitability, and extended survival in mouse models. Understanding the expression and regulation of Kir4.1 will be critical in determining if this channel can be exploited for therapeutic benefit.

Background: Hemimegalencephaly with tuberous sclerosis complex is an uncommon association, usually associated with intractable seizures that begin in the neonatal period or early infancy. Typically, the seizures are managed with medications until the patient is older when surgical treatment is considered safe., Patient Description: We describe a 7-week-old infant with tuberous sclerosis (TSC1 mutation) and hemimegalencephaly who underwent a functional hemispherectomy for status epilepticus. No clinical seizures have occurred since surgery nearly 5 years ago and subsequent weaning of antiepileptic drugs 3 years ago. This is one of the youngest patients with tuberous sclerosis complex treated with a hemispherectomy and one of seven patients described in the literature., Conclusions: Our patient, along with previously reported cases, suggests that a hemispherectomy is a viable option in the very young. With evolution of this surgical process since its inception nearly 6 decades ago, it may now be performed safely in early infancy, engendering the possibility of seizure freedom in most and thus optimizing neurodevelopmental outcome., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rett syndrome (RTT), an X-linked neurodevelopment disorder, occurs in approximately one out of 10,000 females. Individuals afflicted by RTT display a constellation of signs and symptoms, affecting nearly every organ system. Most striking are the neurological manifestations, including regression of language and motor skills, increased seizure activity, autonomic dysfunction, and aberrant regulation of breathing patterns. The majority of girls with RTT have mutations in the gene encoding for methyl-CpG binding protein 2 (MeCP2). Since the discovery of this genetic cause of RTT in 1999, there has been an accelerated pace of research seeking to understand the role of MeCP2 in the brain in the hope of developing a disease-modifying therapy for RTT. In this study, we review the clinical features of RTT and then explore the latest mechanistic studies in order to explain how a mutation in MeCP2 leads to these unique features. We cover in detail studies examining the role of MeCP2 in neuronal physiology, as well as recent evidence that implicates a key role for glia in the pathogenesis of RTT. In the past 20 years, these basic and clinical studies have yielded an extraordinary understanding of RTT; as such, we end this narrative review considering the translation of these studies into clinical trials for the treatment of RTT., Competing Interests: The authors report no conflicts of interest in this work., (© 2015 Cuddapah et al.)

Seizures frequently accompany gliomas and often escalate to peritumoral epilepsy. Previous work revealed the importance of tumor-derived excitatory glutamate (Glu) release mediated by the cystine-glutamate transporter (SXC) in epileptogenesis. We now show a novel contribution of GABAergic disinhibition to disease pathophysiology. In a validated mouse glioma model, we found that peritumoral parvalbumin-positive GABAergic inhibitory interneurons are significantly reduced, corresponding with deficits in spontaneous and evoked inhibitory neurotransmission. Most remaining peritumoral neurons exhibit elevated intracellular Cl(-) concentration ([Cl(-) ]i ) and consequently depolarizing, excitatory gamma-aminobutyric acid (GABA) responses. In these neurons, the plasmalemmal expression of KCC2, which establishes the low [Cl(-) ]i required for GABAA R-mediated inhibition, is significantly decreased. Interestingly, reductions in inhibition are independent of Glu release, but the presence of both decreased inhibition and decreased SXC expression is required for epileptogenesis. We suggest GABAergic disinhibition renders peritumoral neuronal networks hyper-excitable and susceptible to seizures triggered by excitatory stimuli, and propose KCC2 as a therapeutic target., (© 2014 Wiley Periodicals, Inc.)

Malignant gliomas are devastating tumours that frequently kill patients within 1 year of diagnosis. The major obstacle to a cure is diffuse invasion, which enables tumours to escape complete surgical resection and chemo- and radiation therapy. Gliomas use the same tortuous extracellular routes of migration that are travelled by immature neurons and stem cells, frequently using blood vessels as guides. They repurpose ion channels to dynamically adjust their cell volume to accommodate to narrow spaces and breach the blood-brain barrier through disruption of astrocytic endfeet, which envelop blood vessels. The unique biology of glioma invasion provides hitherto unexplored brain-specific therapeutic targets for this devastating disease.

Background: Rett syndrome (RTT), a neurodevelopmental disorder that primarily affects girls, is characterised by a period of apparently normal development until 6-18 months of age when motor and communication abilities regress. More than 95% of individuals with RTT have mutations in methyl-CpG-binding protein 2 (MECP2), whose protein product modulates gene transcription. Surprisingly, although the disorder is caused by mutations in a single gene, disease severity in affected individuals can be quite variable. To explore the source of this phenotypic variability, we propose that specific MECP2 mutations lead to different degrees of disease severity., Methods: Using a database of 1052 participants assessed over 4940 unique visits, the largest cohort of both typical and atypical RTT patients studied to date, we examined the relationship between MECP2 mutation status and various phenotypic measures over time., Results: In general agreement with previous studies, we found that particular mutations, such as p.Arg133Cys, p.Arg294X, p.Arg306Cys, 3° truncations and other point mutations, were relatively less severe in both typical and atypical RTT. In contrast, p.Arg106Trp, p.Arg168X, p.Arg255X, p.Arg270X, splice sites, deletions, insertions and deletions were significantly more severe. We also demonstrated that, for most mutation types, clinical severity increases with age. Furthermore, of the clinical features of RTT, ambulation, hand use and age at onset of stereotypies are strongly linked to overall disease severity., Conclusions: We have confirmed that MECP2 mutation type is a strong predictor of disease severity. These data also indicate that clinical severity continues to become progressively worse regardless of initial severity. These findings will allow clinicians and families to anticipate and prepare better for the needs of individuals with RTT.

Malignant gliomas are highly invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl(-) channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl(-) channels are regulated in the context of migration. Using patch-clamp recordings we show Cl(-) currents are activated by physiological increases of [Ca(2+)]i to 65 and 180nM. Cl(-) currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl(-) channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl(-) currents in a Ca(2+)-dependent fashion, placing Cl(-) channels under the regulation of Ca(2+) entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl(-) channels. Thus endogenous glioma Cl(-) channels are regulated by TRPC1. Cl(-) channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca(2+)]i to the shape and volume changes associated with migrating cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca(2+)](i)), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca(2+)](i) regulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers that spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin. Using simultaneous fura-2 Ca(2+) imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca(2+)](i) and induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca(2+)-dependent, TRAM-34-sensitive K(Ca)3.1 channels, and Ca(2+)-dependent, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and gluconate-sensitive Cl(-) channels. A significant portion of these Cl(-) currents can be attributed to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation of ClC-3, a voltage-gated Cl(-) channel/transporter, because pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca(2+)-activated Cl(-) currents. Western blots show that K(Ca)3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both K(Ca)3.1 and ClC-3 colocalize to the invading processes of glioma cells. Importantly, inhibition of either channel abrogates bradykinin-induced chemotaxis and reduces tumor expansion in mouse brain slices in situ. These channels should be further explored as future targets for anti-invasive drugs. Furthermore, these data elucidate a novel mechanism placing cation and anion channels downstream of ligand-mediated [Ca(2+)](i) increases, which likely play similar roles in other migratory cells in the nervous system.

An important event during apoptosis is regulated cell condensation known as apoptotic volume decrease (AVD). Ion channels have emerged as essential regulators of this process mediating the release of K(+) and Cl(-), which together with osmotically obliged water, results in the condensation of cell volume. Using a Grade IV human glioblastoma cell line, we examined the contribution of calcium-activated K(+) channels (K(Ca) channels) to AVD after the addition of either staurosporine (Stsp) or TNF-α-related apoptosis-inducing ligand (TRAIL) to activate the intrinsic or extrinsic pathway of apoptosis, respectively. We show that AVD can be inhibited in both pathways by high extracellular K(+) or the removal of calcium. However, BAPTA-AM was only able to inhibit Stsp-initiated AVD, whereas TRAIL-induced AVD was unaffected. Specific K(Ca) channel inhibitors revealed that Stsp-induced AVD was dependent on K(+) efflux through intermediate-conductance calcium-activated potassium (IK) channels, while TRAIL-induced AVD was mediated by large-conductance calcium-activated potassium (BK) channels. Fura-2 imaging demonstrated that Stsp induced a rapid and modest rise in calcium that was sustained over the course of AVD, while TRAIL produced no detectable rise in global intracellular calcium. Inhibition of IK channels with clotrimazole or 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) blocked downstream caspase-3 activation after Stsp addition, while paxilline, a specific BK channel inhibitor, had no effect. Treatment with ionomycin also induced an IK-dependent cell volume decrease. Together these results show that calcium is both necessary and sufficient to achieve volume decrease and that the two major pathways of apoptosis use unique calcium signaling to efflux K(+) through different K(Ca) channels.

"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.

Among the most prevalent and deadly primary brain tumors, high-grade gliomas evade complete surgical resection by diffuse invasion into surrounding brain parenchyma. Navigating through tight extracellular spaces requires invading glioma cells to alter their shape and volume. Cell volume changes are achieved through transmembrane transport of osmolytes along with obligated water. The sodium-potassium-chloride cotransporter isoform-1 (NKCC1) plays a pivotal role in this process, and previous work has demonstrated that NKCC1 inhibition compromises glioma invasion in vitro and in vivo by interfering with the required cell volume changes. In this study, we show that NKCC1 activity in gliomas requires the With-No-Lysine Kinase-3 (WNK3) kinase. Western blots of patient biopsies and patient-derived cell lines shows prominent expression of Ste-20-related, proline-alanine-rich kinase (SPAK), oxidative stress response kinase (OSR1), and WNK family members 1, 3, and 4. Of these, only WNK3 colocalized and coimmunoprecipitated with NKCC1 upon changes in cell volume. Stable knockdown of WNK3 using specific short hairpin RNA constructs completely abolished NKCC1 activity, as measured by the loss of bumetanide-sensitive cell volume regulation. Consequently, WNK3 knockdown cells showed a reduced ability to invade across Transwell barriers and lacked bumetanide-sensitive migration. This data indicates that WNK3 is an essential regulator of NKCC1 and that WNK3 activates NKCC1-mediated ion transport necessary for cell volume changes associated with cell invasion.

A hallmark of high-grade cancers is the ability of malignant cells to invade unaffected tissue and spread disease. This is particularly apparent in gliomas, the most common and lethal type of primary brain cancer affecting adults. Migrating cells encounter restricted spaces and appear able to adjust their shape to accommodate to narrow extracellular spaces. A growing body of work suggests that cell migration/invasion is facilitated by ion channels and transporters. The emerging concept is that K(+) and Cl(-) function as osmotically active ions, which cross the plasma membrane in concert with obligated water thereby adjusting a cell's shape and volume. In glioma cells Na(+)-K(+)-Cl(-) cotransporters (NKCC1) actively accumulate K(+) and Cl(-), establishing a gradient for KCl efflux. Ca(2+)-activated K(+) channels and voltage-gated Cl(-) channels are largely responsible for effluxing KCl promoting hydrodynamic volume changes. In other cancers, different K(+) or even Na(+) channels may function in concert with a variety of Cl(-) channels to support similar volume changes. Channels involved in migration are frequently regulated by Ca(2+) signaling, most likely coupling extracellular stimuli to cell migration. Importantly, the inhibition of ion channels and transporters appears to be clinically relevant for the treatment of cancer. Recent preclinical data indicates that inhibition of NKCC1 with an FDA-approved drug decreases neoplastic migration. Additionally, ongoing clinical trials demonstrate that an inhibitor of chloride channels may be a therapy for the treatment of gliomas. Data reviewed here strongly indicate that ion channels are a promising target for the development of novel therapeutics to combat cancer.

Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.

Unlabelled: Delayed auditory feedback (DAF) impacts the speech fluency of normally fluent males more than that of normally fluent females. Understanding this gender difference may contribute to our understanding of gender differences in the prevalence of developmental stuttering. To characterize this gender difference in fluent people, DAF-induced dysfluency was measured in 20 male and 21 female young adults during oral reading and conversation tasks. Stutter-like dysfluencies (SLDs), articulation errors, interjections, reading errors, and speech rate were measured for both speech tasks as the participant spoke without feedback, with non-delayed feedback, and with DAF presented with 5 delay intervals (14 conditions total). DAF induced SLDs (but not other dysfluencies) more frequently during conversation than reading, and this effect was significantly greater for males than females (Gender x Task x Feedback interaction). Males also produced significantly more reading errors than females. DAF reduced speaking rate significantly more while reading than conversing (Task x Feedback interaction). DAF significantly decreased the frequency of interjections and increased the frequency of articulation errors; however, no Gender effects on these variables were observed. Although significant order effects indicated improved fluency across trials, covariance analysis suggested that order effects could not explain other results., Educational Objectives: After reading this article, the reader will be able to (1) Discuss developmental stuttering (DS) and gender differences in DS prevalence. (2) Define delayed auditory feedback (DAF). (3) Evaluate the evidence that gender is linked to DAF effects on fluent people. (4) Summarize the results of new research designed to assess sex differences in DAF effects on speech fluency in normally fluent adults. (5) Evaluate the degree to which evidence from the literature indicating that individual differences in attentional control may help us understand gender difference in DAF effects and possibly in DS prevalence as well.