Medicine (Austin & Northern Health) - Research Publications
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ItemStargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011-04)Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.
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ItemGain-of-function HCN2 variants in genetic epilepsy(WILEY, 2018-02)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 clamp 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.
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ItemModels for discovery of targeted therapy in genetic epileptic encephalopathies(WILEY, 2017-10)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 individuals 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.
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ItemMyoclonus Epilepsy and Ataxia due to KCNC1 Mutation: Analysis of 20 Cases and K plus Channel Properties(WILEY, 2017-05)OBJECTIVE: 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. METHODS: 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-clamp to explore biophysical properties of wild-type and mutant KV 3.1 channels. RESULTS: 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 KV 3.1, increasing channel availability. INTERPRETATION: 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 KV 3.1 subunit-containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics. Ann Neurol 2017;81:677-689.
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ItemSynaptic Zn2+ and febrile seizure susceptibility(WILEY, 2017-01)Zn2+ , the second most prevalent trace element in the body, is essential for supporting a wide range of biological functions. While the majority of Zn2+ in the brain is protein-bound, a significant proportion of free Zn2+ is found co-localized with glutamate in synaptic vesicles and is released in an activity-dependent manner. Clinical studies have shown Zn2+ levels are significantly lower in blood and cerebrospinal fluid of children that suffer febrile seizures. Likewise, investigations in multiple animal models demonstrate that low levels of brain Zn2+ increase seizure susceptibility. Recent work provides human genetic evidence that disruption of brain Zn2+ homeostasis at the level of the synapse is associated with increased seizure susceptibility. In this review, we have explored the clinical, functional and genetic data supporting the view that low synaptic Zn2+ increases cellular excitability and febrile seizure susceptibility. Finally, the review focuses on the potential of therapeutic Zn2+ supplementation for at risk patients.
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ItemCan mutation-mediated effects occurring early in development cause long-term seizure susceptibility in genetic generalized epilepsies?(WILEY, 2018-05)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.
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ItemSelective NaV1.1 activation rescues Dravet syndrome mice from seizures and premature death(NATL ACAD SCIENCES, 2018-08-21)Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.
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ItemPRIMA1 mutation: a new cause of nocturnal frontal lobe epilepsy(WILEY, 2015-08)OBJECTIVE: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene. METHODS: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation. RESULTS: Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system. INTERPRETATION: PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease.
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ItemGabapentin Modulates HCN4 Channel Voltage-Dependence(FRONTIERS MEDIA SA, 2017-08-21)Gabapentin (GBP) is widely used to treat epilepsy and neuropathic pain. There is evidence that GBP can act on hyperpolarization-activated cation (HCN) channel-mediated Ih in brain slice experiments. However, evidence showing that GBP directly modulates HCN channels is lacking. The effect of GBP was tested using two-electrode voltage clamp recordings from human HCN1, HCN2, and HCN4 channels expressed in Xenopus oocytes. Whole-cell recordings were also made from mouse spinal cord slices targeting either parvalbumin positive (PV+) or calretinin positive (CR+) inhibitory neurons. The effect of GBP on Ih was measured in each inhibitory neuron population. HCN4 expression was assessed in the spinal cord using immunohistochemistry. When applied to HCN4 channels, GBP (100 μM) caused a hyperpolarizing shift in the voltage of half activation (V1/2) thereby reducing the currents. Gabapentin had no impact on the V1/2 of HCN1 or HCN2 channels. There was a robust increase in the time to half activation for HCN4 channels with only a small increase noted for HCN1 channels. Gabapentin also caused a hyperpolarizing shift in the V1/2 of Ih measured from HCN4-expressing PV+ inhibitory neurons in the spinal dorsal horn. Gabapentin had minimal effect on Ih recorded from CR+ neurons. Consistent with this, immunohistochemical analysis revealed that the majority of CR+ inhibitory neurons do not express somatic HCN4 channels. In conclusion, GBP reduces HCN4 channel-mediated currents through a hyperpolarized shift in the V1/2. The HCN channel subtype selectivity of GBP provides a unique tool for investigating HCN4 channel function in the central nervous system. The HCN4 channel is a candidate molecular target for the acute analgesic and anticonvulsant actions of GBP.
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ItemGap Junctions Link Regular-Spiking and Fast-Spiking Interneurons in Layer 5 Somatosensory Cortex(FRONTIERS MEDIA SA, 2017-07-17)Gap junctions form electrical synapses that modulate neuronal activity by synchronizing action potential (AP) firing of cortical interneurons (INs). Gap junctions are thought to form predominantly within cortical INs of the same functional class and are therefore considered to act within discrete neuronal populations. Here, we challenge that view and show that the probability of electrical coupling is the same within and between regular-spiking (RS) and fast-spiking (FS) cortical INs in 16-21 days old mice. Firing properties of these two populations were distinct from other INs types including neurogliaform and low-threshold spiking (LTS) cells. We also demonstrate that pre-junctional APs can depolarize post-junctional neurons and increase the probability of firing. Our findings of frequent gap junction coupling between functionally distinct IN subtypes suggest that cortical IN networks are much more extensive and heterogeneous than previously thought. This may have implications on mechanisms ranging from cognitive functions to modulation of pathological states in epilepsy and other neurological disorders.