ORCID Profile
0000-0002-4960-6375
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Neurosciences | Central Nervous System | Biological Physics | Medical Biochemistry and Metabolomics | Analysis of Algorithms and Complexity | Neurocognitive Patterns and Neural Networks | Biomedical Instrumentation | Characterisation of Biological Macromolecules | Medicinal and Biomolecular Chemistry | Physiology | Microelectronics and Integrated Circuits | Biologically Active Molecules | Electrical and Electronic Engineering | Proteins and Peptides | Medical Biotechnology not elsewhere classified | Medical Biochemistry and Metabolomics not elsewhere classified | Neurogenetics | Decision Making | Image Processing | Software Engineering | Central Nervous System | Medical Biochemistry: Proteins And Peptides | Medical Virology | Animal Physiology—Cell | Animal Physiology—Systems | Photonics, Optoelectronics and Optical Communications | Electronic and Magnetic Properties of Condensed Matter; Superconductivity | Regenerative Medicine (incl. Stem Cells and Tissue Engineering) | Nanobiotechnology
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Nervous System and Disorders | Nervous system and disorders | Biological sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in the Physical Sciences | Information processing services | Infectious diseases | Expanding Knowledge in Psychology and Cognitive Sciences | Chemical sciences | Expanding Knowledge in Technology | Scientific instrumentation | Application Tools and System Utilities | Cardiovascular System and Diseases | Medical Instruments | Expanding Knowledge in Engineering | Integrated Circuits and Devices | Expanding Knowledge in the Agricultural and Veterinary Sciences | Blood Disorders |
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Publications
Mechanisms of human inherited epilepsies
Publisher: Elsevier BV
Date: 2009
DOI: 10.1016/J.PNEUROBIO.2008.09.016
Abstract: It is just over a decade since the discovery of the first human epilepsy associated ion channel gene mutation. Since then mutations in at least 25 different genes have been described, although the strength of the evidence for these genes having a pathogenic role in epilepsy varies. These discoveries are allowing us to gradually begin to unravel the molecular basis of this complex disease. In the epilepsies, virtually all the established genes code for ion channel subunits. This has led to the concept that the idiopathic epilepsies are a family of channelopathies. This review first introduces the epilepsy syndromes linked to mutations in the various genes. Next it collates the genetic and functional analysis of these genes. This part of the review is ided into voltage-gated channels (Na+, K+, Ca2+, Cl(-) and HCN), ligand-gated channels (nicotinic acetylcholine and GABA(A) receptors) and miscellaneous proteins. In some cases significant advances have been made in our understanding of the molecular and cellular deficits caused by mutations. However, the link between molecular deficit and clinical phenotype is still unknown. Piecing together this puzzle should allow us to understand the underlying pathology of epilepsy ultimately providing novel therapeutic strategies to complete the clinic-bench-clinic cycle.
Non-Neurotoxic Nanodiamond Probes for Intraneuronal Temperature Mapping
Publisher: American Chemical Society (ACS)
Date: 13-11-2017
Abstract: Optical biomarkers have been used extensively for intracellular imaging with high spatial and temporal resolution. Extending the modality of these probes is a key driver in cell biology. In recent years, the nitrogen-vacancy (NV) center in nanodiamond has emerged as a promising candidate for bioimaging and biosensing with low cytotoxicity and stable photoluminescence. Here we study the electrophysiological effects of this quantum probe in primary cortical neurons. Multielectrode array recordings across five replicate studies showed no statistically significant difference in 25 network parameters when nanodiamonds are added at varying concentrations over various time periods, 12-36 h. The physiological validation motivates the second part of the study, which demonstrates how the quantum properties of these biomarkers can be used to report intracellular information beyond their location and movement. Using the optically detected magnetic resonance from the nitrogen-vacancy defects within the nanodiamonds we demonstrate enhanced signal-to-noise imaging and temperature mapping from thousands of nanodiamond probes simultaneously. This work establishes nanodiamonds as viable multifunctional intraneuronal sensors with nanoscale resolution, which may ultimately be used to detect magnetic and electrical activity at the membrane level in excitable cellular systems.
Channelopathies as a genetic cause of epilepsy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-2003
GABRD encoding a protein for extra- or peri- synaptic GABAA receptors is susceptibility locus for generalized epilepsies
Publisher: Oxford University Press (OUP)
Date: 11-05-2004
DOI: 10.1093/HMG/DDH146
Abnormal Cell Sorting Underlies the Unique X-Linked Inheritance of PCDH19 Epilepsy
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.NEURON.2017.12.005
Abstract: X-linked diseases typically exhibit more severe phenotypes in males than females. In contrast, protocadherin 19 (PCDH19) mutations cause epilepsy in heterozygous females but spare hemizygous males. The cellular mechanism responsible for this unique pattern of X-linked inheritance is unknown. We show that PCDH19 contributes to adhesion specificity in a combinatorial manner such that mosaic expression of Pcdh19 in heterozygous female mice leads to striking sorting between cells expressing wild-type (WT) PCDH19 and null PCDH19 in the developing cortex, correlating with altered network activity. Complete deletion of PCDH19 in heterozygous mice abolishes abnormal cell sorting and restores normal network activity. Furthermore, we identify variable cortical malformations in PCDH19 epilepsy patients. Our results highlight the role of PCDH19 in determining cell adhesion affinities during cortical development and the way segregation of WT and null PCDH19 cells is associated with the unique X-linked inheritance of PCDH19 epilepsy.
P2X7 Is a Scavenger Receptor for Apoptotic Cells in the Absence of Its Ligand, Extracellular ATP
Publisher: The American Association of Immunologists
Date: 09-2011
Abstract: Phagocytosis of apoptotic cells is essential during development and tissue remodeling. Our previous study has shown that the P2X7 receptor regulates phagocytosis of nonopsonized particles and bacteria. In this study, we demonstrate that P2X7 also mediates phagocytosis of apoptotic lymphocytes and neuronal cells by human monocyte-derived macrophages under serum-free conditions. ATP inhibited this process to a similar extent as observed with cytochalasin D. P2X7-transfected HEK-293 cells acquired the ability to phagocytose apoptotic lymphocytes. Injection of apoptotic thymocytes into the peritoneal cavity of wild-type mice resulted in their phagocytosis by macrophages, but injection of ATP prior to thymocytes markedly decreased this uptake. In contrast, ATP failed to inhibit phagocytosis of apoptotic thymocytes in vivo by P2X7-deficient peritoneal macrophages. The surface expression of P2X7 on phagocytes increased significantly during phagocytosis of either beads or apoptotic cells. A peptide screen library containing 24 biotin-conjugated peptides mimicking the extracellular domain of P2X7 was used to evaluate the binding profile to beads, bacteria, and apoptotic cells. One peptide showed binding to all particles and cell membrane lipids. Three other cysteine-containing peptides uniquely bound the surface of apoptotic cells but not viable cells, whereas substitution of alanine for cysteine abolished peptide binding. Several thiol-reactive compounds including N-acetyl-L-cysteine abolished phagocytosis of apoptotic SH-SY5Y cells by macrophages. These data suggest that the P2X7 receptor in its unactivated state acts like a scavenger receptor, and its extracellular disulphide bonds play an important role in direct recognition and engulfment of apoptotic cells.
[25] Confocal Ca2+ imaging of organelles, cells, tissues and organs
Publisher: Elsevier
Date: 1999
Myoclonus epilepsy and ataxia due to KCNC1 mutation: Analysis of 20 cases and K+channel properties
Publisher: Wiley
Date: 05-2017
DOI: 10.1002/ANA.24929
Abstract: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-cl to explore biophysical properties of wild-type and mutant K Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type K MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type K
Generation of Local CA1 γ Oscillations by Tetanic Stimulation
Publisher: MyJove Corporation
Date: 14-08-2015
DOI: 10.3791/52877
SCN1Amutations and epilepsy
Publisher: Hindawi Limited
Date: 06-2005
DOI: 10.1002/HUMU.20178
Abstract: SCN1A is part of the SCN1A-SCN2A-SCN3A gene cluster on chromosome 2q24 that encodes for alpha pore forming subunits of sodium channels. The 26 exons of SCN1A are spread over 100 kb of genomic DNA. Genetic defects in the coding sequence lead to generalized epilepsy with febrile seizures plus (GEFS+) and a range of childhood epileptic encephalopathies of varied severity (e.g., SMEI). All published mutations are collated. More than 100 novel mutations are spread throughout the gene with the more debilitating usually de novo. Some clustering of mutations is observed in the C-terminus and the loops between segments 5 and 6 of the first three domains of the protein. Functional studies so far show no consistent relationship between changes to channel properties and clinical phenotype. Of all the known epilepsy genes SCN1A is currently the most clinically relevant, with the largest number of epilepsy related mutations so far characterized.
'Neonatal' Na(v)1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour
Publisher: Oxford University Press (OUP)
Date: 06-11-2015
DOI: 10.1093/HMG/DDU562
Abstract: Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.
Mapping somatosensory connectivity in adult mice using diffusion MRI tractography and super-resolution track density imaging
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.NEUROIMAGE.2014.07.048
Abstract: In this study we combined ultra-high field diffusion MRI fiber tracking and super-resolution track density imaging (TDI) to map the relay locations and connectivity of the somatosensory pathway in paraformaldehyde fixed, C57Bl/6J mouse brains. Super-resolution TDI was used to achieve 20 μm isotropic resolution to inform the 3D topography of the relay locations including thalamic barreloids and brainstem barrelettes, not described previously using MRI methodology. TDI-guided mapping results for thalamo-cortical connectivity were consistent with thalamo-cortical projections labeled using virus mediated fluorescent protein expression. Trigemino-thalamic TDI connectivity maps were concordant with results obtained using anterograde dye tracing from brainstem to thalamus. Importantly, TDI mapping overcame the constraint of tissue distortion observed in mechanically sectioned tissue, enabling 3D reconstruction and long-range connectivity data. In conclusion, our results showed that diffusion micro-imaging at ultra-high field MRI revealed the stereotypical pattern of somatosensory connectivity and is a valuable tool to complement histologic methods, achieving 3D spatial preservation of whole brain networks for characterization in mouse models of human disease.
Genetic and pharmacological modulation of giant depolarizing potentials in the neonatal hippocampus associates with increased seizure susceptibility.
Publisher: Wiley
Date: 05-11-2013
A targeted resequencing gene panel for focal epilepsy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 30-03-2016
Gabapentin Modulates HCN4 Channel Voltage-Dependence.
Publisher: Frontiers Media SA
Date: 21-08-2017
Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase
Publisher: American Society for Microbiology
Date: 10-2011
DOI: 10.1128/JVI.05022-11
Abstract: Amiloride and its derivative 5-( N -ethyl- N -isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D pol conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82: 1465–1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D pol in vitro , affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D pol by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg 2+ . Docking analysis suggested a binding site for amiloride and EIPA in 3D pol , located in close proximity to one of the Mg 2+ ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.
Generalized epilepsy with febrile seizures plus–associated sodium channel β1 subunit mutations severely reduce beta subunit–mediated modulation of sodium channel function
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1016/J.NEUROSCIENCE.2007.05.038
Abstract: Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in in iduals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch cl studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis.
ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy
Publisher: Oxford University Press (OUP)
Date: 22-04-2016
Abstract: We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.
Mossy fiber sprouting interacts with sodium channel mutations to increase dentate gyrus excitability
Publisher: Wiley
Date: 21-12-2009
DOI: 10.1111/J.1528-1167.2009.02202.X
Abstract: Idiopathic epilepsy is caused by the complex interaction of genetic and environmental factors. The purpose of this study was to use computational approaches to explore the interaction between changes in sodium channel availability caused by mutations and mossy fiber sprouting. We used a previously published biophysically realistic computer model of dentate gyrus neurons and networks. A sensitivity analysis probed the effects of typical mutation-like changes in either single- or multiple-gating parameters. Isolated neuron models were stimulated with current injections, and networks were stimulated with perforant path synaptic input. The gene-environment interaction was studied by including mossy fiber sprouting into these networks. Single neuron responses to current injections were complex, with increased sodium channel availability paradoxically reducing firing rates. In the absence of mossy fiber sprouting, control networks showed strong accommodation supporting the role of the dentate gyrus as a gate. Availability changes alone were not able to drive the networks into ictal states, even though they reduced the effectiveness of the dentate gyrus gate. Interestingly, the presence of electrophysiologic changes substantially increased the ability of mossy fiber sprouting to induce ictal activity. (1) Increased sodium channel availability does not necessarily lead to increased firing rates, (2) network excitability is most sensitive to changes in steady state activation of sodium channels, (3) mutation-induced changes in availability reduce the effectiveness of the dentate gyrus gate, and (4) mutations interact strongly with structural network changes to allow ictal-like activity in the dentate gyrus.
Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy
Publisher: Proceedings of the National Academy of Sciences
Date: 30-10-2007
Abstract: Mutations in the GABA A receptor γ2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a γ2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of γ2(R43Q) was seen in heterozygous mice in the absence of any change in α1 subunit surface expression, ruling out a dominant-negative effect. GABA A -mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.
Febrile seizures: traffic slows in the heat
Publisher: Elsevier BV
Date: 08-2006
DOI: 10.1016/J.MOLMED.2006.06.005
Abstract: Febrile seizures, which occur in young children, have long been known to have a major inherited component. Mutations in some genes that encode sodium channel and GABA(A) receptor subunits have been found in a few families affected by febrile seizures. These mutations account only for a minority of cases, and much remains to be learnt about the molecular architecture of febrile seizures. A rare inherited cause--a mutation in the GABA(A) receptor subunit GABRG2 gene--has been recently shown to cause a temperature-dependent intracellular trafficking defect. This is an important step in unravelling the molecular pathogenesis of this common childhood disorder.
Sodium channel β1 subunit localizes to axon initial segments of excitatory and inhibitory neurons and shows regional heterogeneity in mouse brain
Publisher: Wiley
Date: 14-01-2015
DOI: 10.1002/CNE.23715
Abstract: The β1 subunit of voltage-gated sodium channels, Nav β1, plays multiple roles in neurons spanning electrophysiological modulation of sodium channel α subunits to cell adhesion and neurite outgrowth. This study used immunohistochemistry to investigate Nav β1 subneuronal and regional expression. Nav β1 was enriched at axon initial segments (AIS) and nodes of Ranvier. Nav β1 expression at the AIS was detected throughout the brain, predominantly in the hippoc us, cortex, and cerebellum. Despite expression of Nav β1 in both excitatory and inhibitory AIS, it displayed a marked and fine-grained heterogeneity of expression. Such heterogeneity could have important implications for the tuning of single neuronal and regional excitability, especially in view of the fact that Nav β1 coexpressed with Nav 1.1, Nav 1.2, and Nav 1.6 subunits. The disruption of Nav β1 AIS expression by a human epilepsy-causing C121W genetic mutation in Nav β1 was also investigated using a mouse model. AIS expression of Nav β1 was reduced by approximately 50% in mice heterozygous for the C121W mutation and was abolished in homozygotes, suggesting that loss of Nav α subunit modulation by Nav β1 contributes to the mechanism of epileptogenesis in these animals as well as in patients.
Rare coding variants in genes encoding GABAA receptors in genetic generalised epilepsies: an exome-based case-control study
Publisher: Elsevier BV
Date: 08-2018
P2X7 Receptor Cell Surface Expression and Cytolytic Pore Formation Are Regulated by a Distal C-terminal Region
Publisher: Elsevier BV
Date: 03-2003
Targeting retinal ganglion cell recovery.
Publisher: Springer Science and Business Media LLC
Date: 06-01-2017
DOI: 10.1038/EYE.2016.281
Optogenetic Demonstration of Functional Innervation of Mouse Colon by Neurons Derived From Transplanted Neural Cells
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1053/J.GASTRO.2017.01.005
Abstract: Cell therapy offers the potential to treat gastrointestinal motility disorders caused by diseased or absent enteric neurons. We examined whether neurons generated from transplanted enteric neural cells provide a functional innervation of bowel smooth muscle in mice. Enteric neural cells expressing the light-sensitive ion channel, channelrhodopsin, were isolated from the fetal or postnatal mouse bowel and transplanted into the distal colon of 3- to 4-week-old wild-type recipient mice. Intracellular electrophysiological recordings of responses to light stimulation of the transplanted cells were made from colonic smooth muscle cells in recipient mice. Electrical stimulation of endogenous enteric neurons was used as a control. The axons of graft-derived neurons formed a plexus in the circular muscle layer. Selective stimulation of graft-derived cells by light resulted in excitatory and inhibitory junction potentials, the electrical events underlying contraction and relaxation, respectively, in colonic muscle cells. Graft-derived excitatory and inhibitory motor neurons released the same neurotransmitters as endogenous motor neurons-acetylcholine and a combination of adenosine triphosphate and nitric oxide, respectively. Graft-derived neurons also included interneurons that provided synaptic inputs to motor neurons, but the pharmacologic properties of interneurons varied with the age of the donors from which enteric neural cells were obtained. Enteric neural cells transplanted into the bowel give rise to multiple functional types of neurons that integrate and provide a functional innervation of the smooth muscle of the bowel wall. Circuits composed of both motor neurons and interneurons were established, but the age at which cells are isolated influences the neurotransmitter phenotype of interneurons that are generated.
Oxcarbazepine and its active metabolite, (S)-licarbazepine, exacerbate seizures in a mouse model of genetic generalized epilepsy
Publisher: Wiley
Date: 08-12-2015
DOI: 10.1111/EPI.12866
Abstract: Oxcarbazepine (OXC), widely used to treat focal epilepsy, is reported to exacerbate seizures in patients with generalized epilepsy. OXC is metabolized to monohydroxy derivatives in two enantiomeric forms: (R)-licarbazepine and (S)-licarbazepine. Eslicarbazepine acetate is a recently approved antiepileptic drug that is rapidly metabolized to (S)-licarbazepine. It is not known whether (S)-licarbazepine exacerbates seizures. Here, we test whether OXC or either of its enantiomers exacerbates the number of spike-and-wave discharges (SWDs) in mice harboring the human γ-aminobutyric acid A receptor (GABAA)γ2(R43Q) mutation. OXC (20 mg/kg), (S)-licarbazepine (20 mg/kg), and (R)-licarbazepine (20 mg/kg) all significantly increased the number of SWDs, while their duration was unaffected. The potential for (S)-licarbazepine to exacerbate SWDs suggests that eslicarbazepine acetate should be used with caution in generalized epilepsy. Furthermore, generalized seizure exacerbation for first-, second-, and third-generation carbamazepine-based compounds is likely to occur through a common mechanism.
P2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid
Publisher: Elsevier BV
Date: 05-2012
SCN2A mutation associated with neonatal epilepsy, late-onset episodic ataxia, myoclonus, and pain
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 18-10-2010
Molecular correlates of age-dependent seizures in an inherited neonatal-infantile epilepsy
Publisher: Oxford University Press (OUP)
Date: 05-04-2010
DOI: 10.1093/BRAIN/AWQ057
Abstract: Many idiopathic epilepsy syndromes have a characteristic age dependence, the underlying molecular mechanisms of which are largely unknown. Here we propose a mechanism that can explain that epileptic spells in benign familial neonatal-infantile seizures occur almost exclusively during the first days to months of life. Benign familial neonatal-infantile seizures are caused by mutations in the gene SCN2A encoding the voltage-gated Na(+) channel Na(V)1.2. We identified two novel SCN2A mutations causing benign familial neonatal-infantile seizures and analysed the functional consequences of these mutations in a neonatal and an adult splice variant of the human Na(+) channel Na(V)1.2 expressed heterologously in tsA201 cells together with beta1 and beta2 subunits. We found significant gating changes leading to a gain-of-function, such as an increased persistent Na(+) current, accelerated recovery from fast inactivation or altered voltage-dependence of steady-state activation. Those were restricted to the neonatal splice variant for one mutation, but more pronounced for the adult form for the other, suggesting that a differential developmental splicing does not provide a general explanation for seizure remission. We therefore analysed the developmental expression of Na(V)1.2 and of another voltage-gated Na(+) channel, Na(V)1.6, using immunohistochemistry and real-time reverse transcription-polymerase chain reaction in mouse brain slices. We found that Na(V)1.2 channels are expressed early in development at axon initial segments of principal neurons in the hippoc us and cortex, but their expression is diminished and they are gradually replaced as the dominant channel type by Na(V)1.6 during maturation. This finding provides a plausible explanation for the transient expression of seizures that occur due to a gain-of-function of mutant Na(V)1.2 channels.
The role of neuronal GABAA receptor subunit mutations in idiopathic generalized epilepsies
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.NEULET.2009.02.038
Abstract: Rare GABA(A) receptor gamma2 and alpha1 subunit mutations of pathogenic effect have been described segregating in families with "monogenic" epilepsies. We now report globally on the genetic variation contained within all 16 neuronal GABA(A) receptor subunit genes from the one patient cohort. The cohort consists of GEFS(+), FS, and IGE subgroups as either sporadic cases or index cases from small families, with one index case from one large IGE family. The rarity of mutations and coding variation in general across all of the subunits suggests a low tolerance for mutations affecting GABA mediated neuronal inhibition. Characterization of the broader channelopathy load associated with susceptibility to these common epilepsies mostly with complex genetics will need to be expanded beyond the family of GABA(A) receptor subunits to all families of neuronal ion channels and their interacting molecules by systematic mutation detection associated with functional investigation of their naturally occurring genetic variations.
Synaptic Zn2+and febrile seizure susceptibility
Publisher: Wiley
Date: 28-11-2017
DOI: 10.1111/BPH.13658
Neurons derived from human embryonic stem cells extend long-distance axonal projections through growth along host white matter tracts after intra-cerebral transplantation
Publisher: Frontiers Media SA
Date: 2012
An Arg307 to Gln Polymorphism within the ATP-binding Site Causes Loss of Function of the Human P2X7 Receptor
Publisher: Elsevier BV
Date: 07-2004
Multiple molecular mechanisms for a single GABAA mutation in epilepsy.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 13-02-2013
SEARCHING FOR FUNCTIONAL GENETIC VARIANTS IN NON‐CODING DNA
Publisher: Wiley
Date: 27-02-2008
DOI: 10.1111/J.1440-1681.2008.04880.X
Abstract: 1. The search for DNA sequence variants for complex human polygenic conditions has been a strong focus of recent genetic research. While gene loci have been identified, few variants in the coding sequences of these genes have been found, suggesting that non-coding sequence variation may underlie many complex conditions. 2. Non-coding DNA harbours regulatory elements capable of making changes to gene expression. However, regulatory DNA sequences are currently difficult to recognize and their function is poorly understood, complicating the task of assigning potential functional significance to non-coding variation. 3. Comparative genomics, the study of evolutionary DNA conservation, has enabled the emergent field of non-coding DNA identification in human disease analysis. 4. This brief review will focus on the potential of a relatively high throughput technique based on comparative genomics, that may aid in the identification of functionally important non-coding sequence variation in complex diseases.
Truncation of the GABAA-Receptor γ2 Subunit in a Family with Generalized Epilepsy with Febrile Seizures Plus
Publisher: Elsevier BV
Date: 02-2002
DOI: 10.1086/338710
Discovering the pharmacodynamics of conolidine and cannabidiol using a cultured neuronal network based workflow.
Publisher: Springer Science and Business Media LLC
Date: 15-01-2019
DOI: 10.1038/S41598-018-37138-W
Abstract: Determining the mechanism of action (MOA) of novel or naturally occurring compounds mostly relies on assays tailored for in idual target proteins. Here we explore an alternative approach based on pattern matching response profiles obtained using cultured neuronal networks. Conolidine and cannabidiol are plant-derivatives with known antinociceptive activity but unknown MOA. Application of conolidine/cannabidiol to cultured neuronal networks altered network firing in a highly reproducible manner and created similar impact on network properties suggesting engagement with a common biological target. We used principal component analysis (PCA) and multi-dimensional scaling (MDS) to compare network activity profiles of conolidine/cannabidiol to a series of well-studied compounds with known MOA. Network activity profiles evoked by conolidine and cannabidiol closely matched that of ω-conotoxin CVIE, a potent and selective Cav2.2 calcium channel blocker with proposed antinociceptive action suggesting that they too would block this channel. To verify this, Cav2.2 channels were heterologously expressed, recorded with whole-cell patch cl and conolidine/cannabidiol was applied. Remarkably, conolidine and cannabidiol both inhibited Cav2.2, providing a glimpse into the MOA that could underlie their antinociceptive action. These data highlight the utility of cultured neuronal network-based workflows to efficiently identify MOA of drugs in a highly scalable assay.
Segmentation of the mouse hippocampal formation in magnetic resonance images
Publisher: Elsevier BV
Date: 10-2011
DOI: 10.1016/J.NEUROIMAGE.2011.06.025
Abstract: The hippoc al formation plays an important role in cognition, spatial navigation, learning, and memory. High resolution magnetic resonance (MR) imaging makes it possible to study in vivo changes in the hippoc us over time and is useful for comparing hippoc al volume and structure in wild type and mutant mice. Such comparisons demand a reliable way to segment the hippoc al formation. We have developed a method for the systematic segmentation of the hippoc al formation using the perfusion-fixed C57BL/6 mouse brain for application in longitudinal and comparative studies. Our aim was to develop a guide for segmenting over 40 structures in an adult mouse brain using 30 μm isotropic resolution images acquired with a 16.4 T MR imaging system and combined using super-resolution reconstruction.
Epilepsy, energy deficiency and new therapeutic approaches including diet
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.PHARMTHERA.2014.06.001
Abstract: Metabolic dysfunction leading to epilepsy is well recognised. Dietary therapy, in particular the ketogenic diet, is now considered an effective option. Recent genetic studies have highlighted the central role that metabolism can play in setting seizure susceptibility. Here we discuss various metabolic disorders implicated in epilepsy focusing on energy deficiency due to genetic and environmental causes. We argue that low, uncompensated brain glucose levels can precipitate seizures. We will also explore mechanisms of disease and therapy in an attempt to identify common metabolic pathways involved in modulating seizure susceptibility. Finally, newer therapeutic approaches based on diet manipulation in the context of energy deficiency are discussed.
A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells
Publisher: Elsevier BV
Date: 09-2016
A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis
Publisher: Oxford University Press (OUP)
Date: 17-07-2015
DOI: 10.1093/HMG/DDV278
Abstract: Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.
Increased thalamic inhibition in the absence seizure prone DBA/2J mouse.
Publisher: Wiley
Date: 20-02-2008
Developmental Impact of a Famial GABAA Receptor Epilepsy Mutation
Publisher: Wiley
Date: 28-09-2008
DOI: 10.1002/ANA.21440
Low blood glucose precipitates spike-and-wave activity in genetically predisposed animals
Publisher: Wiley
Date: 22-12-2011
DOI: 10.1111/J.1528-1167.2010.02911.X
Abstract: Absence epilepsies are common, with a major genetic contribution to etiology. Certain environmental factors can influence absence occurrence but a complete understanding of absence precipitation is lacking. Herein we investigate if lowering blood glucose increases spike-wave activity in mouse models with varying seizure susceptibility. Three mouse models were used: an absence seizure model based on the knockin of a human GABA(A) γ2(R43Q) mutation (DBA(R43Q)), the spike-wave discharge (SWD)-prone DBA/2J strain, and the seizure resistant C57Bl/6 strain. Electrocorticography (ECoG) studies were recorded to determine SWDs during hypoglycemia induced by insulin or overnight fasting. An insulin-mediated reduction in blood glucose levels to 4 mm (c.a. 40% reduction) was sufficient to double SWD occurrence in the DBA(R43Q) model and in the SWD-prone DBA/2J mouse strain. Larger reductions in blood glucose further increased SWDs in both these models. However, even with large reductions in blood glucose, no discharges were observed in the seizure-resistant C57Bl/6 mouse strain. Injection of glucose reversed the impact of insulin on SWDs in the DBA(R43Q) model, supporting a reduction in blood glucose as the modulating influence. Overnight fasting reduced blood glucose levels to 4.5 mm (c.a. 35% reduction) and, like insulin, caused a doubling in occurrence of SWDs. Low blood glucose can precipitate SWDs in genetically predisposed animal models and should be considered as a potential environmental risk factor in patients with absence epilepsy.
Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons
Publisher: Elsevier BV
Date: 11-2007
DOI: 10.1016/J.NEURON.2007.09.018
Abstract: Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of in idual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.
Visualization of mouse barrel cortex using ex-vivo track density imaging
Publisher: Elsevier BV
Date: 02-2014
DOI: 10.1016/J.NEUROIMAGE.2013.09.030
Abstract: We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3mm)(3) block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. In idual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the in idual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same s les. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.
Development of a rapid functional assay that predicts GLUT1 disease severity
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 12-2018
DOI: 10.1212/NXG.0000000000000297
Abstract: To examine the genotype to phenotype connection in glucose transporter type 1 (GLUT1) deficiency and whether a simple functional assay can predict disease outcome from genetic sequence alone. GLUT1 deficiency, due to mutations in SLC2A1 , causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function. To test this, we measured glucose transport by GLUT1 variants identified in population controls and patients with mild to severe epilepsies. Controls were reference sequence from the NCBI and 4 population missense variants chosen from public reference control databases. Nine variants associated with epilepsies or movement disorders, with normal intellect in all in iduals, formed the mild group. The severe group included 5 missense variants associated with classical GLUT1 encephalopathy. GLUT1 variants were expressed in Xenopus laevis oocytes, and glucose uptake was measured to determine kinetics (V max ) and affinity (K m ). Disease severity inversely correlated with rate of glucose transport between control (V max = 28 ± 5), mild (V max = 16 ± 3), and severe (V max = 3 ± 1) groups, respectively. Affinities of glucose binding in control (K m = 55 ± 18) and mild (K m = 43 ± 10) groups were not significantly different, whereas affinity was indeterminate in the severe group because of low transport rates. Simplified analysis of glucose transport at high concentration (100 mM) was equally effective at separating the groups. Disease severity can be partly explained by the extent of GLUT1 dysfunction. This simple Xenopus oocyte assay complements genetic and clinical assessments. In prenatal diagnosis, this simple oocyte glucose uptake assay could be useful because standard clinical assessments are not available.
Human epilepsies: interaction of genetic and acquired factors
Publisher: Elsevier BV
Date: 07-2006
DOI: 10.1016/J.TINS.2006.05.009
Abstract: Epilepsies, once regarded as due to demoniacal possession, can have both genetic and acquired causes, with interaction of these factors in many cases. To date, nearly all the genes discovered to be involved in human epilepsies encode subunits of ion channels, both voltage-gated and ligand-gated. Established acquired causes include serious brain trauma, stroke, tumours and infective lesions. Thus, in terms of exploring the neurobiology of "nature and nurture" in disease, the epilepsies are an excellent paradigm. Here, we review the evidence and discuss the possibility that ion channels are a common biological substrate for both genetic and acquired epilepsies. This review is part of the INMED/TINS special issue "Nature and nurture in brain development and neurological disorders", based on presentations at the annual INMED/TINS symposium (inmednet.com/).
HCN channelopathies: pathophysiology in genetic epilepsy and therapeutic implications.
Publisher: Wiley
Date: 06-12-2012
Sodium channels and the neurobiology of epilepsy
Publisher: Wiley
Date: 20-08-2012
DOI: 10.1111/J.1528-1167.2012.03631.X
Abstract: Voltage-gated sodium channels (VGSCs) are integral membrane proteins. They are essential for normal neurologic function and are, currently, the most common recognized cause of genetic epilepsy. This review summarizes the neurobiology of VGSCs, their association with different epilepsy syndromes, and the ways in which we can experimentally interrogate their function. The most important sodium channel subunit of relevance to epilepsy is SCN1A, in which over 650 genetic variants have been discovered. SCN1A mutations are associated with a variety of epilepsy syndromes the more severe syndromes are associated with truncation or complete loss of function of the protein. SCN2A is another important subtype associated with epilepsy syndromes, across a range of severe and less severe epilepsies. This subtype is localized primarily to excitatory neurons, and mutations have a range of functional effects on the channel. SCN8A is the other main adult subtype found in the brain and has recently emerged as an epilepsy gene, with the first human mutation discovered in a severe epilepsy syndrome. Mutations in the accessory β subunits, thought to modulate trafficking and function of the α subunits, have also been associated with epilepsy. Genome sequencing is continuing to become more affordable, and as such, the amount of incoming genetic data is continuing to increase. Current experimental approaches have struggled to keep pace with functional analysis of these mutations, and it has proved difficult to build associations between disease severity and the precise effect on channel function. These mutations have been interrogated with a range of experimental approaches, from in vitro, in vivo, to in silico. In vitro techniques will prove useful to scan mutations on a larger scale, particularly with the advance of high-throughput automated patch-cl techniques. In vivo models enable investigation of mutation in the context of whole brains with connected networks and more closely model the human condition. In silico models can help us incorporate the impact of multiple genetic factors and investigate epistatic interactions and beyond.
A Glu-496 to Ala Polymorphism Leads to Loss of Function of the Human P2X7 Receptor
Publisher: Elsevier BV
Date: 04-2001
Cortical microarchitecture changes in genetic epilepsy.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-03-2015
Can mutation-mediated effects occurring early in development cause long-term seizure susceptibility in genetic generalized epilepsies?
Publisher: Wiley
Date: 16-04-2018
DOI: 10.1111/EPI.14077
Abstract: Epilepsy has a strong genetic component, with an ever-increasing number of disease-causing genes being discovered. Most epilepsy-causing mutations are germ line and thus present from conception. These mutations are therefore well positioned to have a deleterious impact during early development. Here we review studies that investigate the role of genetic lesions within the early developmental window, specifically focusing on genetic generalized epilepsy (GGE). Literature on the potential pathogenic role of sub-mesoscopic structural changes in GGE is also reviewed. Evidence from rodent models of genetic epilepsy support the idea that functional and structural changes can occur in early development, leading to altered seizure susceptibility into adulthood. Both animal and human studies suggest that sub-mesoscopic structural changes occur in GGE. The existence of sub-mesoscopic structural changes prior to seizure onset may act as biomarkers of excitability in genetic epilepsies. We also propose that presymptomatic treatment may be essential for limiting the long-term consequences of disease-causing mutations in genetic epilepsies.
Purinergic receptorsP2RX4andP2RX7in familial multiple sclerosis
Publisher: Hindawi Limited
Date: 13-04-2017
DOI: 10.1002/HUMU.23218
Glucose transporter GLUT12-functional characterization in Xenopus laevis oocytes
Publisher: Elsevier BV
Date: 08-2003
DOI: 10.1016/S0006-291X(03)01417-7
Abstract: We have recently identified and cloned the cDNA of a new member of the glucose transporter family that has been designated GLUT12. GLUT12 possesses the structural features critical to facilitative transport of glucose but the key to understanding the possible physiological roles of this novel protein requires analysis of functional glucose transport. In the current study, we have utilized the Xenopus laevis oocyte expression system to assay transport of the glucose analog 2-deoxy-D-glucose and characterize the glucose transport properties and hexose affinities of GLUT12. Our results demonstrate that GLUT12 facilitates transport of glucose with an apparent preferential substrate affinity for glucose over other hexoses assayed. The results are significant to understanding the potential role and importance of GLUT12 in insulin-sensitive tissues and also cells with high glucose utilization such as cancer cells.
Genetic literacy series: Primer part 2—Paradigm shifts in epilepsy genetics
Publisher: Wiley
Date: 09-05-2018
DOI: 10.1111/EPI.14193
Abstract: This is the second of a 2-part primer on the genetics of the epilepsies within the Genetic Literacy Series of the Genetics Commission of the International League Against Epilepsy. In Part 1, we covered types of genetic variation, inheritance patterns, and their relationship to disease. In Part 2, we apply these basic principles to the case of a young boy with epileptic encephalopathy and ask 3 important questions: (1) Is the gene in question an established genetic etiology for epilepsy? (2) Is the variant in this particular gene pathogenic by established variant interpretation criteria? (3) Is the variant considered causative in the clinical context? These questions are considered and then answered for the clinical case in question.
Single Nucleotide Variations in CLCN6 Identified in Patients with Benign Partial Epilepsies in Infancy and/or Febrile Seizures
Publisher: Public Library of Science (PLoS)
Date: 20-03-2015
Primer Part 1—The building blocks of epilepsy genetics
Publisher: Wiley
Date: 25-05-2016
DOI: 10.1111/EPI.13381
Abstract: This is the first of a two-part primer on the genetics of the epilepsies within the Genetic Literacy Series of the Genetics Commission of the International League Against Epilepsy. In Part 1, we cover the foundations of epilepsy genetics including genetic epidemiology and the range of genetic variants that can affect the risk for developing epilepsy. We discuss various epidemiologic study designs that have been applied to the genetics of the epilepsies including population studies, which provide compelling evidence for a strong genetic contribution in many epilepsies. We discuss genetic risk factors varying in size, frequency, inheritance pattern, effect size, and phenotypic specificity, and provide ex les of how genetic risk factors within the various categories increase the risk for epilepsy. We end by highlighting trends in epilepsy genetics including the increasing use of massive parallel sequencing technologies.
Channelrhodopsins shed light on a new pathway in absence epilepsy
Publisher: Future Medicine Ltd
Date: 2012
DOI: 10.2217/FNL.11.66
Abstract: Evaluation of: Paz JT, Bryant AS, Peng K et al. A new mode of corticothalamic transmission revealed in the Gria4 -/- model of absence epilepsy. Nat. Neurosci. 14(9), 1167–1173 (2011). Absence seizures are a common form of epilepsy characterized by sudden behavioral arrest in conjunction with a stereotypical spike-and-wave discharge on electroencephalography. A reciprocally connected network of thalamocortical neurons that normally controls sleep and other functions, misfires to cause absence epilepsy and is perhaps one of the most studied seizure networks. In a recent paper, Paz and colleagues further dissect this network to understand the basis of seizure initiation in a new animal model of absence epilepsy. Exploiting state-of-the-art ‘optogenetic’ methodology they systematically isolate monosynaptic connections in thalamocortical nuclei to reveal the key pathological mechanism underlying absence seizures in the Gria4 -/- mouse. The main finding is a reduction in the strength of synapses made by excitatory cortical projection neurons onto the reticular thalamic nucleus. The consequent fall in reticular thalamic nucleus inhibitory neuron output results in less feed-forward inhibition of thalamocortical neurons and an increase in thalamic excitability that is, presumably, sufficient to initiate oscillations and absence seizures. The manuscript adds significantly to our understanding of how absence seizures can initiate by implicating, in this case, a thalamic rather than cortical basis. Further, the demonstration of this mode of circuit activity may have significant implications for how the thalamocortical network behaves physiologically.
Hippocampal volume and cell density changes in a mouse model of human genetic epilepsy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-03-2013
A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation
Publisher: EMBO
Date: 25-03-2014
Enhanced in vitro CA 1 network activity in a sodium channel β1(C 121W ) subunit model of genetic epilepsy
Publisher: Wiley
Date: 07-03-2014
DOI: 10.1111/EPI.12568
Abstract: A NaV β1(C121W) mouse model of human genetic epilepsy has enhanced neuronal excitability and temperature sensitivity attributed to a decreased threshold for action potential firing in the axon initial segment. To investigate the network consequences of this neuronal dysfunction and to establish a genetic disease state model we developed an in vitro assay to investigate CA1 network properties and antiepileptic drug sensitivity. CA1 network oscillations were induced by tetanic stimulation and average number of spikes, interspike interval (ISI), duration, and latency were measured in slices from control and NaV β1(C121W) heterozygous mice in the presence and absence of retigabine or carbamazepine. Retigabine was also tested in a thermogenic seizure model. Oscillations were reliably induced by tetanic stimulation and were maintained after severing connections between CA3 and CA1, suggesting a local recurrent circuit. Blocking α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), γ-aminobutyric acid receptor A (GABAA ), Ih , and T-type Ca(2+) channels/receptors reduced the number of spikes. Slices from NaV β1(C121W) heterozygous mice displayed several hallmarks of increased network excitability including increases in duration of the oscillation, the number and frequency of spikes and a decrease in their onset latency. The effect of genotype on network excitability was temperature sensitive, as it was seen only at elevated temperatures. Carbamazepine and retigabine were more effective in reducing network excitability in slices from NaV β1(C121W) heterozygous mice. Retigabine appeared to be more effective in suppressing time to thermogenic seizures in NaV β1(C121W) heterozygous mice compared to wild-type (WT) controls. Hippoc al networks of the NaV β1(C121W) heterozygous mouse model of genetic epilepsy show enhanced excitability consistent with earlier single neuron studies bridging important scales of brain complexity relevant to seizure genesis. Altered pharmacosensitivity further suggests that genetic epilepsy models may be useful in the development of novel antiepileptic drugs that target disease state pathology. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.
A recurrent de novo mutation in KCNC1 causes progressive myoclonus epilepsy
Publisher: Springer Science and Business Media LLC
Date: 17-11-2015
DOI: 10.1038/NG.3144
Oxcarbazepine, not its active metabolite, potentiates GABA(A) activation and aggravates absence seizures
Publisher: Wiley
Date: 2009
DOI: 10.1111/J.1528-1167.2008.01759.X
Abstract: Studies in genetic absence epileptic rats from Strasbourg (GAERS) indicate that enhancement of gamma aminobutyric acid (GABA(A)) receptor activity is a critical mechanism in the aggravation of seizures by carbamazepine (CBZ). We examined whether structural analogs of CBZ, oxcarbazepine (OXC), and its active metabolite, monohydroxy derivative (MHD), also potentiate GABA(A) receptor current and aggravate seizures. In vitro studies in Xenopus oocytes compared the three drugs' effect on GABA(A) receptor currents. In vivo studies compared seizure activity in GAERS after intraperitoneal drug administration. OXC potentiated GABA(A) receptor current and aggravated seizures in GAERS, similarly to the effect of CBZ. Conversely, MHD showed only a minor potentiation of GABA(A) receptor current and did not aggravate seizures. A hydroxyl group at the C-10 position on the CBZ tricyclic structure in MHD reduces GABA(A) receptor potentiation and seizure aggravation. Reports of the aggravation of absence seizures in patients taking OXC may result from circulating unmetabolized OXC rather than MHD.
Pore-Forming Proteins and their Application in Biotechnology
Publisher: Bentham Science Publishers Ltd.
Date: 06-2002
Abstract: Proteins and peptides that form membrane-spanning pores and channels comprise a erse class of molecules ranging from short peptides that are unregulated and create non-selective pathways to large ion channel proteins that are highly regulated and exhibit exquisite selectivity for particular ions. The ersity of regulation and selectivity, together with recent advances in protein "re-engineering" technology, provide a strong framework on which to build custom molecules with wide-ranging biotechnological application. Here we review a selection of pore-forming peptides and proteins from a number of different species to highlight their structural and functional ersity. The current and potential uses of native and re-engineered molecules are discussed together with a novel strategy to re-engineer alpha-hemolysin to create targeted and regulable cell-killing agents termed proimmunolysins. Numerous pore-forming peptides are currently in development as antimicrobial agents with potential application as anti-tumorigenic agents. In addition to their roles as biotherapeutic agents, pore-forming proteins are also being developed as biosensors for a range of different analytes. Recent ex les of this technology include the use of alpha-hemolysin with an adapter molecule to create sensors for organic molecules and gramicidin as a general-purpose sensor for a range of analytes. These approaches promise to deliver a configurable binding site for analytes encoded in a readily measured electrical signal. The number of applications for pore-forming molecules is sure to grow in both quantity and ersity with increased knowledge of the fundamental structure and function of pores.
Selective Na V 1.1 activation rescues Dravet syndrome mice from seizures and premature death
Publisher: Proceedings of the National Academy of Sciences
Date: 03-08-2018
Abstract: Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit Na V 1.1. Cell-based assays showed Hm1a was selective for hNa V 1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with Na V 1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders.
Magneto-optical imaging of thin magnetic films using spins in diamond
Publisher: Springer Science and Business Media LLC
Date: 14-03-2016
DOI: 10.1038/SREP22797
Abstract: Imaging the fields of magnetic materials provides crucial insight into the physical and chemical processes surrounding magnetism and has been a key ingredient in the spectacular development of magnetic data storage. Existing approaches using the magneto-optic Kerr effect, x-ray and electron microscopy have limitations that constrain further development and there is increasing demand for imaging and characterisation of magnetic phenomena in real time with high spatial resolution. Here we show how the magneto-optical response of an array of negatively-charged nitrogen-vacancy spins in diamond can be used to image and map the sub-micron stray magnetic field patterns from thin ferromagnetic films. Using optically detected magnetic resonance, we demonstrate wide-field magnetic imaging over 100 × 100 μm 2 with sub-micron spatial resolution at video frame rates, under ambient conditions. We demonstrate an all-optical spin relaxation contrast imaging approach which can image magnetic structures in the absence of an applied microwave field. Straightforward extensions promise imaging with sub-μT sensitivity and sub-optical spatial and millisecond temporal resolution. This work establishes practical diamond-based wide-field microscopy for rapid high-sensitivity characterisation and imaging of magnetic s les, with the capability for investigating magnetic phenomena such as domain wall and skyrmion dynamics and the spin Hall effect in metals.
Mutant GABA A receptor γ2-subunit in childhood absence epilepsy and febrile seizures
Publisher: Springer Science and Business Media LLC
Date: 05-2001
DOI: 10.1038/88259
Proceedings of the Australian Physiological and Pharmacological Society Symposium: New Frontiers in Muscle Research Gene transfer: manipulating and monitoring function in cells and tissues
Publisher: Wiley
Date: 08-2001
DOI: 10.1046/J.1440-1681.2001.03504.X
Abstract: 1. The ectopic expression of genes has proven to be an extremely valuable tool for biologists. The most widely used systems involve electrically or chemically mediated transfer of genes to immortalized cell lines and, at the other end of the spectrum, transgenic animal models. As would be expected, there are compromises to be made when using either of these broad approaches. Immortalized cell lines have limited "physiological relevance" and transgenic approaches are costly and out of the reach of many laboratories. There is also significant time required for the de novo generation of a transgenic animal. 2. As a viable alternative to these approaches, we describe the use of recombinant adenovirus and Sindbis virus to deliver genes to cells and tissues. 3. We exemplify this approach with studies from our laboratories: (i) an investigation of Ca2+ handling deficits in cardiac myocytes of hypertrophied hearts using infection with recombinant adenovirus encoding either green fluorescent protein (GFP) or the sarcoplasmic/endoplasmic reticulum calcium-ATPase (Serca2a) (ii) a study of the mechanism of macrophage/microglial migration by infection of embryonic phagocytes with a GFP-encoding virus and coculture with brain slices to then track the movement of labelled cells and (iii) we are also exploiting the natural tropism of the Sindbis virus to label neurons in hippoc al brain slices in culture to resolve high-resolution structure and to map neuronal connectivity. 4. Further development of these approaches should open new avenues of investigation for the study of physiology in a range of cells and tissues.
The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.
Publisher: Wiley
Date: 21-07-2016
DOI: 10.1111/EPI.13474
Abstract: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-cl recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm). NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current litude for carbamazepine (31.6 ± 5.9%, n = 9 p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7 p < 0.001). Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.
Genetic generalized epilepsies
Publisher: Wiley
Date: 09-05-2018
DOI: 10.1111/EPI.14042
Abstract: The genetic generalized epilepsies (GGEs) are mainly genetically determined disorders. Although inheritance in most cases appears to be complex, involving multiple genes, variants of a number of genes are known to contribute. Pathogenic variants of SLC2A1 leading to autosomal-dominant GLUT1 deficiency account for up to 1% of cases, increasing to 10% of those with absence seizures starting before age 4 years. Copy number variants are found in around 3% of cases, acting as risk alleles. Copy number variation is much more common in those with comorbid learning disability. Common variant associations are starting to emerge from genome-wide association studies but do not yet explain a large proportion of GGEs. Although currently genetic testing is not likely to yield a diagnosis for most patients with GGEs, it can be of great importance in specific clinical situations. Providers should consider the in idual patient's history in determining the utility of genetic testing.
Method of derivation and differentiation of mouse embryonic stem cells generating synchronous neuronal networks
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.JNEUMETH.2017.08.018
Abstract: Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties. Therefore, strategies to improve correspondence of derived neuronal cultures with primary neurons need to be developed to validate the use of stem cell-derived neuronal cultures as in vitro models. By combining serum-free derivation of ESCs from mouse blastocysts with neuronal differentiation of ESCs in morphogen-free adherent culture we generated neuronal networks with properties recapitulating those of mature primary cortical cultures. After 35days of differentiation ESC-derived neurons developed network activity very similar to that of mature primary cortical neurons. Importantly, ESC plating density was critical for network development. Compared to the previously published methods this protocol generated more synchronous neuronal networks, with high similarity to the networks formed in mature primary cortical culture. We have demonstrated that ESC-derived neuronal networks recapitulating key properties of mature primary cortical networks can be generated by optimising both stem cell derivation and differentiation. This validates the approach of using ESC-derived neuronal cultures for disease modelling and in vitro drug screening.
A quantitative method for measuring innate phagocytosis by human monocytes using real‐time flow cytometry
Publisher: Wiley
Date: 16-10-2013
DOI: 10.1002/CYTO.A.22400
Abstract: Phagocytosis is central to immunity however a rapid and standardized method is much needed for quantitative assessment of the phagocytic process. We describe a real-time flow cytometric method to quantitate the phagocytosis of fluorescent latex beads by human monocytes in serum-free conditions. Effects of buffer composition, temperature, pH, and bead surface on phagocytic rate are described. The innate phagocytic ability of human monocytes from single subjects measured by this method was relatively stable over many months although phagocytosis rate varied as much as two-fold between in iduals. Comparable results were obtained with a simplified method using several mL of whole blood which is suitable for routine clinical application. This method also allows two-color flow cytometric measurement of cytosolic calcium levels during the phagocytic uptake of fluorescent beads.
State transitions through inhibitory interneurons in a cortical network model
Publisher: Public Library of Science (PLoS)
Date: 15-10-2021
DOI: 10.1371/JOURNAL.PCBI.1009521
Abstract: Inhibitory interneurons shape the spiking characteristics and computational properties of cortical networks. Interneuron subtypes can precisely regulate cortical function but the roles of interneuron subtypes for promoting different regimes of cortical activity remains unclear. Therefore, we investigated the impact of fast spiking and non-fast spiking interneuron subtypes on cortical activity using a network model with connectivity and synaptic properties constrained by experimental data. We found that network properties were more sensitive to modulation of the fast spiking population, with reductions of fast spiking excitability generating strong spike correlations and network oscillations. Paradoxically, reduced fast spiking excitability produced a reduction of global excitation-inhibition balance and features of an inhibition stabilised network, in which firing rates were driven by the activity of excitatory neurons within the network. Further analysis revealed that the synaptic interactions and biophysical features associated with fast spiking interneurons, in particular their rapid intrinsic response properties and short synaptic latency, enabled this state transition by enhancing gain within the excitatory population. Therefore, fast spiking interneurons may be uniquely positioned to control the strength of recurrent excitatory connectivity and the transition to an inhibition stabilised regime. Overall, our results suggest that interneuron subtypes can exert selective control over excitatory gain allowing for differential modulation of global network state.
Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus
Publisher: American Society for Clinical Investigation
Date: 02-08-2010
DOI: 10.1172/JCI42219
Missense mutations in the sodium-gated potassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy
Publisher: Springer Science and Business Media LLC
Date: 21-10-2012
DOI: 10.1038/NG.2440
Abstract: We performed genomic mapping of a family with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and intellectual and psychiatric problems, identifying a disease-associated region on chromosome 9q34.3. Whole-exome sequencing identified a mutation in KCNT1, encoding a sodium-gated potassium channel subunit. KCNT1 mutations were identified in two additional families and a sporadic case with severe ADNFLE and psychiatric features. These findings implicate the sodium-gated potassium channel complex in ADNFLE and, more broadly, in the pathogenesis of focal epilepsies.
A roadmap for precision medicine in the epilepsies
Publisher: Elsevier BV
Date: 12-2015
Axon initial segment dysfunction in epilepsy
Publisher: Wiley
Date: 28-05-2010
Interleukin-1 Receptor in Seizure Susceptibility after Traumatic Injury to the Pediatric Brain
Publisher: Society for Neuroscience
Date: 19-07-2017
Differential expression of exon 5 splice variants of sodium channel α subunit mRNAs in the developing mouse brain
Publisher: Elsevier BV
Date: 03-2010
DOI: 10.1016/J.NEUROSCIENCE.2009.12.011
Abstract: Sodium channel alpha subunit genes expressed in the human brain, SCN1A, SCN2A, SCN3A and SCN8A, are subject to alternative splicing of coding exons 5N and 5A. In this study we examined expression of alpha subunit mRNA and exon 5 splicing in the developing mouse brain. Expression levels of Scn1a, Scn2a and Scn8a mRNAs increase postnatally, whereas Scn3a mRNA expression levels decrease. Scn1a mRNA contains only exon 5A, due to the absence of exon 5N in the mouse Scn1a gene. At birth, Scn2a is the only sodium channel alpha subunit mRNA that contains higher or equal amounts of the 5N isoform compared to the 5A isoform in most brain regions. In contrast, the predominant isoform of Scn3a and Scn8a mRNAs in the newborn mouse brain is 5A. 5N/5A ratios for each of the three mRNAs vary across brain regions, with cortex >or= hippoc us>thalamus>cerebellum. In all brain regions and for all three alpha subunits, 5N/5A ratios gradually decrease with age, levelling at a value between 0.1 and 0.2. These findings suggest potential involvement of common factors in the alternative splicing of exon 5 for all three transcripts, and that expression of these factors varies between brain regions and changes during development. Differences in the strength of exon 5N and/or exon 5A splice sites in Scn2a pre-mRNA as compared to Scn1a and Scn8a may underlie the observed differences in 5N/5A ratios in the three alpha subunit mRNAs.
Community-based asthma care: Trial of a 'credit card' asthma self- management plan
Publisher: European Respiratory Society (ERS)
Date: 07-1994
DOI: 10.1183/09031936.94.07071260
Abstract: Although asthma self-management plans are widely recommended as essential in the long-term treatment of adult asthma, there have been few studies examining their use. Our objective was to assess the effect of a "credit card" adult asthma self-management plan in a community experiencing major health problems from asthma, by means of a before and after intervention trial of the efficacy of the "credit card" plan, when introduced through community-based asthma clinics. The participants were 69 Maori people with asthma. The "credit card" plan consisted of written guidelines for the self-management of asthma, based on self-assessment of asthma severity, printed on a plastic card. On one side, management guidelines were based on the interpretation of peak expiratory flow rate (PEFR) recordings, whilst the reverse side was based on symptoms. The outcome measures used were before and after comparison of markers of asthma morbidity and requirement for acute medical treatment and a structured questionnaire assessing the acceptability and use of the credit card plan. Following the introduction of the plan, the mean PEFR increased from 347 to 389 l.min-1, the percentage of nights woken fell from 30.4 to 16.9%, and the number of days "out of action" fell from 3.8 to 1.7%. The requirements for acute medical treatment also fell during the intervention period. Most participants commented favourably on the content and usefulness of the plan. In the situation of worsening asthma, 28% of subjects found the peak flow side of the card most helpful, 7% the symptoms side, and 48% found both sides equally helpful.(ABSTRACT TRUNCATED AT 250 WORDS)
High spatial and temporal resolution wide-field imaging of neuron activity using quantum NV-diamond
Publisher: Springer Science and Business Media LLC
Date: 09-05-2012
DOI: 10.1038/SREP00401
Perturbations in cortical development and neuronal network excitability arising from prenatal exposure to benzodiazepines in mice
Publisher: Wiley
Date: 04-03-2013
DOI: 10.1111/EJN.12167
Abstract: During brain development, many factors influence the assembly and final positioning of cortical neurons, and this process is essential for proper circuit formation and normal brain function. Among many important extrinsic factors that guide the maturation of embryonic cortical neurons, the secreted neurotransmitter GABA has been proposed to influence both their migratory behaviour and their terminal differentiation. The full extent of the short-term and long-term changes in brain patterning and function caused by modulators of the GABA system is not known. In this study, we specifically investigated whether diazepam, a commonly used benzodiazepine that modulates the GABAA receptor, alters neuronal positioning in vivo, and whether this can lead to lasting effects on brain function. We found that fetal exposure to diazepam did not change cell positioning within the embryonic day (E)14.5 mouse cerebral cortex, but significantly altered neuron positioning within the E18.5 cortex. In adult mice, diazepam treatment affected the distribution of cortical interneurons that express parvalbumin or calretinin, and also led to a decrease in the numbers of calretinin-expressing interneurons. In addition, we observed that neonatal exposure to diazepam altered the sensitivity of mice to a proconvulsant challenge. Therefore, exposure of the fetal brain to benzodiazepines has consequences for the positioning of neurons and cortical network excitability.
Augmented currents of an HCN2 variant in patients with febrile seizure syndromes
Publisher: Wiley
Date: 04-2010
DOI: 10.1002/ANA.21909
A Thr357 to Ser Polymorphism in Homozygous and Compound Heterozygous Subjects Causes Absent or Reduced P2X7 Function and Impairs ATP-induced Mycobacterial Killing by Macrophages
Publisher: Elsevier BV
Date: 2006
Prediction by Modeling That Epilepsy May Be Caused by Very Small Functional Changes in Ion Channels
Publisher: American Medical Association (AMA)
Date: 10-2009
DOI: 10.1001/ARCHNEUROL.2009.219
Abstract: To use computer simulation to perform a "genetic sensitivity" analysis to predict which genes are best positioned to increase risk as well as to predict functionally how variants in these genes might increase network excitability. A previously published, biophysically realistic model of the dentate gyrus that included mossy fiber sprouting between granule cells was used to model putative environmental changes associated with temporal lobe epilepsy. Properties of voltage-gated ion channels, either 1 at a time or in combinations, were varied systematically to determine their effect on network excitability. We found that the network is most sensitive to changes in steady-state voltage dependence of activation and relatively insensitive to changes in inactivation. Changes in sodium channels had the greatest effect on excitability, followed by changes in fast-delayed rectifier potassium channels and then N-type calcium channels. We also investigated the effects of simultaneous small changes in several ion channels, modeling a complex genetic background expected for common epilepsies. A combination of 2 or 3 simultaneous voltage shifts in steady-state activation as small as 2 mV could produce large changes in network excitability. Statistical power calculations indicate that changes this small are effectively undetectable with current experimental practices, thus posing new challenges for the functional analysis and validation of epilepsy genes.
A childhood epilepsy mutation reveals a role for developmentally regulated splicing of a sodium channel
Publisher: Elsevier BV
Date: 06-2007
DOI: 10.1016/J.MCN.2007.03.003
Abstract: Seizure susceptibility is high in human infants compared to adults, presumably because of developmentally regulated changes in neural excitability. Benign familial neonatal-infantile seizures (BFNIS), characterized by both early onset and remission, are caused by mutations in the gene encoding a human sodium channel (NaV1.2). We analyzed neonatal and adult splice forms of NaV1.2 with a BFNIS mutation (L1563V) in human embryonic kidney cells. Computer modeling revealed that neonatal channels are less excitable than adult channels. Introduction of the mutation increased excitability in the neonatal channels to a level similar to adult channels. By contrast, the mutation did not affect the adult channel variant. This "adult-like" increased excitability is likely to be the mechanism underlying BFNIS in infants with this mutation. More generally, developmentally regulated NaV1.2 splicing may be one mechanism that counters the normally high excitability of neonatal neurons and helps to reduce seizure susceptibility in normal human infants.
Spike-and-wave discharge mediated reduction in hippocampal HCN1 channel function associates with learning deficits in a genetic mouse model of epilepsy
Publisher: Elsevier BV
Date: 04-2014
DOI: 10.1016/J.NBD.2013.12.007
Abstract: The GABAAγ2(R43Q) mouse is an established model of absence epilepsy displaying spontaneous spike-and-wave discharges (SWD) and associated behavioral arrest. Absence epilepsy typically results from cortico-thalamic networks. Nevertheless, there is increasing evidence for changes in hippoc al metabolism and electrical behavior, consistent with a link between absence seizures and hippoc us-related co-morbidities. Hyperpolarization-activated-cyclic-nucleotide-gated (HCN) channels are known to be transcriptionally regulated in a number of seizure models. Here we investigate the expression and function of these channels in the hippoc us of the genetic epilepsy model. A reduction in HCN1, but not HCN2 transcript, was observed in GABAAγ2(R43Q) mice relative to their littermate controls. In contrast, no change in HCN1 transcript was noted at an age prior to seizure expression or in a SWD-free model in which the R43Q mutation has been crossed into a seizure-resistant genetic background. Whole-cell recordings from CA1 pyramidal neurons confirm a reduction in Ih in the GABAAγ2(R43Q) mouse. Further, a left-shift in half-activation of the Ih conductance-voltage relationship is consistent with a reduction in HCN1 with no change in HCN2 channel expression. Behavioral analysis using the Morris water maze indicates that GABAAγ2(R43Q) mice are unable to learn as effectively as their wildtype littermates suggesting a deficit in hippoc al-based learning. SWD-free mice harboring the R43Q mutation had no learning deficit. We conclude that SWDs reduce hippoc al HCN1 expression and function, and that the reduction associates with a spatial learning deficit.
The mechanism of carbamazepine aggravation of absence seizures
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-08-2006
Abstract: Carbamazepine (CBZ) aggravates many generalized seizures types, particularly absence seizures, but the mechanisms underlying this are poorly understood. GABA signaling within the reticular nucleus (Rt) and the ventrobasal complex (VB) of the thalamus is critical to the neurophysiology of absence seizures. The hypothesis that CBZ aggravates absence seizures by acting at the VB thalamus via a GABA(A) receptor-mediated mechanism was investigated in a genetic rat model, generalized absence epilepsy rats from Strasbourg (GAERS). Seizure activity was quantified by a 90-min electroencephalogram recording postdrug injection. Intracerebroventricular injections of CBZ (15 microg in 4 microl) resulted in seizure aggravation versus vehicle treatment, with a mean increase in seizure time of 40%. This indicates that CBZ acts directly, rather than via a metabolite, on the brain to aggravate seizures. Seizure aggravation also occurred following bilateral microinjection of CBZ (0.75 microg in 0.2 microl) into the VB (53%) but not following injection into the Rt (-9%). However, seizure aggravation was blocked when the GABA(A) receptor antagonist, bicuculline (BIC, 0.04 microg in 0.2 microl), was coinjected with CBZ into the VB. Injection of BIC alone (versus vehicle) into the VB also blocked seizure aggravation following systemic administration of CBZ (15 mg/kg i.p.). In vitro studies in Xenopus oocytes expressing recombinant GABA(A) receptors demonstrated that CBZ produced a dose-dependent potentiation of the GABA current at a physiological relevant concentration range (1-100 microM). These data demonstrate that CBZ acts at the VB thalamus to aggravate absence seizures in GAERS and that activation of GABA(A) receptors is critical to this effect.
Low glycaemic index diet reduces seizure susceptibility in a syndrome-specific mouse model of generalized epilepsy
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.EPLEPSYRES.2013.10.014
Abstract: Clinical evidence suggests that low glycaemic index diets are effective at reducing seizure frequency potentially through the stabilization of blood glucose levels. Here we investigate if diets containing carbohydrates with varying glycaemic index (GI) can modulate seizure susceptibility in a mouse model of generalized epilepsy. Electrocortical recordings were made from mice harboring the GABAAγ2 (R43Q) epilepsy mutation after three weeks on a low-or high-GI diet. Standard rodent diet was used as a control. Occurrence and durations of spike-wave-discharges (SWDs) were measured. An insulin injection was used to reduce blood glucose to levels known to precipitate SWDs in the GABAAγ2 (R43Q) mouse on the low and high-GI diets. SWD occurrence was reduced by approximately 35% in mice on the low-GI compared to high-GI diet. SWD occurrence was not different between high-GI diet and a standard diet suggesting that low-GI diet is protective. Weight gain of mice for all diet groups was identical suggesting that they were equally well tolerated. Under low blood glucose conditions SWD occurrence increased in the low and high-GI diets. Importantly, under low glucose conditions the low-GI diet no longer conferred protection against SWDs. SWDs were reduced in mice on a low GI-diet suggesting it may be an effective and well tolerated therapy for generalized epilepsy. The lack of effect of low-GI diet when glucose levels are reduced suggests that seizure protection in the GABAAγ2 (R43Q) mouse model may be due to the diets ability to stabilize blood glucose levels.
PRIMA1 mutation: A new cause of nocturnal frontal lobe epilepsy
Publisher: Wiley
Date: 03-07-2015
DOI: 10.1002/ACN3.224
Gain-of-function HCN2 variants in genetic epilepsy.
Publisher: Hindawi Limited
Date: 13-11-2017
DOI: 10.1002/HUMU.23357
Abstract: Genetic generalized epilepsy (GGE) is a common epilepsy syndrome that encompasses seizure disorders characterized by spike-and-wave discharges (SWDs). Pacemaker hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are considered integral to SWD genesis, making them an ideal gene candidate for GGE. We identified HCN2 missense variants from a large cohort of 585 GGE patients, recruited by the Epilepsy Phenome-Genome Project (EPGP), and performed functional analysis using two-electrode voltage cl recordings from Xenopus oocytes. The p.S632W variant was identified in a patient with idiopathic photosensitive occipital epilepsy and segregated in the family. This variant was also independently identified in an unrelated patient with childhood absence seizures from a European cohort of 238 familial GGE cases. The p.V246M variant was identified in a patient with photo-sensitive GGE and his father diagnosed with juvenile myoclonic epilepsy. Functional studies revealed that both p.S632W and p.V246M had an identical functional impact including a depolarizing shift in the voltage dependence of activation that is consistent with a gain-of-function. In contrast, no biophysical changes resulted from the introduction of common population variants, p.E280K and p.A705T, and the p.R756C variant from EPGP that did not segregate with disease. Our data suggest that HCN2 variants can confer susceptibility to GGE via a gain-of-function mechanism.
Antisense oligonucleotide therapy reduces seizures and extends life span in an SCN2A gain-of-function epilepsy model.
Publisher: American Society for Clinical Investigation
Date: 12-2021
DOI: 10.1172/JCI152079
SCN1A gain of function in early infantile encephalopathy
Publisher: Wiley
Date: 07-03-2019
DOI: 10.1002/ANA.25438
Abstract: To elucidate the biophysical basis underlying the distinct and severe clinical presentation in patients with the recurrent missense SCN1A variant, p.Thr226Met. Patients with this variant show a well-defined genotype-phenotype correlation and present with developmental and early infantile epileptic encephalopathy that is far more severe than typical SCN1A Dravet syndrome. Whole cell patch cl and dynamic action potential cl were used to study T226M Na T226M channels exhibited hyperpolarizing shifts of the activation and inactivation curves and enhanced fast inactivation. Dynamic action potential cl hybrid simulation showed that model neurons containing T226M conductance displayed a left shift in rheobase relative to control. At current stimulation levels that produced repetitive action potential firing in control model neurons, depolarization block and cessation of action potential firing occurred in T226M model neurons. Fully computationally simulated neuron models recapitulated the findings from dynamic action potential cl and showed that heterozygous T226M models were also more susceptible to depolarization block. From a biophysical perspective, the T226M mutation produces gain of function. Somewhat paradoxically, our data suggest that this gain of function would cause interneurons to more readily develop depolarization block. This "functional dominant negative" interaction would produce a more profound disinhibition than seen with haploinsufficiency that is typical of Dravet syndrome and could readily explain the more severe phenotype of patients with T226M mutation. Ann Neurol 2019 :514-525.
Antisense oligonucleotide therapy for KCNT1 encephalopathy.
Publisher: American Society for Clinical Investigation
Date: 08-12-2022
DOI: 10.1172/JCI.INSIGHT.146090
Abstract: Developmental and epileptic encephalopathies (DEE) are characterized by pharmacoresistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared to mice treated with a control ASO (non-hybridizing sequence). ASO administration at neonatal age was also well-tolerated and effective in controlling seizures and extending the lifespan of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.
Use of Local Field Potentials of Dissociated Cultures Grown on Multi-Electrode Arrays for Pharmacological Assays
Publisher: IEEE
Date: 08-2016
Triheptanoin reduces seizure susceptibility in a syndrome-specific mouse model of generalized epilepsy
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.EPLEPSYRES.2012.09.016
Abstract: Triheptanoin is a triglyceride containing heptanoate, an odd-chained medium fatty acid that is metabolized to produce propionyl-CoA and subsequently C4 intermediates of the citric acid cycle and therefore capable of anaplerosis. These metabolic products are believed to underlie triheptanoin's anticonvulsant effects in rodent seizure models. Here we investigate the anticonvulsive effects of oral triheptanoin in a syndrome-specific genetic mouse model of generalized epilepsy based on the GABA(A)γ2(R43Q) mutation. Mice were fed a diet supplemented with triheptanoin from weaning for three weeks prior to electrocortical recordings. Occurrence and durations of spike and wave discharges (SWDs) were measured. Triheptanoin did not alter body weight or basal blood glucose levels suggesting that it was well tolerated. Triheptanoin supplementation halved the time spent in seizures due to a reduction in both SWD occurrence and duration. An injection of insulin was used to reduce blood glucose, a metabolic stress known to precipitate seizures in the GABA(A)γ2(R43Q) mouse. The reduction in seizure count was also evident following insulin induced hypoglycemia with the triheptanoin treated group having significantly less SWDs than control animals under similar low blood glucose conditions. In summary, triheptanoin may be an effective and well tolerated dietary therapy for generalized epilepsy.
Gap Junctions Link Regular-Spiking and Fast-Spiking Interneurons in Layer 5 Somatosensory Cortex
Publisher: Frontiers Media SA
Date: 17-07-2017
Dynamic action potential clamp predicts functional separation in mild familial and severe de novo forms of SCN2A epilepsy
Publisher: Proceedings of the National Academy of Sciences
Date: 29-05-2018
Abstract: SCN2A , encoding the voltage-gated sodium channel Na v 1.2, has emerged as a major gene implicated in neonatal-, infantile-, and even childhood-onset epilepsies. Many of these epilepsies are also associated with cognitive and behavioral impairments that range in type and severity. The biophysical, neurophysiological, and clinical impacts of SCN2A mutations are poorly understood. Here, we use clinical evaluation and biophysical analyses to explore the mechanisms underpinning distinctive phenotypes produced by SCN2A variants associated with mild familial or severe de novo forms of epilepsy. We show that dynamic cl analysis provides clear benefits over conventional voltage cl for a rapid and definitive prediction of neuron-scale phenotypic consequences, and is well positioned to impact diagnosis and drug discovery in genetic epilepsy.
A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY.
Publisher: Springer Science and Business Media LLC
Date: 12-01-2018
DOI: 10.1038/S41598-017-18045-Y
Abstract: Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/ In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.
Altered kinetics and benzodiazepine sensitivity of a GABA A receptor subunit mutation [γ 2 (R43Q)] found in human epilepsy
Publisher: Proceedings of the National Academy of Sciences
Date: 11-2002
Abstract: The γ-aminobutyric acid type A (GABA A ) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA A receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA A γ 2 -subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA A receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-cl analysis of steady-state currents obtained from α 1 β 2 γ 2 or α 1 β 2 γ 2 (R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABA A receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.
Novel GABRA2 variants in epileptic encephalopathy and intellectual disability with seizures
Publisher: Oxford University Press (OUP)
Date: 10-04-2019
DOI: 10.1093/BRAIN/AWZ079
Models for discovery of targeted therapy in genetic epileptic encephalopathies
Publisher: Wiley
Date: 05-09-2017
DOI: 10.1111/JNC.14134
Abstract: Epileptic encephalopathies are severe disorders emerging in the first days to years of life that commonly include refractory seizures, various types of movement disorders, and different levels of developmental delay. In recent years, many de novo occurring variants have been identified in in iduals with these devastating disorders. To unravel disease mechanisms, the functional impact of detected variants associated with epileptic encephalopathies is investigated in a range of cellular and animal models. This review addresses efforts to advance and use such models to identify specific molecular and cellular targets for the development of novel therapies. We focus on ion channels as the best-studied group of epilepsy genes. Given the clinical and genetic heterogeneity of epileptic encephalopathy disorders, experimental models that can reflect this complexity are critical for the development of disease mechanisms-based targeted therapy. The convergence of technological advances in gene sequencing, stem cell biology, genome editing, and high throughput functional screening together with massive unmet clinical needs provides unprecedented opportunities and imperatives for precision medicine in epileptic encephalopathies.
Two lines of transgenic mice expressing cre-recombinase exhibit increased seizure susceptibility
Publisher: Elsevier BV
Date: 03-2013
DOI: 10.1016/J.EPLEPSYRES.2012.10.005
Abstract: Conditional mouse models based on the Cre-recombinase (Cre)-loxP method are a powerful tool for determining the spatial and temporal function of genes in neuroscience research. The Emx1-Cre conditional model is designed to drive Cre expression in a predominantly excitatory neuron specific manner and the Dlx5/6-Cre mouse expresses Cre predominantly in cortical inhibitory neurons. The mouse models expressing the Cre transgene are healthy, active and have no overt behavioural or brain histological phenotypes. Subcutaneous pentylenetetrazol (scPTZ) is a proconvulsant frequently used to probe neuronal network excitability. In both the Emx1-Cre and Dlx5/6-Cre conditional mouse models the latency to scPTZ-induced seizures was significantly shorter than for their wild-type littermates. This shows that mouse models carrying the Cre transgene alone can have significant behavioural phenotypes. This may act as a confound to the interpretation of data obtained from crosses with loxP-flanked targets especially in the context of epilepsy phenotypes. These data highlight that appropriate control experiments that compare wild-type mice to those that carry the cre-transgene but not the loxP-flanked target are essential when using this method.
Glucose transporter 1 deficiency in the idiopathic generalized epilepsies
Publisher: Wiley
Date: 11-2012
DOI: 10.1002/ANA.23702
Abstract: We examined whether glucose transporter 1 (GLUT1) deficiency causes common idiopathic generalized epilepsies (IGEs). The IGEs are common, heritable epilepsies that usually follow complex inheritance currently little is known about their genetic architecture. Previously considered rare, GLUT1 deficiency, due to mutations in SLC2A1, leads to failure of glucose transport across the blood-brain barrier and inadequate glucose for brain metabolism. GLUT1 deficiency was first associated with an encephalopathy and more recently found in rare dominant families with epilepsy and paroxysmal exertional dyskinesia (PED). Five hundred four probands with IGEs and 470 controls underwent SLC2A1 sequencing. Glucose transport was assayed following expression of SLC2A1 variants in Xenopus oocytes. All available relatives were phenotyped, and SLC2A1 was sequenced. Functionally validated mutations in SLC2A1 were present in 7 of 504 (1.4%) probands and 0 of 470 controls. PED, undiagnosed prior to study, occurred in 1 proband and 3 of 13 relatives with mutations. The IGEs in probands and relatives were indistinguishable from typical IGE. Three cases (0.6%) had mutations of large functional effect and showed autosomal dominant inheritance or were de novo. Four (0.8%) cases had a subtle functional effect 2 showed possible dominant inheritance, and 2 did not. These alleles leading to subtle functional impairment may contribute to complex, polygenic inheritance of IGE. SLC2A1 mutations contribute to approximately 1% of IGE both as a dominant gene and as a susceptibility allele in complex inheritance. Diagnosis of GLUT1 deficiency has important treatment (ketogenic diet) and genetic counseling implications. The mechanism of restricted glucose delivery differs from the current focus on IGEs as ion channel disorders.
Point mutations confer loss of ATP‐induced human P2X7 receptor function
Publisher: Wiley
Date: 04-01-2002
DOI: 10.1016/S0014-5793(01)03311-7
Abstract: Residues considered essential for ATP binding to the human P2X(7) receptor (hP2X(7)R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or site-directed mutants of hP2X(7)R constructs and channel ore activity measured in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X(7)R.
New therapeutic opportunities in epilepsy: A genetic perspective
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.PHARMTHERA.2010.07.003
Abstract: Epilepsy is a common and serious neurological disorder. Despite recent advances in drug therapy, treatment for epilepsy is still largely empirical and rational prescribing based on the mechanism of action in an in idual patient is generally not possible. Genetic studies have identified an increasing collection of disease-causing genes providing a fundamental molecular foundation on which to build this understanding, at least for some forms of epilepsy. The impact of these genetic discoveries is likely to be wide reaching-from the discovery and validation of new drug targets to the potential to enable rational prescribing based on genetic makeup and even further through animal experimentation to tease out molecular and cellular mechanisms that lead to hyperexcitable neuronal networks causing epilepsy. Here we discuss how we can use knowledge of genetic mechanisms to improve treatment strategies now and into the future.
MRI-guided volume reconstruction of mouse brain from histological sections
Publisher: Elsevier BV
Date: 11-2012
DOI: 10.1016/J.JNEUMETH.2012.08.021
Abstract: A method is presented for three-dimensional reconstruction of the mouse brain from histological sections with the guidance of magnetic resonance images (MRI). A major focus of the method is dealing with sections in which anatomical structures have been separated or distorted as a result of histological processing. Although histology has superb resolution with the ability to discriminate cell types and anatomical structures, misalignment between sections and distortion within sections renders 3D reconstruction of the histology volume simply by stacking 2D sections inadequate. In contrast, MRI preserves the spatial and geometric information about structures at a cost of cellular detail. To utilize the information from MRI in reconstructing volumetric histological data, we developed a procedure consisting of a series of segmentation and registration operations. The method is iterative and first identifies the corresponding MRI slices for each histological section. Piecewise rigid registration is then employed to deal with tissue distortion caused by histological processing. Quantitative validation of the method's accuracy was performed on four reconstructed mouse brains by comparing a set of manually selected anatomical landmarks on pairs of MRI and histological volumes. The procedure is highly automated and amenable to high throughput.
Cocaine-mediated synaptic potentiation is absent in VTA neurons from mGlu5-deficient mice
Publisher: Oxford University Press (OUP)
Date: 23-06-2010
Application of rare variant transmission disequilibrium tests to epileptic encephalopathy trio sequence data
Publisher: Springer Science and Business Media LLC
Date: 17-05-2017
DOI: 10.1038/EJHG.2017.61
An Ile-568 to Asn Polymorphism Prevents Normal Trafficking and Function of the Human P2X7 Receptor
Publisher: Elsevier BV
Date: 05-2003
Electron paramagnetic resonance microscopy using spins in diamond under ambient conditions
Publisher: Springer Science and Business Media LLC
Date: 06-09-2017
Temperature elevation increases GABAA-mediated cortical inhibition in a mouse model of genetic epilepsy
Publisher: Wiley
Date: 22-12-2011
Axon initial segment structural plasticity in animal models of genetic and acquired epilepsy
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.EPLEPSYRES.2013.03.004
Abstract: A novel form of neuronal plasticity, occurring at the axon initial segment (AIS), has recently been described. Lengthening of the AIS and movement away from the soma are consequences of changes in neuronal input and result in alterations in neuronal excitability. We hypothesised that AIS plasticity may play a role in epilepsy, due to chronic changes in neuronal activity. Immunohistochemistry and confocal microscopy were used to analyse AIS length and position in pyramidal neurons in deep layer 5 of the somatosensory cortex from 5 mice with genetic epilepsy and 4 controls, and from 3 rats subjected to amygdala kindling and 3 controls. The effect of a subtle alteration of AIS position was modelled computationally. We identified a difference in the position of the AIS in animals with seizures: in mice the AIS was positioned 0.2 μm further away from the soma, and in rats the AIS was positioned 0.6 μm closer to the soma compared with controls. Computational modelling indicated that a subtle alteration in AIS position could result in a change in action potential firing threshold. The identification of AIS plasticity in animal models of epilepsy is significant in furthering our understanding of the pathophysiological mechanisms involved in this disorder.
Genetically Targeted Calcium Sensors Enhance The Study Of Organelle Function In Living Cells
Publisher: Wiley
Date: 14-09-2000
DOI: 10.1046/J.1440-1681.2000.03327.X
Abstract: 1. Understanding the regulation of calcium (Ca2+), the most common of the mineral ions within the human body, has always been of extreme interest to physiologists. While the importance of Ca2+ in contributing to physiological events through regulation of levels has been significantly established, seldom is consideration given to the intricacies of this ion and its mechanics in producing such erse physiological responses in different regions of the cell. 2. The present review will summarize new methodologies used in our laboratories for the study of two major intracellular organelles, mitochondria and the nucleus. These techniques are based predominantly on the use of molecular biological approaches to both create and then target protein-based sensor molecules to specific intracellular locations. 3. The regulation of Ca2+ in the mitochondria and nucleus is of particular interest to us because of the central involvement of these organelles in: (i) cardiac cell responses during ischaemia/reperfusion and (ii) the control of gene expression, respectively.
Toluene inhalation in adolescent rats reduces flexible behaviour in adulthood and alters glutamatergic and GABAergic signalling
Publisher: Wiley
Date: 09-11-2016
DOI: 10.1111/JNC.13858
Abstract: Toluene is a commonly abused inhalant that is easily accessible to adolescents. Despite the increasing incidence of use, our understanding of its long-term impact remains limited. Here, we used a range of techniques to examine the acute and chronic effects of toluene exposure on glutameteric and GABAergic function, and on indices of psychological function in adult rats after adolescent exposure. Metabolomics conducted on cortical tissue established that acute exposure to toluene produces alterations in cellular metabolism indicative of a glutamatergic and GABAergic profile. Similarly, in vitro electrophysiology in Xenopus oocytes found that acute toluene exposure reduced NMDA receptor signalling. Finally, in an adolescent rodent model of chronic intermittent exposure to toluene (10 000 ppm), we found that, while toluene exposure did not affect initial learning, it induced a deficit in updating that learning when response-outcome relationships were reversed or degraded in an instrumental conditioning paradigm. There were also group differences when more effort was required to obtain the reward toluene-exposed animals were less sensitive to progressive ratio schedules and to delayed discounting. These behavioural deficits were accompanied by changes in subunit expression of both NMDA and GABA receptors in adulthood, up to 10 weeks after the final exposure to toluene in the hippoc us, prefrontal cortex and ventromedial striatum regions with recognized roles in behavioural flexibility and decision-making. Collectively, our data suggest that exposure to toluene is sufficient to induce adaptive changes in glutamatergic and GABAergic systems and in adaptive behaviour that may underlie the deficits observed following adolescent inhalant abuse, including susceptibility to further drug-use.
Amiloride Derivatives Inhibit Coxsackievirus B3 RNA Replication
Publisher: American Society for Microbiology
Date: 02-2008
DOI: 10.1128/JVI.01374-07
Abstract: Amiloride derivatives are known blockers of the cellular Na + /H + exchanger and the epithelial Na + channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-( N -ethyl- N -isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.
Lack of response to quinidine in KCNT1-related neonatal epilepsy.
Publisher: Wiley
Date: 04-09-2018
DOI: 10.1111/EPI.14551
Abstract: To evaluate the clinical efficacy and safety of quinidine in patients with KCNT1-related epilepsy of infancy with migrating focal seizures (EIMFS) in the infantile period and to compare with the effect of quinidine on mutant channels in vitro. We identified 4 patients with EIMFS with onset in the neonatal period, pathogenic variants in the KCNT1 gene, and lack of response to AEDs. Patients were prospectively enrolled, treated with quinidine, and monitored according to a predefined protocol. Electroclinical, neuroimaging, and genetic data were reviewed. Two patients had novel variants in the KCNT1 gene that were modeled in Xenopus oocytes with channel properties characterized using electrophysiology recordings. Three of four patients were treated with quinidine early in their disease course, prior to 6 months of age. No significant side effects were noted with quinidine therapy. In addition, there were no significant changes in electroencephalography (EEG)-confirmed seizure burden during therapy, and patients had near hourly seizures before, during, and after treatment. Two patients had previously reported gain-of-function mutations, which demonstrated sensitivity to high levels of quinidine in the oocyte assay. Two patients with novel variants, showed characteristic gain-of-function and were thus predicted to be pathogenic. Of interest, these variants were essentially insensitive to high levels of quinidine. Patients had no reported benefit to quinidine therapy despite age at treatment initiation. Pharmacogenetic results in oocytes were consistent with clinical treatment failure in 2 patients, suggesting that single-dose pharmacologic assessment may be helpful in predicting which patients are exceedingly unlikely to achieve benefit with quinidine. In the 2 patients who had a lack of therapeutic benefit despite sensitivity to high concentrations of quinidine with in vitro oocyte assay, it is likely that the achievable exposure levels in the brain were too low to cause significant in vivo channel blockade.
Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures
Publisher: Elsevier BV
Date: 05-2015
Brain uptake of diazepam and phenytoin in a genetic animal model of absence epilepsy
Publisher: Wiley
Date: 16-04-2010
DOI: 10.1111/J.1440-1681.2010.05362.X
Abstract: 1. Although many studies have assessed changes to brain uptake of anti‐epileptic drugs (AEDs) in chemically and electrically induced seizure models, there are limited data available on changes to brain uptake of AEDs in spontaneous seizure animal models, such as genetic absence epilepsy. 2. In the present study, the brain uptake of diazepam and phenytoin was assessed in a genetic mouse model of absence seizures harbouring a human GABA A receptor γ2‐subunit gene GABRG2 mutation (R43Q) and results were compared with those obtained during acute seizures induced by subcutaneous administration of pentylenetetrazole (PTZ 90 mg/kg). Diazepam and phenytoin were administered intraperitoneally at doses of 2 and 30 mg/kg, respectively, and brain and plasma concentrations were determined 60 min after administration using liquid chromatography–mass spectrometry. 3. Although the brain uptake of phenytoin was significantly reduced following PTZ administration, no changes were observed in phenytoin disposition in the genetic absence epilepsy model. Similarly, the brain uptake of diazepam was significantly enhanced following PTZ administration, but it was not affected in absence epilepsy. 4. The cerebrovascular plasma volume (assessed by administration of the non‐absorbable marker [ 14 C]‐inulin) was not significantly different in saline‐treated compared with PTZ‐treated mice and in wild‐type compared with mutant R43Q mice. 5. These results demonstrate that although the brain uptake of AEDs may be altered in acute seizure models, similar changes to brain uptake may not be observed in the non‐convulsive genetic absence epileptic model.
KCNT1 gain of function in 2 epilepsy phenotypes is reversed by quinidine.
Publisher: Wiley
Date: 04-2014
DOI: 10.1002/ANA.24128
The coma in glaucoma: Retinal ganglion cell dysfunction and recovery
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.PRETEYERES.2018.04.001
Abstract: Retinal ganglion cell (RGC) degeneration causes vision loss in patients with glaucoma, and this has been generally considered to be irreversible due to RGC death. We question this assertion and summarise accumulating evidence that points to visual function improving in glaucoma patients with treatment, particularly in the early stages of disease. We propose that prior to death, RGCs enter periods of dysfunction but can recover with relief of RGC stress. We first summarise the clinical evidence for vision improvement in glaucoma and then detail our experimental work that points to the underlying processes that underpin clinical improvement. We show that functional recovery can occur following a prolonged course of RGC dysfunction and demonstrate how the capacity for recovery can be modified. Detecting RGC dysfunction and augmenting recovery of such 'comatosed' RGCs holds clinical potential to improve early detection of glaucoma and improve visual function.
Quinidine in the treatment of KCNT1-positive epilepsies.
Publisher: Wiley
Date: 18-11-2015
DOI: 10.1002/ANA.24520
A functional correlate of severity in alternating hemiplegia of childhood.
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.NBD.2015.02.002
Abstract: Mutations in ATP1A3, the gene that encodes the α3 subunit of the Na(+)/K(+) ATPase, are the primary cause of alternating hemiplegia of childhood (AHC). Correlations between different mutations and AHC severity were recently reported, with E815K identified in severe and D801N and G947R in milder cases. This study aims to explore the molecular pathological mechanisms in AHC and to identify functional correlates for mutations associated with different levels of disease severity. Human wild type ATP1A3, and E815K, D801N and G947R mutants were expressed in Xenopus laevis oocytes and Na(+)/K(+) ATPase function measured. Structural homology models of the human α3 subunit containing AHC mutations were created. The AHC mutations examined all showed similar levels of reduction in forward cycling. Wild type forward cycling was reduced by coexpression with any mutant, indicating dominant negative interactions. Proton transport was measured and found to be selectively impaired only in E815K. Homology modeling showed that D801 and G947 lie within or near known cation binding sites while E815 is more distal. Despite its effect on proton transport, E815K was also distant from the proposed proton transport route. Loss of forward cycling and dominant negativity are common and likely necessary pathomechanisms for AHC. In addition, loss of proton transport correlated with severity of AHC. D801N and G947R are likely to directly disrupt normal Na(+)/K(+) binding while E815K may disrupt forward cycling and proton transport via allosteric mechanisms yet to be elucidated.
Ion Channels in Genetic Epilepsy: From Genes and Mechanisms to Disease-Targeted Therapies
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 20-12-2018
PBT2 inhibits glutamate-induced excitotoxicity in neurons through metal-mediated preconditioning
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.NBD.2015.02.008
Abstract: Excitotoxicity is the pathological process by which neuronal death occurs as a result of excessive stimulation of receptors at the excitatory synapse such as the NMDA receptor (NMDAR). Excitotoxicity has been implicated in the acute neurological damage from ischemia and traumatic brain injury and in the chronic neurodegeneration in Alzheimer's disease (AD) and Huntington's disease (HD). As a result NMDAR antagonists have become an attractive therapeutic strategy for the potential treatment of multiple neurodegenerative diseases. However NMDAR signaling is dichotomous in nature, with excessive increases in neuronal intracellular calcium through excessive NMDAR activity being lethal but moderate increases to intracellular calcium levels during normal synaptic function providing neuroprotection. Subsequently indiscriminant inhibition of this receptor is best avoided as was concluded from previous clinical trials of NMDAR antagonists. We show that the metal chaperone, PBT2, currently in clinical trials for HD, is able to protect against glutamate-induced excitotoxicity mediated through NMDARs. This was achieved by PBT2 inducing Zn(2+)-dependent increases in intracellular Ca(2+) levels resulting in preconditioning of neurons and inhibition of Ca(2+)-induced neurotoxic signaling cascade involving calpain-activated cleavage of calcineurin. Our study demonstrates that modulating intracellular Ca(2+) levels by a zinc ionophore is a valid therapeutic strategy to protect against the effects of excitotoxicity thought to underlie both acute and chronic neurodegenerative diseases.
Segmentation of the C57BL/6J mouse cerebellum in magnetic resonance images
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.NEUROIMAGE.2012.05.061
Abstract: The C57BL mouse is the centerpiece of efforts to use gene-targeting technology to understand cerebellar pathology, thus creating a need for a detailed magnetic resonance imaging (MRI) atlas of the cerebellum of this strain. In this study we present a methodology for systematic delineation of the vermal and hemispheric lobules of the C57BL/6J mouse cerebellum in magnetic resonance images. We have successfully delineated 38 cerebellar and cerebellar-related structures. The higher signal-to-noise ratio achieved by group averaging facilitated the identification of anatomical structures. In addition, we have calculated average region volumes and created probabilistic maps for each structure. The segmentation method and the probabilistic maps we have created will provide a foundation for future studies of cerebellar disorders using transgenic mouse models.
KCTD12 modulation of GABA(B) receptor function
Publisher: Wiley
Date: 07-06-2017
DOI: 10.1002/PRP2.319
Reduced dendritic arborization and hyperexcitability of pyramidal neurons in a Scn1b-based model of Dravet syndrome
Publisher: Oxford University Press (OUP)
Date: 17-04-2014
DOI: 10.1093/BRAIN/AWU077
Abstract: Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human β1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-cl analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, d ened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease.
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