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Experimental Epileptology

Introduction

The research group of the Department of Neurology and Epileptology is interested to link the molecular mechanisms of defined, mainly genetic, neurological diseases with their clinical symptoms. The main goals are (i) to find out specific disease-causing genetic defects, (ii) understand their molecular and cellular pathomechanisms to ultimately (iii) improve existing or develop new therapies. The research is concentrated on diseases with disturbed neuronal excitability caused by mutations in ion channels, receptors or transporters. These have the advantage that we can examine their gating mechanisms in molecular detail using electrophysiological techniques such as patch clamping.

Genetics of Epilepsy

The current main research focus comprises the genetics, pathophysiology and therapeutic perspectives of idiopathic epilepsies. Epilepsy affects approximately 3% of people during their lifetime. An estimated 40% of all epilepsy patients suffer from so-called "idiopathic epilepsies", which are genetic in origin and not caused by structural or metabolic brain abnormalities. We are interested to identify genetic defects in monogenic and complex genetic forms of epilepsies and related disorders by using classical approaches (linkage studies and sequencing of candidate genes) and next generation genetic techniques in collaboration with other groups in Germany and Europe (whole genome association studies, large scale sequencing of candidate regions, high-throughput sequencing of candidate genes and the whole exome).

Contact for genetics: Yvonne Weber, Holger Lerche

Functional investigations of genetic defects in ion channels

Almost all of the mutations identified so far in idiopathic epilepsy syndromes affect genes encoding ion channels. Numerous ion channels tune the neuronal transmembrane voltage, by opening and closing ("gating") in response to either synaptic neuromediators (ligand-gated channels) or changes in the voltage itself (voltage-gated channels). In this way, genetic mutations affecting these channels can alter neuronal excitability and potentially drive a network of neurons into synchrony to promote a seizure. This conclusion is further supported by the fact that most of the antiepileptic drugs in clinical use today modulate different types of ion channels.

Our group has a long-standing experience with structure-function analysis, the gating properties and pharmacology of ion channels using molecular biological and electrophysiological techniques. We have been functionally characterizing disease-causing mutations of different ion channels including voltage-gated sodium, potassium, calcium and chloride channels as well as ligand-gated channels (GABA receptors, see picture). Most of our previous and a lot of ongoing studies have been carried out in heterologous expression systems, i.e. channels are expressed in a cell line that usually does not contain this molecule but provides the possibility to study its molecular function. We are now exploring the functional implications of disease-related mutations in neurons and determining the specific roles of affected channels in the brain. We use neuronal cultures and genetically altered animal models to assess (i) the cellular and subcellular localization of ion channels, (ii) targeting mechanisms, and (iii) functional analyses of wild type and mutant channels in single neurons and brain slices, by employing established techniques including immunohistochemistry, imaging of fluorescent fusion proteins and electrophysiology.

Contact for functional studies: Snezana Maljevic, Holger Lerche

GABA_A receptors and epilepsy. (A) Proposed transmembrane structure of the GABAA receptor including mutations causing generalized epilepsy with febrile seizures plus, childhood absence epilepsy with febrile seizures (in γ₂-subunit) or juvenile myoclonic or absence epilepsy (in α₁-subunit). The GABAA receptor is a pentamer and its most abundant form in brain contains two α₁-, two β₂-, and one γ₂-subunit. (B) The S326fs328X mutation of the GABA_A receptor α₁-subunit reduces GABA-induced Cl^- current by impairing the surface expression of the GABA_A receptor harbouring the S326fs328X mutant (α₁-mut) subunit. HEK 293 cells were co-transfected with either EGFP-α₁/β₂/γ₂, EGFP-α₁-mut/β₂/γ₂, incubated with a polyclonal antibody against EGFP before they were fixed, permeabilized and their nuclei stained with DAPI (blue). The clear staining of the surface membrane of cells expressing WT receptors (left) was never observed in cells transfected with the mutant DNA (right) (modified after Maljevic et al., 2006). $: splice site mutation.

Cultured neurons, in which potassium channels in the axon (red, KCNQ3 antibodies), dendrites and soma (green, MAP2 antibody), and the nucleus (blue, DAPI) are stained

Brain imaging in the epilepsies

An additional research interest of the department is structural and functional brain imaging. In focal epilepsies, it is important to identify epileptogenic lesions which cannot always be detected with standard magnetic resonance imaging (MRI), so that advanced methods of MRI are explored, for example voxel-based morphometry, a statistical structural evaluation method based on comparison of brains from normal controls and patients. Since memory functions are often affected in epilepsy patients, we use functional MRI to characterize and better understand in particular episodic memory processes generated in the hippocampus.

Contact for imaging studies: Holger Lerche

Funding

BMBF: Netzwerk seltener Erkrankungen IGN NeurONet

(Coordinator: Holger Lerche)

EuroEpinomics: 2011-2014 COGIE (Project Leader: Holger Lerche)

2011-2014 EpiPGX

2008-2013 BMBF: Nationales Genomforschungsnetz (NGFNplus): Epilepsy and Migraine integrated network (EMINet)

2007-2011 DFG: Differential role of the voltage-gated sodium channels Nav1.1 and Nav1.2

2007-2011 EU: ‘Epicure', Integrated project on functional genomics and neurobiology of epilepsy ( www.epicureproject.eu/home.aspx)

2008-2011 BMBF: E-rare program: Benign Familial Neonatal Seizures (BFNS) as disease model for human idiopathic epilepsies (EuroBFNS)

Selected Publications

Original papers:

Genetics, pathophysiology and therapy of idiopathic epilepsies and other paroxysmal diseases of the nervous system

Liao Y, Deprez L, Maljevic S, Claes L, Hristova D, Jordanova A, Ala-Mello S, Bellan-Koch A, Blazevic D, Schubert S, Thomas EA, Petrou S, De Jonghe P, Lerche H. Molecular correlates of age-dependent seizures in an inherited epilepsy of neonates and infants. Brain, in revision.

de Kovel CG, Trucks H, Helbig I, Mefford HC, Baker C, Leu C, Kluck C, Muhle H, von Spiczak S, Ostertag P, Obermeier T, Kleefuß-Lie AA, Hallmann K, Steffens M, Gaus V, Klein KM, Hamer HM, Rosenow F, Brilstra EH, Kasteleijn-Nolst Trenité D, Swinkels ME, Weber YG, Unterberger I, Zimprich F, Urak L, Feucht M, Fuchs K, Møller RS, Hjalgrim H, De Jonghe P, Suls A, Rückert IM, Wichmann HE, Franke A, Schreiber S, Nürnberg P, Elger CE, Lerche H, Stephani U, Koeleman BP, Lindhout D, Eichler EE, Sander T. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain 2009. [Epub ahead of print]

Suls A*, Mullen SA*, Weber YG*, Verhaert K, Ceulemans B, Guerrini R, Wuttke TV, Salvo-Vargas A, Deprez L, Claes LR, Jordanova A, Berkovic SF, Lerche H, De Jonghe P, Scheffer IE. Early-onset absence epilepsy caused by mutations in the glucose transporter GLUT1. Ann Neurol 2009;66:415-9, *contributed equally.

Helbig I, Mefford HC, Sharp AJ, Guipponi M, Fichera M, Franke A, Muhle H, de Kovel C, Baker C, von Spiczak S, Kron KL, Steinich I, Kleefuss-Lie AA, Leu C, Gaus V, Schmitz B, Klein KM, Reif PS, Rosenow F, Weber Y, Lerche H, Zimprich F, Urak L, Fuchs K, Feucht M, Genton P, Thomas P, Visscher F, de Haan GJ, Møller RS, Hjalgrim H, Luciano D, Wittig M, Nothnagel M, Elger CE, Nürnberg P, Romano C, Malafosse A, Koeleman BP, Lindhout D, Stephani U, Schreiber S, Eichler EE, Sander T. 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy. Nat Genet 2009;41:160-2.

Weber YG, Storch A, Wuttke TV, Brockmann K, Kempfle J, Maljevic S, Margari L, Kamm C, Schneider SA, Huber SM, Pekrun A, Roebling R, Seebohm G, Koka S, Lang C, Kraft E, Blazevic D, Salvo-Vargas A, Fauler M, Mottaghy FM, Münchau A, Edwards MJ, Presicci A, Margari F, Gasser T, Lang F, Bhatia KP, Lehmann-Horn F, Lerche H. GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak. J Clin Invest. 2008;118:2157-68.

Suls A, Dedeken P, Goffin K, Van Esch H, Dupont P, Cassiman D, Kempfle J, Wuttke TV, Weber Y, Lerche H, Afawi Z, Vandenberghe W, Korczyn AD, Berkovic SF, Ekstein D, Kivity S, Ryvlin P, Claes LR, Deprez L, Maljevic S, Vargas A, Van Dyck T, Goossens D, Del-Favero J, Van Laere K, De Jonghe P, Van Paesschen W. Paroxysmal exercise-induced dyskinesia and epilepsy is due to mutations in SLC2A1, encoding the glucose transporter GLUT1. Brain 2008;131:1831-44.

Wuttke TV, Jurkat-Rott K, Paulus W, Garncarek M, Lehmann-Horn F, Lerche H. Peripheral nerve hyperexcitability due to dominant-negative KCNQ2 mutations. Neurology 2007;69:2045-53.

Hunter*, Maljevic M*, Shankar A, Siegel A, Olson L, Weissman B, Holt P, Lerche H§, Escayg A§. Subthreshold changes of voltage-dependent activation of the K_V7.2 channel in neonatal epilepsy. Neurobiol Dis 2006;24:194-201. *contributed equally, §corresponding authors.

Maljevic S, Krampfl K, Cobilanschi J, Tilgen N, Beyer S, Weber YG, Schlesinger F, Ursu D, Melzer W, Cossette P, Bufler J, Lerche H*, Heils A*. A mutation in the GABA_A receptor alpha₁-subunit is associated with absence epilepsy. Ann Neurol 2006;59:983-7. *corresponding authors.

Wuttke TV, Seebohm G, Bail S, Maljevic S, Lerche H. The new anticonvulsant Retigabine favors voltage-dependent opening of the Kv7.2 (KCNQ2) channel by binding to its activation gate. Mol Pharm 2005;67:1009-17.

Lerche H, Biervert C, Alekov AK, Schleithoff L, Lindner M, Klingler W, Bretschneider F, Mitrovic N, Jurkat-Rott K, Bode H, Lehmann-Horn F, Steinlein OK. A reduced K+ current due to a novel mutation in KCNQ2 causes neonatal convulsions. Ann Neurol 1999;46:305-12.

Sodium channelopathies of skeletal muscle

Jurkat-Rott K, Mitrovic N, Hang C, Kouzmenkine A, Iaizzo P, Herzog J, Lerche H, Nicole S, Vale-Santos J, Chauveau D, Fontaine B, Lehmann-Horn F. Voltage sensor sodium channel mutations cause hypokalemic periodic paralysis type 2 by enhanced inactivation and reduced current. Proc Natl Acad Sci USA 2000;97:9549-54.

Mitrovic N, George AL, Rüdel R, Lehmann-Horn F, Lerche H. Mutant channels contribute < 50% to Na⁺ current in paramyotonia congenita muscle. Brain 1999;122:1085-92.

Lerche H, Mitrovic N, Dubowitz V, Lehmann-Horn F. Paramyotonia congenita: The R1448P sodium channel mutation in adult human skeletal muscle. Ann Neurol 1996;39:599-608.

Lerche H, Heine R, Pika U, George AL Jr, Mitrovic N, Browatzki M, Weiss T, Rivet-Bastide M, Franke C, Lomonaco M, Ricker K, Lehmann-Horn F. Human sodium channel myotonia: Slowed channel inactivation due to substitutions for a glycine within the III-IV linker. J Physiol 1993;470,13-22.

Brain Imaging

Roebling R, Przybylski C, Maier C, Lerche H, Grön G. Retrieval mode-dependent evaluation of the subsequent memory effect of hippocampal encoding, submitted.

Weber YG, Roebling R, Kassubek J, Hoffmann S, Rosenbohm A, Wolf M, Steinbach P, Jurkat-Rott K, Walter H, Reske SN, Lehmann-Horn F, Mottaghy FM, Lerche H. Comparative analysis of brain structure, metabolism and cognition in myotonic dystrophy 1 and 2. Neurology, in revision.

Roebling R, Scheerer N, Uttner I, Gruber O, Kraft E, Lerche H. Evaluation of cognition, structural, and functional MRI in juvenile myoclonic epilepsy. Epilepsia 2009 [Epub ahead of print]

Roebling R, Lerche H. Painful seizures associated with a lesion in the midcingulate cortex. J Neurol 2009;256:1012-4.

Invited reviews:

Weber YG, Lerche H. Genetics of paroxysmal dyskinesias. Curr Neurol Neurosci Rep 2009;9:206-11.

Maljevic S, Wuttke T, Lerche H. Nervous system K_V7 disorders: break down of a subthreshold brake. J Physiol 2008;586:1791-801.

Wuttke T, Lerche H. Novel anticonvulsant drugs targeting voltage-dependent ion channels. Exp Opin Invest Drugs 2006;15:1167-77.

Lerche H, Weber YG, Jurkat-Rott K, Lehmann-Horn F. Ion channel defects in idiopathic epilepsies. Curr Pharm Des 2005;11:2737-52.

Full list of Publications

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