Gene Expression Underlying Changes in Network Excitability

Chapter 28

Katarzyna Lukasiuk

Raymond Dingledine

Daniel H. Lowenstein

Asla Pitkänen

Introduction

Remarkable progress in the fields of molecular biology and molecular genetics has added a new dimension to epilepsy research. Just few years ago, studies on the basic mechanisms of epilepsy were limited primarily to anatomic, electrophysiologic, and pharmacologic analyses. However, molecular approaches have provided a more direct and specific means for studying biochemical events in the brain, leading to the recognition that seizures and other forms of injury can induce complex changes in gene expression. These changes are likely to regulate the molecular architecture of neuronal cells and circuits that contribute to longer-term process of epileptogenesis as well as seizure initiation, spread, and cessation. The application of molecular biology to investigate epilepsy can be divided into two broad categories: (a) studies on the genetic or inherited basis of epilepsies (which will not be discussed here) and (b) studies on the role of alterations in expression levels of specific genes or ensembles of genes during epileptogenesis and in epilepsy. This chapter focuses mainly on recent results of global analyses of gene expression in humans and in experimental models of epileptogenesis and epilepsy that provide new understanding of cellular and metabolic processes occurring in the brain affected by the disease.

Basic Principles of Gene Expression

This section provides a brief review of the main steps involved in the conversion of genetic information encoded by DNA into proteins (for a detailed review, see Drlica⁴⁴).

DNA Replication

The genetic code for all cellular proteins is contained in DNA. In its native state, DNA exists as a double helix of deoxyribonucleic acids bound together by phosphate and hydrogen bonds. The hydrogen bonding between DNA strands ensures that the base adenosine (A) will pair with thymidine (T), whereas guanine (G) pairs with cytosine (C). Replication (or duplication) of DNA requires unraveling and separation of the complementary strands of DNA, attachment of DNA-binding proteins, annealing of small primer fragments, and creation of new complementary strands via DNA polymerase enzymes. Because each strand is replicated once, the process is termed semiconservative. DNA replication occurs prior to cell division or mitosis, but not in nondividing cells. The combination of highly specialized enzymes and the ability to “proofread” during the synthesis of a new strand of DNA makes this a remarkably reliable process. Even nondividing cells must constantly maintain the integrity of the DNA by using a host of scanning, editing, and repair enzymes. The double-stranded nature of DNA helps to ensure that a mutation in one strand will be rapidly detected and repaired by the cell.

Transcription

During transcription, the enzyme RNA polymerase recognizes and attaches to a specific region at the starting point of a gene. RNA polymerase synthesizes a single-stranded RNA molecule that is complementary to the DNA template, with the exception that ribonucleic acids are substituted for deoxyribonucleic acids and the base uracil (U) is substituted for thymidine in the coding scheme. Transcription continues until the enzyme reaches a termination sequence in the gene. Once the full-length gene is transcribed, segments that do not code for a specific portion of a protein (termed introns) are excised, and only the exons, which contain specific information for the coding of proteins, remain. In addition, a long tail of adenosine bases is attached to the RNA, thereby identifying it as “messenger” RNA (mRNA). Messenger RNA is then used to translate the genetic code from ribonucleic acids into amino acids and proteins.

The mechanisms controlling the recognition and attachment of RNA polymerase to the starting point of a gene are critical determinants of the pattern of gene expression in a cell. Specific DNA-binding proteins known as transcription factors can attach to a regulatory element upstream from the starting point of the gene. Depending on the type of transcription factor, this may either promote or inhibit binding of the RNA polymerase and subsequent transcription. Similarly, other upstream and downstream regions known as enhancer or repressor sites can also influence transcriptional activity. These transcription factors are critical links in the process of stimulus-induced regulation of gene expression.

Translation

Transcribed mRNA is translated into protein by an interpretive process involving ribosomes, transfer RNA (tRNA), and a variety of other proteins that facilitate the process, such as initiation factors and elongation factors. Protein synthesis begins with the attachment of the mRNA to a ribosome near the “start codon.” A codon is a triplet of RNA bases that codes for either a specific amino acid or a start or stop to the translation procedure. A tRNA, which carries a particular amino acid, then joins the complex based on the complementary nucleotide sequence of the mRNA (codon) and tRNA (anticodon). The polypeptide chain is created by sequentially bringing pairs of

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tRNAs together on the ribosome using the mRNA as a template. A peptidyl transferase reaction forms a new peptide bond between the two amino acids on the tRNAs. This process continues until the end of the mRNA encoding sequence is reached, and the newly formed polypeptide is released in the cell with the aid of specific release factors.

Aspects of Gene Expression Unique to the Central Nervous System

When the techniques of modern molecular biology are applied to the study of epilepsy and neural injury, certain special aspects of the central nervous system (CNS) must be taken into consideration. Although these unique characteristics do not necessarily limit the use of molecular methods in neurobiology, they do provide insight into the complexities of gene regulation that are unique to the signal processing and cellular repair mechanisms of the CNS. This section discusses several of these aspects.

One of the most notable aspects of neurons is that they are postmitotic.⁹² Most mature neurons, therefore, are required to maintain their functional integrity for, ideally, the lifetime of the organism. To do this, neurons use many “housekeeping” functions. These functions include replacing and repairing cytoskeletal structures, replenishing membrane and membrane-associated proteins, synthesizing and packaging neurotransmitters, and maintaining integrity of synaptic contacts. If cell injury occurs, genes are activated in an attempt to restore function and to protect from further injury.¹⁷^,²⁷^,⁵⁴^,¹⁰⁹^,¹¹¹^,¹²⁴^,¹⁴⁵ If cell death occurs, in certain systems new synaptic contacts are made by the surviving presynaptic neurons, presumably in response to the lost functional connectivity of the dead neuron.¹¹⁰

In addition to being postmitotic, neurons have a considerable amount of phenotypic diversity and plasticity. Phenotypic diversity refers to the unique characteristics of various types of neurons, such as differences in neuron size and shape, classes of neurotransmitters and peptides released, firing conditions and frequencies, and responses to stress and injury.¹⁰⁸^,¹⁷⁴ Phenotypic plasticity refers to the ability of an individual neuron to alter its function. There are numerous levels of control of phenotypic plasticity. Neurons can transiently modify the quantity of neurotransmitters they release in response to both external stimuli and intracellular signaling mechanisms, as has been suggested in certain forms of long-term potentiation.¹¹^,¹¹⁶ Phenotypic plasticity is a dynamic process that is highly dependent on the molecular aspects of cellular physiology. Neurons can modulate the relative amounts and properties of proteins they express through changes in gene expression, editing of mRNA transcripts, and a variety of posttranslational modifications. For example, in response to repeated stimulation, neurons can synthesize new ion channels and membrane receptors that may contribute to long-lasting changes in membrane excitability.⁴²^,⁵⁷^,⁹⁸^,¹¹⁸^,¹²⁶ Similarly, following stimulation or seizures, neurons may modify the response characteristics of specific voltage-dependent ion channels and membrane receptors and alter expression of mRNAs coding for various synapse-modifying proteins.⁴¹^,¹¹⁹ Phenotypic plasticity is an ongoing and constant component of neuronal and glial behavior and is an essential ingredient in the ability of the CNS to interact with the environment.

An especially interesting example of phenotypic diversity in neurons comes from the observation that protein synthesis may be highly localized in specific regions of the cell that are distant from the nucleus and regulated independently from translation occurring in the cell body. In most cells, translation of mRNA into protein occurs within the cell body, and newly synthesized proteins are then transported to their appropriate site based on intracellular signaling mechanisms. Most neurons are polarized cells with three fairly distinct compartments: (a) the dendritic processes, (b) a central regulatory soma, and (c) axonal projections ending with presynaptic terminals. Each of these regions has specific functions and interactions with its microenvironment.³³ mRNA can be differentially distributed and translated in dendritic processes.¹³^,³³ Furthermore, ribosomes necessary for carrying out protein translation have also been located in dendrites. This localized distribution of mRNA and ribosomes suggests that dendrites have the capacity to support highly localized and site-specific protein synthesis. This additional level of phenotypic plasticity may prove to be an important element in the synaptic modifications implicated in activity-dependent learning. It is interesting that there is no evidence that a similar phenomenon of localized protein synthesis occurs in axons.³³ This implies that the neuron may rely on intracellular transport mechanisms, such as axoplasmic flow and microtubule systems, to deliver newly synthesized protein to the axon terminal.

Epilepsy and Alterations in Gene Expression

An “Intelligent Guess” = Candidate Gene Approach

Changes in gene expression leading to network reorganization and neuronal hyperexcitability are crucial to understanding epilepsy and have been studied with traditional methods. Several candidate genes encoding proteins having a role in brain excitability have been tested. Obviously, much attention has been given to genes coding for proteins directly influencing electrical properties of neurons, such as receptors and channels. The other group of genes has been those coding for proteins involved in remodeling of neuronal networks, such as growth factors and their receptors, synaptic proteins, or other structural proteins. However, such studies have concentrated mostly on seizure-induced alteration and only within hours after seizures. These data were reviewed recently⁴⁹^,¹⁷⁵ and are only briefly mentioned here. Less information is available on alteration in expression of selected genes during late epileptogenesis and in epileptic tissue. In this chapter, we briefly review only data on changes in the expression of genes that are hypothesized to influence brain excitability directly or indirectly. Data from both human tissue and experimental models of epileptogenesis and epilepsy are included, but data on immediate effect of seizures or status epilepticus, reflecting influence of increased neuronal activity on gene expression, are omitted.

Traditional Methods Most Commonly Used for Studies of Gene Expression Levels

The reverse transcription polymerase chain reaction (RT-PCR) is a powerful technique for making many copies of DNA from minute quantities of RNA targets used as starting material. For gene expression analysis, the first step is synthesis of complementary DNA (cDNA) on the basis of mRNA extracted from the studied tissue using the enzyme reverse transcriptase. The PCR is then used to amplify cDNA representing the gene of interest. PCR uses an enzymatic reaction to repeatedly copy the original DNA fragment present in the reaction. The PCR reaction mixture includes four basic components: (a) the target DNA, (b) oligonucleotide “primers,” which are designed to hybridize the sequence of DNA to be amplified, (c) an excess of individual nucleotides (deoxyadenosine 5′-triphosphate [dATP], deoxycytidine 5′-triphosphate

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[dCTP], thymidine 5′-triphosphate [dTTP], deoxyguanosine 5′-triphosphate [dGTP]), and (d) a DNA polymerase that is extremely stable at a high temperatures. With the use of an automated heating block, this mixture is cycled through a series of temperature changes that are designed to allow the polymerase to repeatedly duplicate the target DNA. Each cycle begins with a high temperature (e.g., 94°C) to separate the strands of target DNA (“strand separation phase”). The temperature is then dropped (i.e., 42°C–60°C, depending on reaction conditions) to allow the oligonucleotides to anneal (“primer annealing phase”). Then the temperature is increased to 72°C, which is the optimal temperature for Taq polymerase activity, so that the DNA replication occurs using the oligonucleotides as the primers (“primer extension phase”). Repeated cycling through these steps results in amplification of the DNA in a highly efficient fashion.

A Northern blot is a method for detecting the presence of a particular mRNA transcript in a sample. RNA is isolated from tissue or cells using standard extraction procedures and separated by size via gel electrophoresis. The size-separated RNA is transferred to a membrane, and the membrane is then exposed to a radiolabeled probe. If the mRNA of interest is in the sample, the probe will specifically hybridize to the mRNA and produce a signal at the predicted size on the autoradiogram.

In situ hybridization provides a means of determining the pattern of mRNA expression at the cellular level. Whereas Northern analysis uses mRNA derived from homogenized tissue, in situ hybridization uses fixed tissue sections so that the cellular anatomy is preserved. Otherwise, the technique uses the same concepts described for Northern analysis. The tissue sections are mounted on slides and prepared in a way that allows the labeled probe to have access to the cellular compartments and to hybridize with the mRNA. Radioactively labeled probe is then detected with X-ray film. To obtain cellular detail, the slides are coated with photographic emulsion, such that the radiolabeled probe will expose the emulsion directly overlying the cells. The slides are then “developed” in the same manner as film, and silver grains in the emulsion seen as black specks reflect the presence of this signal, signifying the presence of the specific mRNA transcript. In case of fluorescently labeled probe, the results of hybridization are analyzed using standard fluorescent microscopy techniques.

Immediate-Early Genes

In recent decades, a class of genes termed immediate-early genes (IEGs) has been identified that appears to function as a vital link between acute activity-dependent events and long-term changes in gene expression.¹⁵^,³¹^,⁶³^,⁸⁸^,⁸⁹^,¹⁰²^,¹⁵⁰^,¹⁵⁴^,¹⁷⁵ By definition, induction of expression of IEG mRNAs is rapid and does not depend on protein synthesis. Protein products of IEGs often serve as transcription factors regulating the transcription of other genes (e.g., c-fos, c-jun, c-myc, jun-B, krox-20, krox-24, fra-1, zif/268, etc.).

Expression of IEGs in neurons has been shown to be induced by a wide range of stimuli, including membrane depolarization due to both physiologic¹⁴^,¹⁵^,⁵⁵^,⁶⁷^,⁷⁰^,⁸³^,⁸⁴^,⁸⁵^,⁸⁶ and nonphysiologic stimulation,³⁷^,⁴¹^,⁴²^,⁸²^,⁸⁸^,¹⁰⁵^,¹¹³^,¹²⁷^,¹³³^,¹⁴⁷^,¹⁵²^,¹⁶¹ mechanical trauma,⁴³ and ischemia.¹⁴⁰ Studies using seizures induced by kindling, electroconvulsive seizures, electrolytic lesions, and chemoconvulsants have also shown that IEGs are markedly induced in specific CNS regions such as the hippocampus and cerebral cortex. Much effort has been invested in understanding molecular pathways leading to induction of IEGs expression following seizures, and the results of these studies are reviewed in detail elsewhere.¹³⁹^,¹⁷⁵

For the purposes of this review, an important question is, what are the target genes for transcription factors (IEGs and other) participating in the development of epilepsy? Several target genes that can influence neuronal excitability and their relation to transcription factors governing their expression have been described. For example, expression of the metabotropic γ-aminobutyric acid receptor GABA_BR1a and GABA_BR1b isoforms in hippocampal neurons is mediated by the cAMP response element–binding protein (CREB), which binds to unique cAMP response elements in the alternative promoter regions. CREB is then critical for transcriptional mechanisms that control GABA_BR1 subunit levels in vivo.¹⁵³ In addition, the activating transcription factor-4 (ATF4) differentially regulates GABA_BR1a and GABA_BR1b promoter activity.¹⁵³

The DREAM transcription factor that is expressed widely in the nervous system can be induced by seizures.²⁹^,¹²³ Its target genes include genes related to neuronal plasticity such as c-fos and preprodynorphin.²⁸^,²⁹

The AP-1 transcription factor (which is composed of proteins belonging to the Fos and Jun families) has been shown to regulate expression of nerve growth factor (NGF) after hilus lesion–induced seizures⁴⁸ and proenkephalin and prodynorphin after kainic acid–induced seizures.⁹⁶^,¹⁴³

There are also IEGs upregulated during epileptogenesis that lack transcription factor functions and can have lasting effect on neuronal excitability. One of these proteins is homer-1, which codes for a scaffold protein anchoring metabotropic glutamate receptors to the cytoskeleton and regulates pyramidal neuron excitability.²⁴^,¹⁴¹^,¹⁴⁸

Neurotransmitter Receptors and Ion Channels

Alterations in the expression of mRNAs encoding neurotransmitter receptors and ion channels are likely candidates for affecting network excitability. Table 1 gives a summary of such changes in experimental and human epilepsy.

In the mature nervous system, GABA_A receptors function as ligand-gated chloride channels that confer fast inhibitory synaptic transmission. Several studies have demonstrated abnormal GABA_A receptor function in epileptic tissue.²²^,⁶⁴^,⁸⁰ Because receptor properties depend on subunit composition,¹¹³^,¹⁶⁹ altered expression of GABA_A-receptor subunits can explain functional abnormalities. In fact, changes in mRNA expression for selected GABA_A-receptor subunits have been reported in epilepsy models induced by kainic acid¹⁶² and pilocarpine²³^,¹⁴⁶ and electrically induced status epilepticus (SE),¹⁰³^,¹³⁴ as well as following hippocampal kindling.¹³⁴

Metabotropic GABA_B-receptor expression has been studied in epileptic animals. Changes in the expression of mRNA encoding GABA_B receptors occur in the hippocampus of patients with temporal lobe epilepsy (TLE)⁶⁰ as well as in kainic acid–induced epilepsy,⁵⁹ hippocampal kindling,¹³⁴ and epilepsy following electrically-induced SE.¹³⁴

Glutamate, the major excitatory neurotransmitter in the brain, acts on ionotropic and metabotropic receptors. Sixteen genes encoding for ionotropic receptors that belong to three functional families (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA], kainate, and N-methyl-D-aspartic acid [NMDA] receptors) have been identified.³⁸ Alterations in the expression of AMPA receptor subunits have been demonstrated in human epilepsy⁶¹^,¹²¹ as well as in electrically-⁹⁰^,¹⁷³ and pentylenetetrazole-induced kindling.⁴⁶ Changes in kainate receptor subunits occur in human epileptic tissue⁶⁸ and the kainate model of epilepsy¹⁶³ as well as in amygdala and hippocampal kindling.⁷⁵^,⁹¹ Changes in the expression of NMDA subunits were observed in patients with TLE,⁷^,¹²¹^,¹²²^,¹³⁰ in rats kindled with amygdala⁹⁴ or hippocampal stimulation,¹⁰⁰^,¹⁰¹ and following intra-amygdala injection of kainic acid.¹⁴⁴

Table 1 Summary of studies using conventional methods to demonstrate alterations in the expression of genes encoding neurotransmitter receptors, transporters, and ion channels

Authors	Model/disorder	Structure	Gene
*Ionotropic GABA receptors*
Rice et al. (1996)¹⁴⁶	Pilocarpine-induced SE	CA1–CA3	↓α2, α5 at 1–2 mo
Tsunashima et al. (1997)¹⁶²	Kainic acid–induced SE	Hippocampus	↓α2, α3, α5, β1, β3, γ2, δ; ↑ α1, α4, β2 at 6–24 hr ↓α5, δ; ↑α1, α3, β1, β2 at 7–30 d
Brooks-Kayal et al (1998)²⁶	Pilocaprine-induced SE	Dentate gyrus	↓α1, β1; ↑ α4, β3 chronically
Lauren et al. (2003)¹⁰³	Amygdala stimulation model of TLE	CA3 CA1	↓α2; ↑β3 in epileptic rats ↓α4; ↑β3 in epileptic rats
Nishimura et al. (2005)¹³⁴	Hippocampal kindling Electrically induced SE	Dentate gyrus CA3 Dentate gyrus	↓δ; ↑α4, β1–3, γ2 at 7–30 d ↓α2 and δ1–3 ↓α5 at 24 hr ↓δ, α5; ↑α4, β1–3, γ2 at 7–30 d
*Metabotropic GABA receptors*
Furtinger et al. (2003)⁵⁹	Kainic acid–induced SE	Hippocampus	↓ GABA_B-1, GABA_B-2 acutely
Furtinger et al. (2003)⁶⁰	Human TLE	Dentate gyrus	↑ GABA_B-1 GABA_B-2
Nishimura et al. (2005)¹³⁴	Hippocampal kinding and electrically induced SE	Dentate gyrus	↑ GABA_B-2 acutely and chronically
*Ionotropic glutamate receptors*
Wong et al. (1993)¹⁷³	Seizures		↓↑GluR1
Garcia-Ladona et al. (1994)⁶¹	Human TLE	Hippocampus	↑ GluR1
Kamphuis et al. (1994)⁹⁰	Kindling	Hippocampus	↑ GluR1 flip up to 4 wk
Kamphuis et al. (1995)⁹¹	Hippocampal kindling	Dentate gyrus	↓ GluR7 at 28 d
Bayer et al. (1995)⁷	Human TLE	Dentate gyrus	↑ NR2
Kraus et al. (1994, 1996)¹⁰⁰^,¹⁰¹	Hippocampal kindling	Hippocampus	↓ NR1 splice variant at 28 d
Grigorenko et al. (1997)⁶⁸	Human TLE	Hippocampus	↑ GluR6
Mathern et al. (1997)¹²¹	Human TLE	Dentate gyrus	↑ NR2
Rafiki et al. (1998)¹⁴⁴	Intra-amygdalar kainic injection	Hippocampus	↑ NR1-2a, NR-2b, NR1-3a, NR1-3b, NR1-3b at 21 d–4 mo
Mathern et al. (1997)¹²¹	Human TLE	Hippocampus	↑ GluR1
Mathern et al. (1999)¹²²	Human TLE	Dentate gyrus CA2/3	↑ NR2A, NR2B ↑ NR2B, NR1; ↓ NR2A
Kikuchi et al. (2000)⁹³	Amygdala kindling	Frontal and temporal cortex Piriform cortex	↑ NR1 at 4 wk ↓ NR1 at 4 wk
Ekonomou et al. (2001)⁴⁶	Pentylenetetrazole-induced kindling	CA1 and dentate gyrus cortex	↓ GluR2, GluR3 at 1 wk ↑ GluR2 at 1 mo
Neder et al. (2002)¹³⁰	Human TLE	Sclerotic hippocampus Nonsclerotic hippocampus	↓ NR1 ↑ NR1
Hikiji et al. (1993)⁷⁵	Amygdala kindling	CA3	↑ KA1 at 28 d
Ullal et al. (2005)¹⁶³	Kainic acid–induced SE	Hippocampus	↓ GluR7 at 3 mo; ↑ GluR5 at 9 mo
*Metabotropic glutamate receptors*
Akbar et al (1996)¹	Amygdala kindling	Hippocampus	↓ mGluR5; ↑ mGluR1 at 24 hr
Blumcke et al. (2000)¹⁹	Kindling Kainic acid–induced SE	Hippocampus	↑ mGluR1
*Glutamate transporters*
Akbar et al. (1998)²	Genetically epilepsy-prone rats	Cortex, striatum, and hippocampi	↓ GLT-1
Ingram et al. (2000)⁷⁸	GAERS rats	Thalamus Cortex	↑ GLT-1 ↑ GLAST
Doi et al. (2000)⁴⁰	Fe(3+)-induced epileptogenesis		↑ EAAC-1 up to 30 d ↓ GLAST in epileptic animals
Ingram et al. (2001)⁷⁹	EL mice	CA3, parietal cortex	↓ GLT-1
Crino et al. (2002)³⁵	Pilocarpine-induced SE	Dentate gyrus	↑ EAAC1 in epileptic animals
Proper et al. (2002)¹⁴²	Human TLE	Hippocampus	↑ GLT-1, EAAC1
*Voltage-gated sodium channels*
Aronica et al. (2001)⁴	Hippocampal stimulation	Hippocampus	↑ NaCh II and III up to 3 mo
Whitaker et al. (2001)¹⁷²	Human TLE	CA3 CA4	↓ NaCh II ↑ NaCh III
Ellerkmann et al. (2003)⁴⁷	Pilocarpine-induced SE	Dentate gyrus	↓ Nav 1.2, Nav 1.6 up to 30 d
Klein et al. (2004)⁹⁵	WAG/Rij absence epileptic rats	Cortex	↑ Nav 1.1 and Nav 1.6
*Calcium channels*
Hendriksen et al. (1997)⁷²	Hippocampal kindling	Hippocampus CA3	↑ VDCC-α1A, -α1D, -α1E; ↓ -α1E during initial kindling ↑ VDCC-α1B in fully kindled animals
Vigues et al. (1999)¹⁶⁸	Kainic acid–induced SE	Hippocampus	↑ Q-type α1A up to 7 d
Talley et al. (2000)¹⁵⁸	GAERS rats	Thalamus	↑ T-type α1H, α1G
Blalock et al. (2001)¹⁸	Kindling	CA1	↓ L-type α 1D
*Hyperpolarization-activated cyclic nucleotide–gated cation channels (HCNs)*
Brewster et al. (2002)²⁵	Rat febrile seizures	CA1 CA3	↓ HCN1; ↑ HCN2 at 1 wk ↑ HCN2 at 1 wk
Bender et al. (2003)¹²	Human TLE	Dentate gyrus	↑ HCN1
	Pilocarpine-induced SE
CA1, CA1 subfield of the hippocampus proper; CA3, CA3 subfield of the hippocampus proper; CA4, CA4 subfield of the hippocampus proper; EAAC, excitatory amino acid carrier; EAAT, excitatory amino acid transporter; GABAR, γ-aminobutyric acid receptor; GAERS, genetic absence epilepsy rats from Strasbourg; GLAST, glutamate/aspartate transporter; GluR, glutamate receptor; GLT, glutamate transporter; HCN, hyperpolarization-activated cyclic nucleotide–gated cation channel; KA1, kainic acid receptor subunit 1; mGluR, metabotropic glutamate receptor; NR, N-methyl-D-aspartate receptor subunit; SE, status epilepticus; TLE, temporal lobe epilepsy; VDCC, voltage-dependent calcium channel.

Table 2 Summary of global analyses of gene expression

Authors	Organism	Model/disorder	Tissue	Platform	Number of regulated genes	Time points
Sandberg et al. (2000)¹⁴⁹	Mouse	Pentylenetetrazole-induced seizures	Cortex, cerebellum, midbrain, hippocampus	Affymetrix	>50	1 hr
French et al. (2001)⁵⁶	Mouse	ECS-induced seizures	CA1	Genome Systems	14	1 hr
Newton et al. (2003)¹³²	Rat	ECS-induced seizures	Hippocampus, choroid plexus	In-house microarray	Up to 86 (at 6 hr)	2 and 6 hr
Nedivi et al. (1993)¹³¹	Rat	KA-induced SE	Dentate gyrus	Differential cloning	52	6 hr
Hevroni et al. (1998)⁷⁴	Rat	KA-induced SE	Dentate gyrus	Differential cloning	362	6 hr
Tang et al. (2002)¹⁵⁹	Rat	KA-induced SE	Striatum, cortex	Affymetrix	187	1 d
Hunsberger et al. (2005)⁷⁷	Rat	KA-induced SE	Hippocampus	In-house microarray		1 d
Lukasiuk et al. (2003)¹¹⁵	Rat	Amygdala stimulation model of TLE	Hippocampus, temporal lobe	ResearchGenetics	282	1 d, 4 d, 14 d
Hendriksen et al. (2001)⁷²	Rat	Angular bundle stimulation–induced SE	Hippocampus	SAGE	79	8 d
Elliot et al. (2003)⁵⁰	Rat	Pilocarpine-induced TLE	Dentate gyrus	Affymetrix	129	14 d
Becker et al (2003)⁹	Rat, human	Pilocarpine-induced SE Human TLE	Dentate gyrus, CA1	Affymetrix	50–700, depending on the experimental group	3 d, 14 d, chronically epileptic
Liang et al. (2001)¹⁰⁶	Mouse	Electrical kindling	Hippocampus	Differential display	26	0.5 h, 1 d, 1 wk, and 1 mo
Potschka et al. (2002)¹⁴¹	Rat	Amygdala kindling	Hippocampus	MPSS	264	2 hr
Gu et al. (2004)⁶⁹	Rat	Amygdala kindling	Temporal lobe	Affymetrix	>200	24 hr
Arai et al. (2003)³	Rat	Ihara rat	Hippocampus	SAGE	21	2 mo old
Becker et al. (2002)⁸	Human	Pharmacoresistant TLE	Hippocampus	Atlas (Clontech)	21	Chronic epilepsy
Bo et al. (2002)²⁰	Rat	Epilepsy-prone P77MC rat	Cortex	Atlas (Clontech)	15	Chronic epilepsy
Kim et al. (2003)⁹⁴	Human	Intractable epilepsy	Cortical dysplasia		4	Chronic epilepsy
Crino et al. (2001)³⁴	Human	Intractable epilepsy	Cortical dysplasia	In-house microarray	4	Chronic epilepsy
CA1, CA1 subfield of the hippocampus proper; ECS, electroconvulsive seizures; KA, kainic acid; MPSS, massively parallel signature sequencing; SAGE, serial analysis of gene expression; SE, status epilepticus; TLE, temporal lobe epilepsy.

There is little information about the long-term changes in mRNA expression of metabotropic glutamate receptors in

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epilepsy. Kindling induced a transient decrease in metabotropic glutamate receptor mGluR5 and an increase in mGluR1.¹ Increase in mGluR1 was also observed following intraperitoneal kainate injections.¹⁹

Sodium channels are of particular importance for epilepsy because many epilepsies are associated with mutation in these channels, and several antiepileptic drugs (AEDs) have their site of action on sodium channels.⁹⁷ Data on the mRNA expression of sodium channels in epileptogenesis and epilepsy are sparse. Changes in mRNA levels for sodium channels were found in WAG/Rij absence epileptic rats,⁹⁵ after induction of SE with pilocarpine⁴⁷ or electrical stimulation of the hippocampus,⁴ and in the human epileptic hippocampus.¹⁷²

Various types of calcium channels contribute to increased excitability in epilepsy.⁵³^,⁸¹^,¹³⁶^,¹⁵⁵ Accordingly, protein levels of subunits have been demonstrated to be altered in both experimental and human epilepsy.³⁹^,¹⁶⁴ The little that is known about the expression of mRNA indicates changes in Q, R, and L-type voltage-sensitive Ca²⁺ channels in kindling¹⁸^,⁷² and in Q-type channels in the kainic acid model.¹⁶⁸ It has been suggested that the epileptic phenotype in adult genetic absence epilepsy rats from Strasbourg (GAERS) may relate to elevation in the levels of mRNA encoding T-type calcium channels.¹⁵⁸

Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) mediate the hyperpolarization-activated (I_H) currents in the brain.¹³⁵ Long-lasting changes in function of HCNs have been implicated in epileptogenesis.²⁵^,³⁰ It has been suggested that the normal or modified HCN channels might be involved in epileptogenic or protective mechanisms in the epileptic hippocampus.¹⁷¹ Increase in HCN1 expression was observed in hippocampi of patients with TLE and in the pilocarpine model of chronic TLE in rats.¹² Alterations in HCN mRNA expression was also found in the rat model of febrile seizures.²⁵

Other Genes Involved in Regulation of Neuronal Excitability

Apart from ion channels and receptors, there are also other known mechanisms that can influence neuronal excitability, including the effects of transporters, peptide neurotransmitters, growth factors, and other neuromodulators.⁵^,⁶⁵^,⁹⁹^,¹⁶⁰^,¹⁶⁶ Some of these have been studied in epilepsy, but data on long-term regulation of expression of genes coding for members of these pathways are sparse.

Extracellular glutamate levels are controlled by glutamate transporters that remove glutamate from synaptic cleft. Upregulation of glial glutamate transporter GLT-1 and neuronal excitatory amino acid transporter EAAC1 has been reported in patients with TLE.¹⁴² As summarized in Table 1, altered expression of mRNA encoding glutamate transporters also has been reported in several models of experimental epilepsy, including genetically epileptic-prone rat,² EL mice,⁷⁹ GAERS rats,⁷⁸ Fe³⁺-induced epilepsy,⁴⁰ and pilocarpine-induced epilepsy.³⁵

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Neuropeptide Y (NPY) has a significant role in regulating seizure activity. Its presynaptic Y2 receptors are capable of suppressing seizure activity by inhibiting glutamate release from mossy fibers.¹⁶⁷ On the other hand, Y1 receptor antagonists have anticonvulsive potential.⁶² Marked increases of NPY mRNA in hippocampal granule cells and interneurons were found in Ihara’s epileptic rats and spontaneously epileptic rats.¹⁵⁷ In epileptic human hippocampus, NPY mRNA was increased in hilar interneurons of sclerotic and nonsclerotic specimens. Thus, together with upregulation of Y2 receptors, downregulation of Y1 receptors in the hippocampus of patients with Ammon’s horn sclerosis may be an endogenous anticonvulsant mechanism.⁵⁸

Neurotrophins can influence neuronal network excitability, and their expression following seizures has been extensively studied. Little information about their mRNA expression is available in other epilepsy-related situations. Increased expression of brain-derived growth factor (BDNF) mRNA has been observed in the neocortex of animals in which spontaneous seizures were induced following injection of tetanus toxin. At later times there was an upregulation of BDNF mRNA at the injection site and downregulation in a surrounding cortical zone. This could contribute to plastic changes in epileptic neuronal circuits and may be associated with the inhibitory surround that hampers seizure spread but facilitates the persistence of a chronic epileptic focus.¹⁰⁷ Epileptic patients with hippocampal sclerosis show increased granule cell mRNA levels for BDNF, nerve growth factor (NGF), and neurotrophin 3 (NT-3).¹²⁰^,¹²⁸

Global Analysis of Gene Expression

Studies of gene expression in epilepsy using traditional molecular biology methods have focused on a limited number of preselected genes at a time. Recent technological developments allow the analysis of gene expression at the level of nearly the whole transcriptome, which provides an unbiased insight into molecular events that occur in the brain during epileptogenesis and epilepsy. The data derived from these studies can be expected to highlight the most prominent metabolic pathways or other phenomena that underlie reorganization of the epileptogenic circuitry and, eventually, guide our efforts at identifying candidate targets for antiepileptogenic treatments.

As described earlier, numerous studies have demonstrated up- or downregulation of a variety of neuropeptides, transporters, ion channels, and receptors in the epileptic state, as well as a host of immediate early genes and other potential effector genes. These studies demonstrate that seizures trigger transcriptional programs that can be long lasting. Unfortunately, they have not guided us to a satisfactory understanding of the molecular events underlying epileptogenesis, partly because the only genes that have been examined so far are those for which prior physiologic or anatomic study suggested their involvement. It is expected that a large array of genes may be involved in epileptogenesis, including genes involved in cell growth control, cell adhesion and migration, intracellular signal transduction, apoptosis, protein turnover, cytoskeleton, and others. Lack of information on the molecular basis of epileptogenesis directly limits the development of treatments that might prevent epilepsy from developing after, for example, traumatic brain injury.

Experiments in which global analysis of gene expression was used to study epileptogenesis and epilepsy have, to a large extent, confirmed data previously gathered with conventional methods.³⁴^,¹¹⁴^,¹¹⁶ However, large-scale studies of gene expression have provided insight into other, often unexpected metabolic pathways that may be of great importance for epileptogenesis and epilepsy. Data described in this chapter are summarized in Table 2.

Methods Used for Global Analysis of Gene Expression

Several methods have been developed for the analysis of the whole transcriptome. Here we focus on those that have been successfully used in epilepsy research.

Differential display is an RT-PCR–based method used to identify differentially expressed genes. First, mRNAs from brain tissue specimen are reverse transcribed, and then they are amplified using nonspecific primers. The array of bands obtained from a series of such amplifications is separated on a sequencing gel and compared with analogous arrays from different samples. Any bands unique to a particular sample are differentially expressed. The bands can be purified from the gel and sequenced.

Serial analysis of gene expression (SAGE) is a method for comprehensive analysis of gene expression patterns.¹⁶⁵ It allows the quantitative and simultaneous analysis of a large number of transcripts without the need for creating a probe for each transcript. The principle of SAGE relies on the production of small cDNA tags on the basis of studied mRNA and their subsequent sequencing, counting, and assignment to corresponding genes using bioinformatic tools. The frequency of a specific tag is related to the abundance of the corresponding mRNA in the cell and allows for a comparison of mRNA expression of both known and novel genes in different samples.

Massively parallel signature sequencing (MPSS) is similar to SAGE and is capable of analyzing gene expression without a priori knowledge of the transcript sequence and mRNA abundance. MPSS also relies on the production of short tags. However, due to the combination of in vitro cloning of cDNA molecules on the surface of microbeads with non–gel-based high-throughput signature sequencing, a single MPSS experiment can generate >10⁷ tags (100 times more than that in a SAGE experiment).²⁶

DNA microarrays are increasingly popular in studies of gene expression. They enable evaluation of the expression levels of thousands of genes at the same time and thereby provide global insight into transcriptional events taking place in a studied phenomenon. The principle of DNA microarray technology is based on the hybridization of labeled cDNA synthesized on the basis of mRNA derived from the tissue of interest, applied to a microarray consisting of a large number of gene probes placed with high density on a solid surface. The strength of the hybridization signal to the particular gene probe reflects the abundance of the respective mRNA in the sample. There are two main types of microarrays: (a) spotted microarrays and (b) Affymetrix Gene Chips. For spotted microarrays, the probe (oligonucleotides or cDNA) is usually spotted on a glass surface. The sample cDNA for hybridization is typically fluorescently labeled. These microarrays offer the possibility of using cDNA from two different conditions (e.g., control vs. treated) labeled with different fluorochromes for simultaneous hybridization to the same chip. Differences in expression are detected by comparing the signal from the two fluorochromes used for labeling the examined samples. Most commercial and academic microarrays belong to this category. In the case of GeneChips produced by Affymetrix (Affymetrix, Inc., Santa Clara, CA), the oligonucleotide probes are synthesized directly onto the surface. The use of perfect and mismatch probe pairs and several probes for each gene increases the specificity and reproducibility of the quantitative results. Some methodologic issues concerning the use of microarrays in the brain have recently been extensively reviewed.⁶^,¹¹⁴^,¹⁵¹

The recent development of high-density cDNA or oligonucleotide microarrays has brought a new level of sensitivity and specificity to large-scale monitoring of gene expression. This technology has been most productively used in yeast, for which the sequence of every gene is known and available for analysis on microarrays. Rapid progress has occurred in the

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identification of human and mouse genes; a substantial proportion of all human and mouse genes are already available for study.

Changes in Pattern of Gene Expression in Response to Brief Seizures

One of the first studies using cDNA arrays to evaluate se-izure-induced gene expression was performed by Sandberg and colleagues.¹⁴⁹ The study examined whether neurobehavioral differences in mice from different strains are linked to differences in gene expression. In addition to defining the basal gene expression in different brain areas, it also examined gene expression in response to seizures in two mouse strains that display differences in susceptibility to seizure-induced damage: 129vEv (susceptible) and C57BL/6 (resistant). The seizures were evoked by subcutaneous application of pentylenetetrazole, and animals were sacrificed 1 hour after seizure. Two Affymetrix GeneChips, Mu11KsubA and Mu11KsubB, were used for gene profiling. The two mouse strains differed in their transcriptome response to seizures. Twenty-four genes were differentially expressed between the two mouse strains in all brain areas examined, and >50 genes were differentially expressed in specific brain areas.

French et al.⁵⁶ sought to identify the genes involved in plasticity in the hippocampus by using electroshock-evoked seizures as a model. Animals were sacrificed 1 hour after application of current via corneal electrodes. Control animals were exposed to the electrodes without stimulation. Samples from area CA1 of the hippocampus were profiled using the Mouse GEM1 microarray (Genome Systems). Of 9,000 genes that were present on the microarray, only 14 genes were found to have significant changes in expression following seizures. Nine were downregulated and five were upregulated. Subsequent validation of the expression changes with in situ hybridization, however, failed to detect any mRNA expression of six of the candidate genes. Upregulation in area CA1 was confirmed for only one gene (nerve growth factor–induced clone B [NGFI-B]), and the authors were unable to confirm differential expression of any other candidate genes. Three genes were upregulated in the dentate gyrus. These findings suggest that, in contrast to the dentate gyrus, seizures do not appear to be accompanied by large modulation of gene expression in area CA1 of the hippocampus in the model used.

The influence of electroconvulsive seizures (ECS) on the profiles of gene expression has also been extensively studied in relation to effect of ECS on mood. Newton et al.¹³² studied the expression of neurotrophic growth factors and related signaling molecules in the rat hippocampus in response to electroconvulsive seizures using a custom microarray containing 645 gene probes. They reported regulation of several genes involved in growth factor signaling and angiogenesis, which could have an important role in the molecular action of ECS.

Changes in Patterns of Gene Expression as an Immediate Consequence of Status Epilepticus

One of the first attempts to describe global changes in gene expression following SE was done by Nedivi and coworkers.¹³¹ They used differential cloning to search for plasticity-related genes. Gene expression was studied in the dentate gyrus 6 hours after SE induced by intraperitoneal injection of kainic acid. Granule cells of the dentate gyrus are resistant to kainic acid–induced neurotoxicity and display plastic changes such as axonal sprouting, growth of basal dendrites, and changes in the number and morphology of dendritic spines. About 5% of the screened genes had a change in level of expression following the seizures (candidate plasticity genes [CPG]). Fifty-two clones derived from genes showing altered expression were partially sequenced. Seventeen of those genes were previously known, with some having a link to neuronal plasticity. Five transcription factors with known role in neuronal plasticity were identified (c-jun, zif/268, c-fos, fosB, and cAMP responsive element modulator [CREM]). The product of another gene, TIMP (tissue inhibitor of metalloproteinase), is a component of the TIMP/matrix metalloproteinase [MMP] system, which influences neuronal plasticity by controlling enzymatic activities of metalloproteinases and regulating local proteolysis of extracellular matrix.⁴⁵ An interesting group of genes are involved in the functioning of vesicles (such as clathrin heavy chain, dynorphin, secretogranin) or in encoding the components of synapse (catechol-O-methyltransferase [COMT], syndecan).

Further work by Nedivi et al. identified a total of 362 candidate plasticity genes.⁷⁴ The sequencing and characterization of unknown CPGs revealed a role in the neuronal plasticity for some of them. For example, CPG15 (also known as neuritin) is an activity-induced molecule that promotes dendritic growth of projection neurons and neurite outgrowth and arborization in culture.¹²⁹ Its involvement in experience-induced neuronal plasticity has been shown in well-established models of barrel cortex plasticity and visual cortex plasticity.⁷¹^,¹⁰⁴ The other gene, CPG2, influences synaptic properties by regulation of endocytosis of glutamate receptors.³²

Tang et al.¹⁵⁹ used Affymetrix microarrays to study the genomic response in the rat striatum and cortex to a variety of brain-damaging insults including kainate-induced SE. Subcutaneous injection of kainic acid resulted in upregulation of 187 genes and downregulation of 89 genes in the parietal cortex 24 hours later. Expression of many of these genes was also changed by ischemic stroke, intracerebral hemorrhage, and hypoglycemia or hypoxia. The protein products of the regulated genes belong to a variety of functional classes, including stress-related proteins, proteases, cytoskeletal proteins, and receptors. The function of a number of genes responding to kainate treatment implicates their involvement in remodeling of neuronal networks (see earlier discussion). For example, c-jun, fos, tissue inhibitor of metalloproteinases 1 (TIMP-1), and syndecan participate in regulation of dendritic spine morphology and hippocampal long-term potentiation (LTP).⁵¹^,⁸⁷

Recently, response to kainic acid–induced SE has been studied also by Hunsberger et al.⁷⁷ In this study, gene expression profiles of the rat hippocampi were characterized at 24 hours after intraperitoneal application of kainate using custom cDNA array. Kainic acid–induced genes were classified into multiple functional classes such as angiogenesis, cell cycle and proliferation, cell death, extracellular matrix signaling, kinases and phosphatases, neuroprotection, neurotransmitter signaling, and transcription factors.⁷⁷ Thirty-six novel kainic acid–regulated genes were found in addition to those that were discovered in previous studies, including transcription factors (CCAAT/enhancer binding protein δ [C/CEBPδ], glycoprotein 38 [gp38], and ankyrin repeat and SOCS box protein 13 [ASB13]) and those involved in neurotransmitter signaling (fibroblast growth factor receptor 4 [Fgfr4], G protein–coupled receptor 18, and L-myc-1 proto-oncogene protein [Lmyc1] transcription factor).

Lukasiuk and colleagues examined gene expression profiles during epileptogenesis at 1 day after SE induced by electrical stimulation of the amygdala using Research Genetics Rat Array (Research Genetics, Huntsville, AL) containing approximately 5,000 genes and expressed sequence tags (ESTs). One day after the induction of SE, changes were observed in the expression of 37 genes in the hippocampus (8 downregulated and 29 upregulated) and 29 genes in the temporal lobe (13 downregulated and 16 upregulated). Products of some of those genes are involved in normal metabolism, including cytochrome function, protein synthesis, and carbohydrate and amino acid metabolism. Some of these changes might reflect rescue efforts from metabolic

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disturbances. Others, such as downregulation of predominantly neuronal synaptophysin or 14–3-3 gamma, might reflect ongoing neurodegeneration.¹¹⁴^,¹¹⁵

Changes in Pattern of Gene Expression During Late Epileptogenesis

Hendriksen et al.⁷³ investigated the pattern of gene expression in the hippocampus of rats undergoing epileptogenesis that was induced by SE evoked by electrical stimulation of the angular bundle. Four hours after the stimulation, SE was stopped with pentobarbital. The hippocampus contralateral to the stimulation site was isolated 8 days after stimulation and used for gene profiling with SAGE methodology. During the latency period, rats were under continuous electroencephalographic (EEG) monitoring, and only the animals that did not express any seizure activity during monitoring were included in the study. Sequencing >10,000 tags for each group revealed 92 differentially regulated tags. Genes with altered expression were associated with expression of ribosomal proteins, protein processing, axonal growth, and glial proliferation. The authors classified 6 genes as related to axonal growth and regeneration and therefore as likely involved in the remodeling of neuronal network, including cyclophilin, glycoprotein 65, α-tubulin, Thy-1 antigen, thymosin α-10, and an expressed sequence tag highly similar to tubulin β chain.

Elliot et al.⁵⁰ characterized the pattern of gene expression in the pilocarpine model of rat epilepsy. SE was induced by an intraperitoneal injection of pilocarpine hydrochloride and terminated after 2 hours of convulsive SE with diazepam. Animals were sacrificed 14 days later. In this study, the authors focused on the analysis of gene expression in the dentate gyrus of the hippocampus. Expression levels were analyzed using the Affymetrix chips, and changes in level of expression were found for 129 genes. Genes altered during epileptogenesis represented a wide range of functions, with a high number of genes involved in response to injury and cell survival. The authors hypothesized that epileptogenesis shares some features with normal development of the nervous system. Therefore, they compared gene expression in the dentate gyrus of the hippocampus during development and epileptogenesis. A total of 37 genes had an altered level of expression during both epileptogenesis and development. According to functional annotations performed by authors, genes altered during epileptogenesis have functions related mainly to injury and survival, morphology, signaling, metabolism, and cell cycle. Our bioinformatic analysis of this data set that was undertaken to detect overrepresentation of functional gene groups revealed statistically significant overrepresentation of genes involved in RNA metabolism, neurogenesis, cytoskeleton organization and biogenesis, development and cell differentiation, and nerve maturation.¹¹⁶ Some of these genes may be of particular interest in the context of remodeling of neuronal network. For example, internexin neuronal intermediate filament protein α (Inexa) may play a role in neuronal regeneration in response to injury. Bhlhb3 (basic helix-loop-helix domain–containing protein class B3) has been implicated in the regulation of neuronal differentiation during development and adaptive neuronal plasticity and neurite outgrowth in the adult. Finally, protein phosphatase 1 regulatory subunit 9B (Ppp1r9b) and Ras homolog enriched in brain (Rheb) play a role in synaptic plasticity. Another gene, Synthenin, is abundant during the period of development characterized by intense growth and synapse formation and stabilization and may play a role in determining the formation and maturation of synapses.⁷⁶ Vascular growth factor (VGF) plays a key role in neuronal differentiation and survival and regulates synaptic function in hippocampal neurons, neurotrophic tyrosine kinase receptor type 2 (Ntrk2) is a receptor for brain-derived neurotrophic factor, and CD24 antigen (Cd24) may be involved in neuronal migration during development.

Becker et al.⁹ also studied gene expression in the dentate gyrus and the CA1 region of the hippocampus at 3 and 14 days after pilocarpine-induced SE with Affymetrix microarrays. Increased expression of >400 genes in the dentate gyrus and 700 genes in the CA1 was observed 3 days after SE, whereas a lower number of genes was regulated at 14 days after SE: >50 genes in the dentate gyrus and 400 genes in the CA1. The authors suggested that several genes from their data set might be involved in stress reaction and structural reorganization of the hippocampus. Regulated immediate early genes include transcription factors (JE-immediate early gene, c-fos), tissue plasminogen activator (tPA; a component of the plasminogen system), as well as mitogen-activated protein kinase 1 (MEKK1) and cell division control protein 2a (cdc2a) kinases. Some genes may be linked to structural plasticity such as microtubule-associated protein 2 (map2), pentraxin, TIMP-2, glial fibrillary acidic protein (GFAP), or Thy-1 cell surface antigen. Others, like Ras-related protein Rab-3 (rab3), regulating synaptic membrane exocytosis (Rim) Iα, neurexin III-α, and neurexin I-β, are associated with the synapse and can influence synaptic plasticity.

Lukasiuk et al. analyzed changes in gene expression in rats undergoing epileptogenesis after SE induced by electrical stimulation of the amygdala.¹¹⁵ Tissue was sampled in the hippocampus and temporal lobe at 4 and 14 days after induction of SE. Four days after stimulation, 10 genes were upregulated and 2 were downregulated in the hippocampus. In the temporal lobe, 57 genes were upregulated and 98 were downregulated. Fourteen days after stimulation, there were 3 genes upregulated and 11 downregulated in the hippocampus and 17 genes upregulated and 15 downregulated in the temporal lobe. Many genes regulated during epileptogenesis are involved in basic metabolism, including energy metabolism, protein synthesis and degradation, and signal transduction. Further analysis revealed statistically significant overrepresentation of functional gene classes. At 4 days after SE there was an upregulation of genes involved in cytoskeleton organization and biogenesis (Ywhag [tyrosine 3-monooxygenase/tryp-tophan 5-monooxygenase activation protein, γ polypeptide], Lasp1 [LIM and SH3 protein 1], Dlc1 [deleted in liver cancer 1], and stathmin 1) and in cell differentiation (vav 1 oncogene, selenoprotein 15, Ywhag, interferon-related developmental regulator 1 [Ifrd1], bone morphogenetic protein 4, dual-specificity phosphatase 6).¹¹⁶ At 14 days after SE, genes involved in electron transport and secretion were overrepresented. One of the genes that may be of particular importance for plastic changes was munc13–4, which encodes a protein mediating synaptic vesicle exocytosis from glutamatergic synapses.

Changes in Pattern of Gene Expression in Epileptic Tissue

Global analysis of gene expression has been used for characterization of epileptic tissue both in animal models and in surgically resected human tissue.

Several studies have dealt with global alterations in gene expression in human temporal lobe epilepsy. Becker et al.⁸ studied patterns of gene expression in the resected human epileptic hippocampus with Ammon’s horn sclerosis and compared them to unlesioned nonepileptic hippocampal tissue using the Atlas Human Neurobiology array. Nine genes were upregulated and 12 were downregulated in the sclerotic hippocampi. Subsequent real-time quantitative RT-PCR analysis of gene expression in laser-microdissected cells enabled identification of the genes that were differentially regulated in the neuronal and glial cell populations. For example, ataxin-3 was upregulated in neurons and GFAP (glial fibrillary acidic protein) in

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astrocytes. On the other hand, calmodulin expression did not differ in individual neurons sampled from sclerotic or unlesioned hippocampus, suggesting that a decrease in its expression in tissue extracts is due to the loss of calmodulin-expressing neurons.

In their further work, Becker et al. characterized and compared gene expression profiles in rat and human epileptic tissue using Affymetrix microarrays.⁹ In rats, epilepsy was induced by pilocarpine-evoked SE. Gene expression was studied and compared in the CA1 and DG regions of the rats exhibiting either low or high seizure frequency. In animals with high seizure frequency, 96 genes were altered in the CA1 and 74 in the dentate gyrus. In the group with low seizure frequency, 89 genes were altered in the CA1 and 57 in the dentate gyrus. In general, fewer alterations in gene expression were detected in epileptic animals than in rats that were sacrificed for analysis at 3 or 14 days after pilocarpine application (epileptogenesis phase). Genes induced in chronically epileptic animals included neurotransmitter receptor genes (D1A dopamine receptor), channels (Kv3.2 potassium channel), and genes important for synaptic transmission such as pentraxin, Rim, and neurexins. Our analysis of this data set revealed overrepresentation of genes belonging to several functional groups, including synapse organization, response to stress, and the wounding or immune response.¹¹⁶ In addition, the authors compared gene expression profiles in hippocampi of epileptic rats and hippocampi of human epileptic patients. Eighteen genes were similarly regulated in epileptic animals and in humans, demonstrating remarkable interspecies similarity in gene expression profiles. Coregulated genes are involved in cell–matrix interactions, cell growth and differentiation, cellular metabolism, neuronal signaling, and regulation of transcription.

Lukasiuk and colleagues studied patterns of gene expression of the hippocampi and temporal lobes of rats that developed spontaneous seizures following SE induced by electrical stimulation of the amygdala.¹¹⁵ In the hippocampus, 12 genes were upregulated and 30 genes were downregulated. In the temporal lobe, 58 genes were upregulated and 4 genes were down-regulated. It is interesting that there was little overlap between the genes regulated in epileptic animals in comparison to animals that did not have spontaneous seizures at the same time after stimulation. The regulated genes have a variety of functions, including those related to basic metabolism, cell cycle, and signal transduction.

Gene expression profiling also has been used to study genetically epileptic rats. Bo et al.²⁰ searched for differentially expressed genes in the cerebral cortex of genetically epilepsy-prone P77MC rats using Atlas Expression Array. They found 13 genes elevated and 2 genes downregulated in P77MC rats compared to normal Wistar rats. These genes were involved in protein synthesis, metabolism, membrane transport, cytoskeleton, and ion channels.

Arai et al.³ studied genetically epileptic Ihara rats that have genetically programmed micodysgenesis in the hippocampus. They used SAGE to compare the expression pattern of IERs with that of control Wistar rats at 2 months of age, that is, prior to the expected manifestation of spontaneous seizure activity. They identified 21 differentially regulated genes. The genes upregulated in IER rats were involved in protein synthesis, metabolism, membrane transport, the cytoskeleton, and ion channels. Genes coding for intracellular components and neurotransmission-related proteins were downregulated. Most of these genes had not been previously implicated in epileptogenesis or epilepsy.³

Kim et al.⁹⁴ studied cortical dysplasia and compared gene expression in dysplastic with nondysplastic cortex. Three genes were upregulated and four genes were downregulated in dysplastic cortex. It is interesting that the majority of the genes showing altered expression were associated with apoptosis.

An earlier study of cortical dysplasia by Crino at al.³⁴ using targeted cDNA array revealed that GluR4, NR2B, and NR2C subunit mRNAs were increased and NR2A and GABA_ARβ1 subunit mRNAs were decreased in dysplastic neurons, whereas GABA_ARα1, -Rα2, and -Rβ2 as well as GluR mRNA levels were reduced in both dysplastic and heterotopic neurons.

Changes in Pattern of Gene Expression During Kindling

Apart from SE-induced epileptogenesis, kindling has also been used as a model to study plasticity-associated changes in trans-criptome. During kindling, repetitive application of an initially subconvulsive electrical stimulus results in development of epileptiform activity and finally generalized seizures in response to the same stimulus.⁶⁶ One advantage of this model is the absence of severe brain damage that is present in SE models. Therefore, genes that change expression level during kindling are presumably related to changes in excitability, and the data set is less likely to be contaminated by alterations related to neurodegeneration.

Liang and Seyfried¹⁰⁶ used RT-PCR differential display (DD) to study hippocampal tissue at 0.5 hour, 1 day, 1 week, and 1 month after rapid kindling. Out of 30,000 bands analyzed, 50 were differentially displayed. Northern blot analysis confirmed changes in 26 of these genes. Fourteen of them were known genes and 12 were novel. Only 5 genes had changed expression level for at least 1 day. Kinase αCaMK II, phosphatase PP2A, and GTPase-like protein Cyr 61 were downregulated, whereas King12 was upregulated for up to 1 day after kindling. Only RGS4, a GTPase-activating protein, was persistently downregulated up to 1 month after rapid kindling.

MPSS was used to study gene expression in the rat amygdala kindling model by Potschka et al.¹⁴¹ Kindling by repeated electrical stimulation of the amygdala resulted in the differential expression of 264 genes in the hippocampus compared to sham controls. The most strongly induced gene was Homer 1A, an immediate-early gene involved in the modulation of glutamate receptor function. Because kindling induces overexpression of Homer 1A in the hippocampus and mice overexpressing Homer 1A exhibited retardation in kindling, this may represent an intrinsic antiepileptogenic mechanism in counteracting progression of the disease.¹⁴¹

Gu et al.⁶⁹ studied the effect of amygdala kindling on gene expression in the temporal lobe with Affymetrix microarrays. A number of genes were regulated during kindling, and the authors clustered >200 of them into 15 protein pathways or functional classes. These functional classes could reflect ongoing changes leading to increased excitability and included G-protein signaling, synaptic transmission, CA²⁺-dependent kinases, ion channels, transcription factors, neurofilaments, microtubules, surface-linked signal transduction, and others.

Influence of Antiepileptic Drugs on Pattern of Gene Expression

Large-scale molecular profiling studies have provided opportunities to discover new mechanisms of action of AEDs that extend beyond their antiepileptic effects and can mediate their effects on neuronal recovery after epileptogenic insult. Such analyses have been performed with tissues exposed to valproic acid, lamotrigine, or phenytoin. Bosetti et al.²¹ administered 200 mg/kg of valproic acid to rats for 30 days and reported that 87 of 8,799 genes on a U34A Affymetrix oligonucleotide microarray were downregulated and 35 were upregulated. The regulated genes affect a variety of molecular pathways, including synaptic transmission; ion channels and transport; G-protein signaling; lipid, glucose, and amino acid metabolism; transcriptional and translational regulation; phosphoinositol

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cycle; protein kinases and phosphatases; and apoptosis. The actions on these pathways might explain some of the effects of valproic acid on psychiatric conditions as well as on the recovery process after epileptogenic brain insults. One of the effects might be on angiogenesis and cell proliferation. It is interesting that valproic acid (0.25–1 mM) was recently reported to inhibit endothelial cell proliferation and migration as well as reduce angiogenesis in vitro and in vivo, effects that are probably mediated by the inhibition of histone deacetylase and decreased expression of endothelial nitric oxide synthase.¹²⁵ Suppressed expression of the angiogenic factors vascular endothelial growth factor and fibroblast growth factor is also proposed to contribute to the antiangiogenic actions of valproate.¹⁷⁶ The effect on cellular proliferation has sparked interest in valproate as an antineoplastic agent.¹⁶ Whether trauma- or seizure-induced proliferation of neuronal and glial cells is affected by valproic acid is unknown.

Wang and colleagues¹⁷¹ exposed hippocampal primary cell cultures to 0.1 mM lamotrigine for 1 week. Of 1,200 genes on the Atlas Rat 1.2 array, 8 genes were upregulated and 6 were downregulated. Regulated genes included subunits of ion channel receptors, kinases, phosphatases, and proteins involved in cell survival. The functional significance of the data has not yet been explored.

The effects of phenytoin on gene expression in human dermal fibroblasts were studied by Swamy et al.¹⁵⁶ Of the 18,000 elements on the cDNA microarrays, 1,500 genes were differentially expressed after exposure of cultures to 20 μg/mL phenytoin for up to 48 hours. The major growth factors and their receptors involved in wound healing were upregulated, as were the genes encoding proteins involved in extracellular matrix degradation (e.g., matrix metalloproteinase-1). These findings suggest a molecular basis for some peripheral effects of phenytoin, including its beneficial effects on wound healing. They are also consistent with earlier studies demonstrating that in an experimental model of wound healing, as well as in stroke patients with peripheral ulcers, phenytoin facilitates fibroblast infiltration and neovascularization.³⁶^,¹³⁷ Further investigation of the effects of phenytoin on genes encoding proteins that contribute to the reorganization of the extracellular matrix and blood vessels in neuronal tissue after brain trauma seems very worthwhile.

Gu et al.⁶⁹ studied the effect of levetiracetam on gene expression during amygdala kindling. Levetiracetam had no effect on gene expression in control rats. However, application of levetiracetam during kindling not only suppressed kindling, but also influenced gene expression patterns in the temporal lobe. Expression of some epilepsy-related genes such as neuropeptide Y, thyrotropin-releasing hormone, and DFAP was partially normalized by levetiracetam treatment. Nevertheless, a significant number of genes remained altered by kindling even on levetiracetam treatment.

Caveats in Large-Scale Profiling of Epileptic Tissue

Despite the rising number of reports of global analysis of gene expression in epileptogenesis and epilepsy, the most essential molecular players in these processes are still unspecified. The first analysis of various data sets available a few years ago led to the conclusion that there are very few common features among different studies.¹¹⁴ Differences may relate to technical issues associated with large-scale gene expression analysis. Methods of global analysis of gene expression have limited sensitivity, especially when brain tissue is studied. It has been suggested, for example, that GeneChips reliably detect no more than 30% of the hippocampal transcriptome.⁵² In addition, in the case of SAGE or other methods using large-scale sequencing, rare transcripts may not be detected. Furthermore, the brain is obviously not a homogeneous tissue and consists of multiple cell types. Therefore, if a particular transcript is regulated in only one cell type, this alteration may be “diluted” in extracts from the tissue and remain below the level of detection. In addition, techniques of global analysis of gene expression do not provide information regarding cellular localization of the detected changes in gene expression. In case of neurodegenerative diseases, some changes can be related to the disappearance of selected neuronal populations, as demonstrated by Becker et al.⁸ Finally, investigators have used different animal models or human tissues as well as analyzed the pattern of gene expression at different stages of disease development and in different brain areas. As Lukasiuk et al. showed, the changes in gene expression induced by SE are dynamic and change with time.¹¹⁵ Furthermore, changes in the expression of the majority of genes are often specific to the a brain area, and there is limited overlap between genes regulated, for example, in the hippocampus and the temporal lobe.⁹^,¹¹⁵ Thus, there is a great variability in genes considered to be regulated depending on the experimental model, time point, and experimental procedure used in the study. Finally, the microarray approach will not identify posttranslational events that may be critical to the process of epileptogenesis.

Global Analysis of Gene Expression in Epileptogenesis and Epilepsy—Attempt at a Synthesis

To reassess the information in data sets available from epileptogenic or epileptic tissue, Lukasiuk et al. used emerging bioinformatics methods that were designed for the global analysis of gene expression to search (a) highly represented functional gene classes (gene ontology [GO] terms) within data sets and (b) individual genes that appear in several data sets and, therefore, might be of particular importance for the development of epilepsy due to different etiologies.¹¹⁶ The investigators focused on two well-described models of brain insult that induce the development of spontaneous seizures in experimental animals: (a) SE and (b) traumatic brain injury (TBI). A few papers describing gene expression in rat and human epileptic tissue were included for comparison.

The analysis revealed that various epileptogenic insults induce statistically significant changes in gene expression in functionally linked genes that were predefined as GO terms (Fig. 1). The representation of statistically significant GO terms describing biologic process was time dependent. Within hours after epileptogenic brain insult, the GO term “regulation of transcription” was overrepresented. It is interesting that this GO term did not become significant at any later time point. Many of the transcription factors detected by the analysis can contribute to ongoing neurodegeneration or, more generally, to cell stress. On the other hand, changes in the expression of some transcription factors might induce succeeding events that are presumably crucial for epileptogenesis, such as neuronal plasticity. Genes under the GO term “neurogenesis” become apparent at 1 day after injury. In epileptic tissue collected from humans or animals with spontaneous recurrent seizures (i.e., the end result of epileptogenic process), the presence of the GO terms related to “synaptic organization” (BDNF), “plasticity” (BDNF), and “transmission” (apolipoprotein E [APOE], synaptosomal-associated protein 25 kDa [SNAP25]) emerged. This supports the idea that neuronal plasticity occurs for a longer period of time after an insult, and can progress even after epilepsy is diagnosed.¹³⁸ It is interesting that several GO terms, including “immune response,” “cell motility,” “response to stress,” “response to wounding,” and “ion homeostasis,” are represented across different time points after an insult. Numerous individual genes change their expression in more than two models of epileptogenesis (Fig. 2). Alterations in their expression are time specific. A closer look at altered individual genes as well as GO terms indicated an involvement of many

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processes previously linked with epileptogenesis such as cell death and survival, neuronal plasticity, and immune response. It is interesting, however, that only a few genes are involved in defining the electrical properties of neurons and glia. Perhaps the most striking finding was that the predominant function of genes with altered expression is related to the immune response. Although inflammation and immune response in brain trauma and epilepsy are receiving more attention, they have not been considered to have a key role in epileptogenesis and epilepsy.

FIGURE 1. Summary of biological process gene ontology (GO) terms that are overrepresented after epileptogenic brain insults (status epilepticus or traumatic brain injury). Overrepresented functional gene classes were detected by meta-analysis of GO terms in gene lists that were generated from the data provided in selected papers using GOstat software (available at http://gostat.wehi.edu.au).¹⁰ Only GO terms overrepresented in at least two lists within a time window are presented. For details of the analysis see Lukasiuk et al.¹¹⁶ For an extended version of the results see http://www.uku.fi/aivi/neuro/research_epilepsy.shtml or http://www.nencki.gov.pl/labs/epg.htm.¹¹⁶

FIGURE 2. Functional classification of individual genes that appear in several data sets describing global analysis of gene expression following epileptogenic insult (status epilepticus or traumatic brain injury). transd, transduction. To find a particular gene with altered expression, data provided in selected manuscripts were reannotated and analyzed using a purpose-build relational database on a MySQL server platform. For an extended version of the results see http://www.uku.fi/aivi/neuro/research_epilepsy.shtml or http://www.nencki.gov.pl/labs/epg.htm.¹¹⁶

Summary and Conclusions

Alterations in gene expression that underlie cellular modifications leading to epileptogenesis and epilepsy have been extensively studied by using conventional molecular biology methods as well as by global analysis of gene expression. Most studies have focused on the immediate effects of seizures or SE on gene expression and therefore describe the effects of increased neuronal activity or neuronal injury on gene expression. An overall review of the studies indicates that gene lists obtained by global analysis include very few of the genes studied previously with traditional methods, such as those having a role in regulation of neuronal excitability such as receptors or channels. These observations suggest that receptor or voltage-gated channelopathies may just be some of the phenomena that occur in serial and parallel manner (possibly interacting) with other cellular changes (e.g., inflammation) that underlie the development of epilepsy and change in excitability, and these should be investigated to greater extent.

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	Epilepsy: A Comprehensive Textbook 2nd Edition
	© 2008 Lippincott Williams & Wilkins

Epilepsy: A Comprehensive Textbook2nd Edition

Epilepsy: A Comprehensive Textbook
2nd Edition