Functional genomics in experimental and human temporal lobe epilepsy: powerful new tools to identify molecular disease mechanisms of hippocampal damage

T. Sutula and A. Pitkanen (Eds.) Progress in Brain Research, Vol. 135 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 14

Functional genomics in experimental and human temporal lobe epilepsy: powerful new tools to identify molecular disease mechanisms of hippocampal damage Albert J. Becker *, Otmar D. Wiestler and lngmar Bltimcke Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany

Abstract: The human genome project is a milestone for molecular genetic studies on complex, sporadic disorders in the human central nervous system (CNS). Functional analysis and tissue-/cell-specific expression profiles will be of particular importance anticipating the magnitude of expressed genes in the brain and their dynamic epigenetic modifications. The recent progress in microarray technologies allows expression studies for a large number of genes. In combination with laser-microdissection and quantitative reverse transcription-polymerase chain reaction technologies, such large-scale expression analyses can be successfully addressed in well-defined tissue specimens or cellular subpopulations. Complex, sporadic diseases, such as temporal lobe epilepsy (TLE), are challenging for functional genomics. Issues of particular importance in this field include molecular mechanisms of neurodevelopmental abnormalities, neuronal plasticity and hyperexcitability as well as neuronal cell damage in affected CNS areas. The availability of anatomically well-preserved surgical specimens, i.e. hippocampus obtained from epilepsy patients with Ammon's horn sclerosis or focal lesions not affecting the hippocampus proper as well as comparisons with experimental TLE models may help to elucidate specific molecular-pathological mechanisms during epileptogenesis and in chronic conditions of the disease.

Introduction

Temporal lobe epilepsy Epilepsy is a common neurological disorder characterized by recurrent spontaneous seizures that affects about 2 - 3 % of the population worldwide. A substantial fraction of epileptic patients does not respond to antiepileptic drug therapy. In most of these patients, seizures originate in the mesial temporal lobe (Elger and Schramm, 1993). Several lines of evidence sug-

* Correspondence to: A.J. Becker, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud Str. 25, 53105 Bonn, Germany. Tel.: +49228-287-9108; Fax: +49-228-287-4331; E-mail: albert_becker @uni-bonn.de

gest the hippocampal formation to be critically involved in temporal lobe epilepsy (TLE): Recordings from intracerebrally implanted electrodes demonstrate that the first electrographic abnormalities in temporal lobe seizures often appear within this structure (Van Roost et al., 1998). Surgical removal of the amygdala and hippocampal formation considerably diminishes or abolishes seizures in most pharmacoresistant TLE patients (Zentner et al., 1995). TLE pathogenesis involves a variety of developmental, metabolic and/or hypoxic alterations, while it lacks significant genetic inheritance (Jackson et al., 1998). A central question addresses the intriguing issue whether the alterations observed in the chronic epileptic state resemble an end stage of the disease after long-term additive pathophysiological events, maintenance of an initial etiologic episode or a combination of both. Data from human and experimental

162 TLE suggest that recurrent seizures, but not necessarily status epilepticus, progressively affect the hippocampal formation (Cavazos et al., 1994; Kalviainen et al., 1998; Salmenpera et al., 1998). While numerous molecular genetic alterations of cellular injury have been identified in hippocampal neurons following status epilepticus and within epileptogenesis (Lynch et al., 1996; Coulter and DeLorenzo, 1999), molecular pathways associated with neuronal damage and recurrent brief seizure episodes, i.e. the chronic state of human TLE, are less characterized. With this review we will address the question whether pathogenetic casades similar to those induced by status epilepticus are active in the chronic state of TLE and how functional genomics can gain our understanding of region- and cell-specific epileptogenesis. How can such delicate experiments be successfully applied in human tissue, in particular since proper controls are, for obvious reasons, not available? Neuropathological evaluation of surgical specimens is an important strategy to address this obstacle. The majority of resected mesial temporal lobe structures can be classified in two groups, i.e. Ammon's horn sclerosis versus focal lesions not affecting the hippocampus proper. The comparative analysis between both groups of patients, i.e. with respect to specific cell types and/or anatomical regions, may help to identify pathogenetic mechanisms specifically associated with each epileptogenic lesion. Ammon's horn sclerosis

Approximately 60% of TLE patients present with severe unilateral atrophy of either the fight or left hippocampus (fight/left = 1.08/1, n = 293, data obtained from the archives of the Department of Neuropathology, University of Bonn Medical Center). Histopathologically, the hippocampal formation shows segmental neuronal loss in CA1 and CA4, whereas CA2 and dentate gyms granule cells appear more resistant (Bliamcke et al., 1999a). Dense fibrillary astrogliosis and sclerosis of the tissue are observed in all segments with prominent neuronal cell loss. This macroscopic aspect has been first described in 1880 and classified as Ammon's horn sclerosis (AHS) (Sommer, 1880; Margerison and

Corsellis, 1966). Neuronal cell loss is also observed in hippocampal segments others than CA1 and CA4 (Kim et al., 1990; BliJmcke et al., 1996b). In the dentate gyms, specific cytoarchitectural abnormalities have been described in AHS, which may reflect seizure associated postnatal neurogenesis and persistence of Cajal-Retzius-like interneurons (Bltimcke et al., 1996a, 1999b, 2001; Nakagawa et al., 2000). Along with hippocampal cell loss, the entorhinal cortex and amygdala complex is affected in most patients (Pitkanen et al., 1998; Yilmazer-Hanke et al., 2000). Lesion-associated TLE

A second group, representing approximately 3040% of TLE patients exhibit focal lesions within the temporal lobe, which usually do not involve the hippocampus proper. This group covers lowgrade glio-neuronal neoplasms, i.e. gangliogliomas and dysembryoplastic neuroepithelial tumors (DNT), low-grade astrocytomas and oligodendrogliomas as well as glio-neuronal malformations, i.e. focal cortical dysplasia (Wolf and Wiestler, 1993; Bltimcke et al., 1999a). These lesions share predominant localization within the temporal lobe and frequent association with chronic, intractable seizures combined with benign biological behavior and rare recurrence after surgical removal. Gangliogliomas and DNTs are composed of a neoplastic glial and dysplastic neuronal cell population (Bltimcke et al., 1999a). Some of these tumors were found together with a malformative lesion pointing towards a maldevelopmental origin (Wolf and Wiestler, 1993; Wolf et al., 1994; Blfimcke et al., 1999a). Recently, a novel mutation in the TSC2 gene was selectively detected within the glial component of a ganglioglioma suggesting that the glioma portion derives from clonal evolution (Becker et al., 2001). In contrast to the characteristic pattern of neuronal cell loss in AHS, no significant neuropathological alterations are observed in the hippocampal formation of lesionassociated epilepsy (Bltimcke et al., 2000). A small subgroup of patients presents with dual pathology, i.e. AHS in addition to focal lesions (Bliamcke et al., 1999a).

163 The use of animal models to study functional genomics of TLE

A major obstacle for the systematic analysis of surgical specimens obtained from patients with pharmacoresistant TLE is the lack of non-epileptic, agematched controls. Comparative analysis between two groups of TLE patients, i.e. AHS versus lesionassociated TLE can be applied with respect to different clinico-pathological features, i.e. extent of structural changes and duration/severity of seizures. Rarely, biopsy samples from tumor patients without epileptic seizures can be obtained as truly nonepileptic controls. Autopsy specimens suffer from a variable post mortem delay and are therefore not appropriate for delicate molecular biological studies, such as mRNA expression analysis. Another strategy for the evaluation of epilepsyassociated changes follows the comparison between human epilepsy tissue and experimental animal models. In particular, alterations observed in both human and different experimental models appear more likely to be of pathogenic relevance (Bltimcke et al., 2000). Since surgical specimens are usually obtained at a late stage of the disease, experimental data based on human samples may not allow to distinguish between primary pathogenetic lesions and secondary changes. Animal models provide the possibility to analyze epileptogenesis and epilepsyassociated structural and molecular changes. As an example, studies on seizure-induced neuronal apoptosis are almost exclusively restricted to animal models, because terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL-staining) can be employed only for a limited time interval after the excitotoxic event (Tuunanen et al., 1999; Venero et al., 1999). It is extremely challenging to collect groups of human surgical specimens within appropriate post seizure intervals. Commonly used animal models for focal limbic epilepsies are kainate-, pilocarpine- and kindlinginduced chronic seizures (Mello et al., 1993; Ben-Ari and Cossart, 2000; Sutula, 2001). Induction of status epilepticus by intracerebral or intraperitoneal injection of epileptogenic compounds induces delayed segmental neuronal cell loss in the hippocampal formation. The segmental pattern of cell loss partially resembles that in human AHS with less severe neu-

ronal loss of CA1 in animals. On the other hand, subconvulsive electrical kindling of the amygdala or tractus perforans produces sustained hippocampal seizure activity, which usually results in less significant histopathological alterations compared to application of epileptogenic compounds (Clusmann et al., 1992; Bertram and Lothman, 1993). The degree of histopathological changes also depends on the severity and frequency of seizures, in particular following status epilepticus (Bertram and Lothman, 1993; Cavazos et al., 1994; Ebert and L/Sscher, 1995). In summary, different experimental paradigms have been established with neuropathological changes similar to TLE patients, i.e. pilocarpine or kainate injections modeling AHS, whereas kindling associated epileptogenesis resembles the epileptogenic hippocampus in patients with focal lesions. With increasing availability of transgenic mice carrying targeted mutations in epilepsy-related candidate genes, such models will play a significant role to study epileptogenesis and epilepsy-associated structural and molecular alterations. However, it is pivotal to note that mouse strains bear different susceptibilities for kainic acid-induced excitotoxic neurodegeneration. With respect to studies on functional genomics, mouse strain specific mRNA expression levels as well as developmentally regulated and regionally different gene expression profiles have to be considered (Schauwecker and Steward, 1997; Wen et al., 1998; Cantallops and Routtenberg, 2000; Sandberg et al., 2000). Functional genomics of TLE specimens

Microarray technology offers the opportunity to analyze human gene expression profiles on a genome wide level (Brown and Botstein, 1999; Lipshutz et al., 1999). In recent years, different expression array technologies have been developed (Table 1) including cDNA nylon arrays (Wellmann et al., 2000), large-scale oligonucleotide (Lipshutz et al., 1999) and glass microscope slide DNA arrays (Brown and Botstein, 1999) (Fig. 1). Besides such large-scale 'chip' approaches, designed arrays and real-time reverse transcription-polymerase chain reaction (RTPCR) quantification represent useful tools to substantiate hypothesis based expression studies (Bartosiewicz et al., 2000; Miyajima et al., 2001).

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165 TABLE 1 Differentexpressionanalysis tools are outlined Type

Approach

Scale

Label type

Methodicalfeature

System

Publication

Atlas-Array filters GeneChip microarrays cDNA microarrays PIQOR Real-time RT-PCR In-situ hybridization

inductive inductive inductive inductive deductive deductive

low density high density high density low density low density singlegene

radioactive fluorescent fluorescent fluorescent fluorescent fluorescent, radioactive

cDNA,nylon oligonucleotide cDNA cDNA relativequantitation oligonucleotides, RT-PCRproducts

Clontech Affymetrix P. Brown Memorec Applera

Wellmann et al. (2000) Lipshutz et al. (1999) Brown and Botstein (1999)

Major strategies for chip analysis include comparisons between identified cell populations, brain regions or groups of neuropathologically characterized individuals (Sandberg et al., 2000). The careful selection of appropriate controls or matched pairs of samples plays a pivotal role for expression profiling experiments. Especially when starting from hippocampal biopsy specimens of pharmacoresistant TLE patients, there are a number of problems inherent in such an approach. Besides significant expression differences between human subjects due to individual genetic background, temporal lobe epilepsy patients may exhibit considerable heterogeneity with respect to drug treatment, progression of the disease and frequency, type and intensity of seizures. These problems should be addressed by a careful matching of patient subjects with respect to clinical criteria and by increasing numbers of studied individuals per group, for which real-time RT-PCR may provide a particularly economical method. In complex, sporadic brain disorders, such as temporal lobe epilepsy a variety of molecular pathways and genes are involved and differentially regulated during the long medical history of the disease (Table 2) (BRimcke et al., 1999a). The bioinformatic analysis of large scale or even transcriptome expression (i.e. the level of each mRNA detectable in the genome) is a major challenge (Mimics, 2001). Introduction of certain reference or housekeeping genes enables a systematic comparison between different sets of experiments, including complex estimations, such as cluster analysis (Eisen et al., 1998; Bassett et al., 1999). However, the identification of reliable housekeeping genes may considerably change with experimental paradigms as has been shown to be

Fink et al. (1998) Lie et al. (2000); Chen et al. (2001)

relevant for the pilocarpine epilepsy model (Waha et al., 1998; Chen et al., 2001). At the present time, a major limitation of microarray technology is the need of sufficient amounts of region/cell specific mRNA. For the majority of microarray systems, approximately 50 Izg of total RNA are recommended for reverse transcription (Duggan et al., 1999). Due to these amounts of required starting material, such strategies do not provide information about cell specific patterns of gene regulation. Furthermore, certain expression alterations may reflect changes in the composition of neuronal tissue, if pronounced degeneration of specific cell types or reactive cell infiltration is observed. Such problems have to be considered for expression studies in TLE since patients with AHS exhibit segmental neuronal cell loss, reactive astrogliosis as well as structural and molecular reorganization in the hippocampal formation (Bltimcke et al., 1999a). Since altered mRNA levels between hippocampi of patients with AHS and control individuals may then reflect simply altered tissue composition, expression array analyses from TLE tissue have to be supplemented by a detailed analysis of expression alterations at the cellular level. Several approaches have been taken to establish expression analysis with very low amounts of input mRNA including RT-PCR and antisense mRNA (aRNA) amplification techniques, aRNA amplification has been used to generate sufficient amounts of aRNA for array hybridization starting from individual cells (Eberwine et al., 1992; Phillips and Eberwine, 1996; Luo et al., 1999). An advantage of this approach is the opportunity to screen large numbers of genes starting from minute amounts of mRNA. This linear amplification technique may se-

166 TABLE 2 Pathogenic mechanisms potentially involved in TLE

1 2 3 4 5

TLE-associated pathomechanism

Candidate genes

Apoptosis Cytoarchitectural malformations Axonal reorganization Cellular hyperexcitability Gliosis

iNOS, PIN, JIP-1, JNK, c-JUN, caspases, heat shock, ubiquitin, bcl, bax Reelin, CDK5, p35, TSC1, TSC2 Extracellular matrix molecules and receptors, cadherin, neurotransmitter-receptors Voltage dependent Ca2+, Na +, K+-channels, neurotransmitter-receptors Connexins, extracellular matrix molecules and receptors, K+-inward rectifiers

lect for certain populations of mRNAs, a problem which may be overcome by a novel strategy combining linear amplification and a template switch effect for microarray probe preparation (Wang et al., 2000). Compared to these techniques, real-time RT-PCR allows to monitor reaction dynamics of the PCR amplification and relative efficiency of target as well as reference gene amplification for every cycle. It provides detailed information regarding the linear dynamic range of the reactions (Fink et al., 1998). However, RT-PCR will usually be restricted to a limited number of genes. RT-PCR combined with laser microdissection (Fink et al., 1998; Schtitze and Lahr, 1998; Lahr, 2000) of hippocampal subfields will provide a reproducible tool to confirm and/or localize differentially regulated genes of interest to respective hippocampal cell populations. In addition to the confirmation of differential gene expression levels with RT-PCR, experimental errors introduced by false annotation of spotted sequences have to be controlled (Knight, 2001). These obstacles underline the need for alternative strategies to analyze expression profiles and to confirm their regional and cellular origin. Large-scale expression studies offer the unique opportunity to identify novel pathways potentially involved in sporadic diseases, such as TLE and AHS. It is important to note that the choice of the expression-monitoring tool, i.e. real-time RT-PCR or microarrays, strongly influences the experimental design. Simultaneous, transcriptome wide expression analysis describes an inductive approach. Expression profiles are compared between a variety of physiological and/or pathophysiological states. The result is an indefinite number of differentially expressed genes, which is used to build up a hypothesis for further experiments. However, for certain genes, it might be difficult to transfer differential expression into functional consequences. Using a deductive or

'top down' approach, expression analysis of a limited number of genes requires a certain hypothesis to be verified or disproven (Bassett et al., 1999).

Molecular pathways of seizure-induced hippocampal damage Necrosis and apoptosis have been shown as two independent pathways of excitotoxic neuronal damage (Ankarcrona et al., 1995; Van Lookeren Campagne et al., 1995). While necrosis results from cellular swelling, bursting and lysis, apoptosis follows a programmed mode of active cellular degeneration (Nicotera et al., 1997). Hippocampal apoptosis has been described in several epilepsy models. Following pilocarpine and kainate-induced status epilepticus, neuronal apoptosis is pronounced in CA3 and CA1 neurons, whereas dentate gyrus granule cells are more resistant (Mello et al., 1993; Ben-Ari and Cossart, 2000). The molecular pathogenesis of neuronal apoptosis has been associated with a glutamate receptor-mediated pronounced intracellular Ca 2+ increase (Choi, 1987; Wahlestedt et al., 1993). Subsequently, excitotoxicity proceeds via stimulation of various intracellular signaling cascades including the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein kinases (Gupta et al., 1996; Martin et al., 1996; Kawasaki et al., 1997; Schwarzschild et al., 1997) and the formation of nitric oxide (NO) with caspase-mediated apoptosis (Bruno et al., 1993; Leist et al., 1997; Montecot et al., 1998). Mice with a targeted mutation of the JNK3 isoform selectively expressed in the nervous system show an increased resistance to kainic acid-induced cell loss (Yang et al., 1997). Kainate-induced expression of JNK-1 relates to increased apoptosis in hippocampal neurons, while serine-73 phosphorylation of c-Jun is associated with resistance to cell

167 death (Schauwecker, 2000). In the kainate model, an inverse correlation is observed between the hippocampal distribution of kainic acid receptors and the pattern of neuronal cell loss, i.e. low receptor density in the highly vulnerable segment CA1 and vice versa in the dentate gyrus (Sperk et al., 1983). A striking relationship occurs between expression of the endogenous protein inhibitor of neuronal nitric oxide synthase (PIN), a cytoplasmic inhibitor of the JNK signal transduction pathway designated JNK interacting protein-1 (JIP-1) and the gene for the apoptosis-executing protease caspase-3 to patterns of hippocampal vulnerability after kainate-induced seizures (Dickens et al., 1997; Jaffrey and Snyder, 1996; Becker et al., 1999). In the dentate gyrus, no delayed cell loss is observed although high kainate receptor densities are encountered in this area (Sperk et al., 1983). Here, PIN and JIP-1 mRNA signals increase significantly, whereas caspase-3 expression remains at basal levels. In CA1 with extensive neuronal cell loss and low kainate receptor density, weaker expression of JIP-1 and PIN vs. induction of caspase-3 are observed compared to the dentate gyrus (Sperk et al., 1983; Becker et al., 1999). This selective regulation may serve as example for the capacity of downstream apoptotic signaling cascades to interfere with excitotoxic apoptotic stimuli in different hippocampal subfields. Functional pathways involved in seizure-associated apoptosis include expression of the TP53 tumor suppressor (Sakhi et al., 1994; Liu et al., 1999) and the tissue plasminogen activator gene (Tsirka et al., 1995). Certain lines of evidence suggest that single intermittent seizures, resembling the chronic state of TLE, induce apoptosis. Severe neuronal cell loss is observed after repeated kindling seizures (Cavazos et al., 1994). Also, apoptosis occurs in the dentate gyrus following intermittent kindling stimulation in the ventral CA1 region (Bengzon et al., 1997). There is evidence that a limited number of brief repeated kindling seizures do not alter total amygdaloid or hilar neuronal cell numbers, but may induce degeneration of certain neuronal subpopulations (Pretel et al., 1997; Tuunanen et al., 1997; Tuunanen and Pitkiinen, 2000). However, novel data suggest that neurodegenerative pathways in the chronic TLE state may be similar to those which occur early during TLE pathogenesis. Expression of bcl-2, bcl-xL, bax, caspase-3 and

caspase-1 proteins shows alterations in resected temporal lobe structures from patients with long-term pharmacoresistant TLE (Henshall et al., 2000). The molecular signals predisposing hippocampal neurons to enhanced or reduced susceptibility for seizureinduced damage in the chronic TLE state have not yet been fully characterized. Potential candidates include a variety of ionotropic and metabotropic neurotransmitter receptors.

Neurodegeneration or neuroprotection: role of neurotransmitter receptors Several lines of evidence suggest that recurrent spontaneous seizures in human as well as experimental chronic TLE are caused by alterations in the balance between inhibitory and excitatory neurotransmitter systems (Meldrum et al., 1999; Ben-Aft and Cossart, 2000; Chapman, 2000; Kullmann et al., 2000). Changes in neurotransmitter receptor expression, subunit composition and their functional consequences may not only contribute to enhanced seizure susceptibility but also predispose or protect neuronal cells for/from cellular damage. This has been demonstrated for excitatory ionotropic and metabotropic glutamate receptors as well as for inhibitory ionotropic GABAA receptor pathways (Jacobs et al., 2000; Meldrum, 2000; Coulter, 2001). With respect to epilepsy-associated neuronal damage, neuroprotection as well as novel pharmacological treatment strategies, metabotropic glutamate receptors (mGluRs) have emerged as interesting target molecules. The mGluR family consists of at least 8 different subtypes (Nicoletti et al., 1996). Activation of class I mGluRs (i.e. mGluR1 and mGluR5) results in excitatory membrane depolarization followed by release of Ca 2+ from intracellular stores, which appears to be mediated by inositol phosphate hydrolysis. Class II (mGluR2 and mGluR3) and class III (mGluR4, mGluR6-8) mGluRs operate mainly via a G-protein-mediated inhibition of adenylate cyclase (Nicoletti et al., 1996). Immunohistochemical studies and in-situ hybridization revealed distinct preor postsynaptic localization of mGluR isoforms in rat (Baude et al., 1993; Shigemoto et al., 1997) and human hippocampus (Bliimcke et al., 1996c; Lie et al., 2000). Recent molecular, pharmacological and physiological data point to a role for specific mGluR

168 subtypes in the generation and propagation of epileptiform activity (Mayat et al., 1994; Attwell et al., 1995; Holmes et al., 1996; Aronica et al., 1997; Merlin et al., 1998). In particular, agonists of excitatory class I mGluRs exert significant convulsant properties, whereas class I antagonists can prevent excitotoxic neuronal damage (Mukhin et al., 1996; Strasser et al., 1998; O'Leary et al., 2000). Furthermore, kainate and kindling models revealed enhanced expression of class I mGluRs as well as increased phosphoinositide hydrolysis (Nicoletti et al., 1987; Akbar et al., 1996). Expression alterations of excitatory class I (mGluR1 and mGluR5) and inhibitory class III (mGluR4) metabotropic glutamate receptors were observed in chronic TLE. mRNA expression and protein distribution analysis of mGluR1 and mGluR5 revealed a striking induction of mGluRlc~ in the hippocampal dentate gyms with an almost identical regional distribution in kainic acid-treated and amygdala-kindled, chronic epileptic animals as well as in human TLE specimens (Bltimcke et al., 2000). This expression alteration may significantly predispose cells with enhanced mGluRlc~ expression to neuronal excitability. An opposite functional effect may be the result of regional and cellular induction of the mGluR4 subtype in chronic TLE. In contrast to control hippocampus obtained from nonepileptic controls, i.e. patients suffering from diffuse infiltrating malignant gliomas, most TLE specimens showed a significant increase of mGluR4 protein and mRNA expression within the dentate gyms and residual CA4 neurons (Lie et al., 2000). With respect to a neuroprotective potential of mGluR4 in various cell culture models, mGluR4 induction may constitute a cellular mechanism to antagonize excitatory hippocampal activity and critical intracellular Ca 2+ overload (Gasparini et al., 1999; Bruno et al., 2000). In the pilocarpine animal model, the acute status epilepticus is frequently followed by a silent period of weeks before chronic spontaneous limbic seizures occur (Coulter, 2000). In the hippocampus, sprouting of zinc containing mossy fibers can be observed during this adaptation phase (Cavazos et al., 1991; Mello et al., 1993). GABAA receptors of dentate gyms granule cells show enhanced sensitivity to blockade by zinc in chronic TLE. Expression profiling of single cells and functional analysis revealed major alterations in subunit composition of

the GABAA receptor (Brooks-Kayal et al., 1998). The enhanced sensitivity of dentate gyms granule cell GABAA receptors to blockade by zinc in chronic TLE may be due to decreased expression of the c~l subunit of the GABAA receptor. The combination of increased zinc sensitive GABAA receptors and sprouted zinc-containing mossy fiber terminals may result in a failure of inhibition and concomitant enhanced seizure propensity triggering chronic TLE and cellular damage (Brooks-Kayal et al., 1998). Enhanced potency of GABA in activating GABAA receptors as well as reduced c~2 and c~5 GABA subunit expression in CA1 and additional changes in subunit expression in other hippocampal neurons have also been described using a combined approach of expression arrays and electrophysiology (Rice et al., 1996; Gibbs et al., 1997; Becker et al., 1998; Coulter, 1999; Coulter and DeLorenzo, 1999).

Perspectives for functional genomics in human TLE The plethora of functional cascades involved in the pathogenesis of chronic TLE is a challenging feature of this complex, sporadic disease. With the opportunity to study large-scale gene expression using novel microarray technologies, we may discover novel pathways of TLE associated hyperexcitability, neuronal damage or functional/stmctural plasticity. Due to the complexity of human TLE tissue, TLE animal models and the expression array data, epilepsy researchers using array technology would benefit from intemet platforms for functional genomics providing access to brief annotations of specific genes as well as links to known biochemical pathways and interactions at the transcriptional level to verify and extend their observations. Currently, experimental conditions and potential problems of expression data are discussed intensively (Geschwind, 2001; Lockhart and Barlow, 2001) and first internet platforms for expression array data such as Gene Expression Omnibus (GEO) and ArrayExpress are established. Uniform expression data formats and integrated analysis tools will also be essential for a successful application of a functional genomics approach in human and experimental TLE.

169

Acknowledgements T h e authors thank S. N o r m a n n for e x c e l l e n t technical assistance. W e like to a c k n o w l e d g e the grateful support and contribution of our clinical c o l l e a g u e s Profs. E l g e r and S c h r a m m to the interdisciplinary e p i l e p s y p r o g r a m . Our w o r k is g e n e r o u s l y supported by D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( S F B - T R 3 ) , B M B F ( G e n o m i c N e t w o r k s , S P l l ) and the B O N F O R p r o g r a m o f the U n i v e r s i t y of B o n n M e d i c a l Center.

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