Mechanisms of epileptogenesis and potential treatment targets

Mechanisms of epileptogenesis and potential treatment

Asla Pitkänen, Katarzyna Lukasiuk Prevention of epileptogenesis after brain trauma is an unmet medical challenge. Recent molecular profi ling studies Lancet Neurol 2011; 10: 173–86
have provided an insight into molecular changes that contribute to formation of ictogenic neuronal networks, Department of Neurobiology,

A I Virtanen Institute for
including genes regulating synaptic or neuronal plasticity, cell death, proliferation, and infl ammatory or immune
Molecular Sciences, University
responses. These mechanisms have been targeted to prevent epileptogenesis in animal models. Favourable eff ects of Eastern Finland, and
have been obtained using immunosuppressants, antibodies blocking adhesion of leucocytes to endothelial cells, Department of Neurology,
gene therapy driving expression of neurotrophic factors, pharmacological neurostimulation, or even with Kuopio University Hospital,
Kuopio, Finland (Prof
conventional antiepileptic drugs by administering them before the appearance of genetic epilepsy. Further studies
A Pitkänen MD); and The Nencki
are needed to clarify the optimum time window and aetiological specifi city of treatments. Questions related to Institute of Experimental
adverse events also need further consideration. Encouragingly, the recent experimental studies emphasise that the Biology, Polish Academy of
complicated process of epileptogenesis can be favourably modifi ed, and that antiepileptogenesis as a treatment Sciences, Warsaw, Poland
indication might not be an impossible mission.
Correspondence to:Prof Asla Pitkänen, A I Virtanen Introduction
Defi nitions
Epilepsy is one of the world’s oldest recognised The term epileptogenesis is most often associated with Finland, PO Box 1627, FIN-70 211 disorders, fi rst described by Hippocrates in the 5th the development of symptomatic (acquired) epilepsy that Kuopio, Finland
century BC.1 At present, around 50 million people presents with an identifi able structural lesion in the brain.4 worldwide have active epilepsy with continuing seizures Some studies suggest that epileptogenesis also occurs in that need treatment, and 30% of patients are drug genetic epilepsies, in which it is regulated, for example, by refractory.2 Nearly 90% of epilepsy cases are in low- developmental programming of gene expression leading income countries, and in India, for example, the total to abnormal circuitry during maturation.5cost for an estimated 5 million cases of epilepsy has Currently, the terms epileptogenesis or latency period been shown to be equivalent to 0·5% of the gross are used synonymously as operational terms to refer to a national product.2 Europe has been estimated to have period that begins after the occurrence of insult (eg, 6 million patients with active epilepsy, and the annual traumatic brain injury [TBI] or stroke), or even during the European health costs associated with epilepsy are over insult (prolonged febrile seizure, status epilepticus [SE], €20 billion.3 In addition to the cost, the social burden or encephalitis), and ends at the time of the appearance of associated with the disease and the two-to-three-times the fi rst spontaneous seizure. Epileptogenesis refers to a increased risk of death mean that there is an urgent dynamic process that progressively alters neuronal need to fi nd ways to prevent the disease in individuals excitability, establishes critical interconnections, and at risk.
perhaps requires intricate structural changes before the cient ways to prevent fi rst spontaneous seizure occurs.6 These changes can epileptogenesis are genetic counselling or prevention include neurodegeneration, neurogenesis, gliosis, axonal of primary epileptogenic injury, for example, by wearing damage or sprouting, dendritic plasticity, blood–brain a helmet while riding a bike. In 2011, the prevention of barrier (BBB) damage, recruitment of infl ammatory cells epilepsy in patients at risk after acquired injury remains into brain tissue, reorganisation of the extracellular an unmet medical need worldwide. However, there matrix, and reorganisation of the molecular architecture have been recent developments in the modelling of of individual neuronal cells.7epileptogenesis after genetic or acquired conditions in Importantly, recent experimental and patient data mice and rats, which increase the clinical relevance of suggest that molecular and cellular changes triggered these models. By use of these animal models, large- by an epileptogenic insult can continue to progress after scale molecular profi ling studies have provided clues to the epilepsy diagnosis, even though they might the mechanisms that can contribute to formation of qualitatively and quantitatively diff er at various phases seizure-generating (ictogenic) neuronal circuits. Finally, of the epileptic process.8,9 These neurobiological data several laboratories have made attempts to target these raise the question of whether the term “epileptogenesis” mechanisms in clinically relevant experimental study should be extended to also include disease progression.10 designs, and some of these have shown favourable Thus, not only the prevention or delay of epilepsy but antiepileptogenic eff ects. We review and discuss these also seizure modifi cation (less frequent or shorter studies to identify unsolved problems needing attention seizures, milder seizure type, change from drug- before the current proof-of-principle studies are taken resistant to drug-responsive) and even cure would be to preclinical antiepileptogenesis trials or even to considered to be clinically relevant endpoints for the clinic.
antiepiletogenesis studies. Consequently, the window Vol 10 February 2011
for any search for treatment targets and for the initiation select the candidate mechanism to be tested in vivo in of antiepileptogenic treatments would extend beyond proof-of-principle experiments?
the latency phase to also cover the epilepsy phase
(fi gure). Moreover, because epilepsy can link to several Transcriptomics
comorbidities such as memory or emotional impairment,
The introduction of methods to analyse gene expression comorbidity modifi cation is one aspect that could be at the whole transcriptome level in the mid-1990s raised monitored in antiepileptogenesis studies.
expectations for the prompt discovery of molecular In line with emerging neurobiological data, we use the mechanisms of epileptogenesis, which would allow term epileptogenesis to cover both the latency phase and researchers to single out targets for antiepileptogenic the epilepsy phase, and we discuss the implications for therapies (table 1).11–21 This hope has not yet been target identifi cation and treatment.
fulfi lled. Only a few studies have been designed to specifi cally study the latency period or time period after Identifi cation of molecular mechanisms
If we consider epileptogenesis to be the result of circuitry Furthermore, the analysis of transcriptomic data to reorganisation that can occur either at the synaptic or identify common epileptogenic mechanisms in network level, a critical question is: what molecular diff erent preparations is a challenge. This relates to use pathways are involved in epileptogenic plasticity and how of diff erent array platforms, normalisation algorithms, can we identify them? Because they are likely to be or cutoff points for selecting regulated genes, use of multiple and diverse, what reasoning should be used to diff erent animal species and strains,22 analysis of diff erent brain structures,12,15,22 use of variable insults to trigger epileptogenesis, selection of timepoints for Intervention points for antiepileptogenesis tissue sampling after the insult, and characterisation of epilepsy phenotype at the time of sampling.15,17 Consequently, when we compared the lists of genes regulated during epileptogenesis, only 46 (7·4%) of 624 regulated genes were found to have abnormal regulation in more than one study. Such genes with a known function are summarised in table 2. 17 (37%) of these 46 genes were regulated in both SE and TBI models, indicating similarity in molecular events during epileptogenesis between diff erent conditions.
Only a few reports have studied changes in the transcriptome throughout epileptogenesis from early after the insult to the chronic phase.11,12,15,17,18,23 Individual genes show diff erent expression profi les. Some genes are regulated throughout the latent phase and also after epilepsy diagnosis, whereas others are only transiently regulated. We can also observe waves of orchestrated gene expression, because clusters of genes show similar Altered gene expression by injury and consequent seizures patterns of expression changes over time. These Epigenetic and post-transcriptional modulation of gene expression (histone observations might be relevant for new therapeutic modification, DNA methylation, siRNA, miRNA) by injury and consequent seizures strategies. First, the timing could be a crucial factor for a successful intervention if abnormalities had to be targeted at the time of occurrence. Second, because Figure: Mechanisms and intervention points during epileptogenesis
some types of molecular dysfunction that are present in Epileptogenesis includes both the latency period between the insult and occurrence of seizures, and the progression the latent period persist into the chronic phase, it might of epilepsy. In an optimum case, treatment results in cure associated with the reversal of epileptogenic pathological be reasonable to extend antiepileptogenic interventions changes. The natural course of epileptogenesis and the screening of mechanisms of epileptogenesis can be beyond the time of epilepsy diagnosis. In the latter infl uenced by genetic predisposition, epigenetic mechanisms, and the use of AEDs. Data available (table 1) suggest that injury-induced gene expression depends on the time of sampling. Additionally, diff erent patterns of gene scenario, a notable factor is that many antiepileptic expression can be observed, including genes constantly regulated following insult and those regulated only in drugs (AEDs; eg, levetiracetam, phenytoin, lamotrigine, specifi c time windows, which results in dynamic changes in the transcriptome over time. These data suggest that valproate), which would be administered in parallel with the target for antiepileptogenesis can vary over time. Moreover, polytherapy might be favoured over monotherapy. antiepileptogenic treatments, can also modify gene Similarly, the expression of a biomarker might vary at the time of investigation. Arrows indicate the potential timepoints for therapeutic interventions. Favourable eff ects have been found when treatments have been given expression.24,25 Finally, because some regulated genes either at early or later phases of the latency period, and even at the time of established epilepsy (table 1). can contribute to post-insult recovery that occurs in Pretreatment could be a clinically relevant intervention point, for example, before surgical interventions that carry a parallel with epileptogenesis (eg, after TBI), it is risk of brain ischaemia or haemorrhage. Status epilepticus or encephalitis are conditions in which antiepileptogenic important not to sacrifi ce their benefi cial eff ects while treatment can be started during the insult (ie, as co-administration with AEDs; insult-modifying treatment). BM=biomarker. AEDs=antiepileptic drugs. siRNA=small interfering RNA. miRNA=microRNA. Vol 10 February 2011
The next question is whether bioinformatics tools have or epilepsy be tested.26–36 Unfortunately, this is a very helped the analysis. Although over the past few years the laborious path, and relatively few of the leads obtained accessibility, quality, and user-friendliness of data mining from arrays have been systematically followed. Examples tools have improved, allowing more in-depth and include studies on the role of cystatin C (CST3),26,31,36 sophisticated interpretation of microarray data, the basic urokinase-type plasminogen activator (PLAU),32,33 secreted knowledge about the proteins encoded by genes aff ected phospho protein 1 (SPP1; formerly osteopontin),37 tweety by epileptogenesis is often lacking. It is not surprising homolog 1 (TTYH1),30 sodium channel type 7 subunit A that microarray data have triggered further studies on the (SCN7A),34 transforming growth factor β (TGFB) identifi ed proteins or pathways. Only after gaining signalling,27 prostaglandin G/H synthase 2 (PTGS2;
additional data on their function in the normal and formerly cyclo-oxygenase 2),38,39 ferritin (FTH or FTL),40 For DAVID Bioinformatic
diseased brain (including analysis of human tissue from complement activation,28 and proteolysis35 in Resources version 6.7 see
epilepsy surgery) can their involvement in epileptogenesis epileptogenesis. None of the genes identifi ed has yet led Status epilepticus (chemically induced)
Status epilepticus (induced by electrical stimulation)
Traumatic brain injury
Functional gene classes are shown as provided by authors of original publications. EEG=electroencephalogram. CA1=cornu ammonis 1. CA3=cornu ammonis 3. SAGE=Serial Analysis of Gene Expression. DAVID=Database for Annotation, Visualization and Integrated Discovery. *Indicates studies on which we did additional analyses: gene identifi ers or accession numbers were converted to offi analysed by use of the Biological Function FAT annotation chart option of DAVID Bioinformatic Resources version 6.7.20,21 †Note that platforms containing diff erent gene sets were used in various studies: CodeLink (Applied Microarrays, Tempe, AZ); Aff ymetrix (Santa Clara, CA); Illumina (San Diego, CA); Research Genetics (Huntsville, AL); Incyte Genomics (Palo Alto, CA).
Table 1: Gene functions most frequently regulated during epileptogenesis induced by status epilepticus or traumatic brain injury Vol 10 February 2011
to rigorous testing of antiepileptogenic approaches in brain-derived neurotrophic factor (BDNF) and preclinical studies.
neurotrophic tyrosine kinase receptor type 2 (NTRK2). Their concentrations are altered in experimental and/ Serendipity
or human epileptic tissue, and genetically modifi ed Interpretation of transcriptome alterations at the level NTRK2 regulates excitability in vivo in mice, whereas of functional gene groups or signalling pathways seems some studies suggest that a BDNF polymorphism more rewarding than focusing on individual genes might play a part in human epilepsy.41 Recently, Paradiso when attempting to pinpoint epileptogenic mechanisms. and colleagues42 tested the hypothesis that limiting This approach has highlighted gene groups with tissue damage and enhancing repair by neurotrophins relatively unspecifi c functions, such as those regulating alleviates epileptogenesis. These investigators triggered signal transduction or transcription, which can underlie SE with pilocarpine and 4 days after SE, rats received a any molecular process. Importantly, more specifi c unilateral hippocampal injection of a vector expressing functional gene groups that contribute to the generation fi broblast growth factor 2 (FGF-2) and BDNF. On the c network alterations already linked to basis of 20-day video electroencephalogram (EEG) epileptogenesis have also been detected. These include monitoring, there was no evidence that the treatment infl ammation, immune response, reaction to wounding, lowered the proportion of rats that developed epilepsy. synaptic transmission and plasticity, ion transport, However, a clear seizure-modifying eff ect was seen and channel and receptor function, and neurotransmitter FGF-2 and BDNF duotherapy reduced both the metabolism. To search for more specifi c targets, one can frequency and severity of spontaneous seizures. This match the transcriptome data with search terms in was associated with a normalised pattern of literature databases.
neurogenesis as well as preserved dendritic inhibition of granule cells by surviving hilar somatostatin As one tries to match the “omics” data with the literature Erythropoietin also has neurotrophic eff ects, in addition database and extract specifi c targets from the articles to its role in antiapoptotic, antioxidant, and anti-published in that category, there is evidence that within the “epileptogenesis and plasticity” category, erythropoietin itself has not been revealed as a target by neurotrophins show remarkable changes during molecular profi ling on the basis of the data available to epileptogenesis in diff erent animal models, especially date, its functions cover several diff erentially regulated Genes (n)
cial gene symbol*
C1QA, GABRD†, NPY, GRIA2, SLC6A1, SYT4, NPTX2†, APOE, GRIN2C, CAMK2G, GABRA5, GABARAP KCNC2, GABRD†, NPY, GRIA2, SCN3B, GRIN2C, CAMK2G, SCN2A, GABRA5, CAMK2B†, CACNG2, KCNK1 GABRD†, NPY, GRIA2, SLC6A1, SYT4, NPTX2†, APOE, GRIN2C, GABRA5, GABARAP PTPN6†, PPP1R9B, PTGS2, APOE, GRN†, CLU, CD81, IL6R†, SPARCC1QA, PTPN6†, C1QB, GRIN2C, CLU, IL6R†, CTSB, C1QC C1QA, C1QB, CLU, IL6R†, CTSS, C1QC, CD74, B2M PTGS2, NPY, SLC6A1, S100B†, GABRA5, IL6R†, CALB1 PTGS2, APOE, CLU, DNAJC5†, IL6R†, CTSB, CD74 C1QA, C1QB, CLU, IL6R†, C1QC, CD74 PTGS2, GRIA2, SLC6A1, APOE, GRIN2C, CALB1 C1QA, C1QB, CLU, C1QC, CD74 PTGS2, SLC6A1, S100B†, GABRA5, CALB1 C1QA, C1QB, CLU, IL6R†, C1QC RPS27, NPY, S100B†, CD81, CD74 PPP1R9B, APOE, CD81, IL6R†, CD74 C1QA, C1QB, CLU, C1QC PTGS2, APOE, GRIN2C, CALB1 C1QB, PTGS2, SLC6A1, IL6R† Genes that were regulated in at least two studies presented in table 111–19 were assigned to functional classes using the Biological Function FAT option of DAVID Bioinformatic Resources 6.7.20,21 DAVID=Database for Annotation, Visualization and Integrated Discovery. *See HUGO Gene Nomenclature Committee website for full gene names. †Genes For the HUGO Gene
regulated by both status epilepticus and traumatic brain injury.
Nomenclature Committee
website see http://www.
Table 2: Genes belonging to diff erent functional gene classes Vol 10 February 2011
gene classes revealed by transcriptomics.43 Chu and co- reduced the behavioural severity of seizures workers44 induced SE in rats with lithium-pilocarpine compared with vehicle alone.
and administered erythropoietin starting immediately The third NSAID that has been tested is SC58236, a after SE cessation for 7 days. The proportion of rats that selective inhibitor of PTGS2.38 SE was triggered by developed epilepsy in the treatment group was no electrical stimulation of the angular bundle and allowed diff erent to that in the vehicle group. However, the to continue for 4 h. SC58236 treatment was then started seizure frequency and duration as assessed by video and continued for 7 days. Animals underwent monitoring were reduced in the erythropoietin group continuous video-EEG monitoring for up to 35 days compared with the vehicle group. This was associated after SE. SC58236 treatment did not delay the latency to with reductions in BBB damage, neurodegeneration, the occurrence of spontaneous seizures or the microglial activation, development of ectopic granule proportion of rats that developed epilepsy. It did not cells in the hilus, and gliosis.
aff ect seizure duration and had no eff ect on the severity of neuro Infl ammation and immune response For most of the other functional categories, it is diffi to extract a single specifi c target. For example, in the Infl ammatory cell adhesioncategory of infl ammation and immune response, various Fabene and colleagues53 showed that integrin α4/β1 and compounds that inhibit diff erent infl ammatory pathways P-selectin glycoprotein ligand 1 are the mediators of have been used (table 3).38,42,44–61 Lukasiuk and Sliwa50 leucocyte adhesion to endothelial cells in cerebral blood investigated the eff ect of tacrolimus on SE-induced vessels after pilocarpine-induced SE. This was proposed to epileptogenesis. Tacrolimus is an immunosuppressant result in increased leucocyte extravasation, cerebral that binds to intracellular immunophilins. The infl ammatory response, leakage of the BBB, impaired K+ tacrolimus–immunophilin complex inhibits the activity buff ering, and epileptogenesis. They hypothesised that of calcineurin, resulting in the inability of T cells to preventing leucocyte adhesion by using an integrin-α4-respond to activation by antigen-presenting cells. specifi c monoclonal antibody (α4 MAb) after SE would Consequently, no functional cytokine response occurs.62 prevent epileptogenesis. To address this question, they Tacrolimus was started 24 h after SE and continued for induced SE in C57BL/6 mice and administered α4 MAb 2 weeks. On the basis of 4-week continuous video-EEG starting at 1 h after SE. Treatment was continued every monitoring, no positive eff ects were observed on the other day for 20 days. On the basis of video-EEG monitoring animals that developed epilepsy, latency to the fi rst for 5–20 days after SE, the latency to the appearance of seizure, seizure frequency, or seizure type.
spontaneous seizures was similar in the α4 MAb and Non-steroidal anti-infl ammatory drugs (NSAIDs) have vehicle groups. Also, the duration of seizures was not been used in preclinical antiepileptogenesis trials on the altered by treatment. However, the seizure frequency as basis of their ability to inhibit PTGS2. PTGS2 inhibition assessed during α4 MAb therapy was reduced from about reduces activation of prostanoid pathways, resulting in 0·8 to 0·2 seizures per day. Importantly, mice treated with reduced microglial activation, leucocyte infi ltration, α4 MAb had less severe BBB damage at the acute phase suppressed cytokine release and oxidative stress, and (18–24 h after SE) and reduced chronic neurodegeneration reduced neurodegeneration.62 The fi rst NSAID tested in (30 days after SE). In addition, their exploratory behaviour epileptogenesis models was celecoxib. Jung and was better preserved than in the vehicle group. Thus, colleagues51 induced SE with lithium-pilocarpine in adult unlike other treatments targeted to alleviate infl ammatory rats and started celecoxib 1 day after SE, and then response, α4 MAb treatment had both seizure-modifying continued the treatment for 42 days. On the basis of video and comorbidity-modifying eff ects on epileptogenesis.
monitoring of seizures, the treatment did not reduce the
proportion of rats that developed epilepsy. However, Epigenomics
celecoxib treatment decreased the seizure frequency and There is some evidence that gene expression triggered by
duration. In addition, celecoxib reduced hippocampal epileptogenic brain insults occurs in temporally
neurodegeneration and microglial activation, and coordinated waves. This has been proposed to be
inhibited both the generation of ectopic granule cells in orchestrated by regulation of transcription by specifi c
the hilus and new glia in CA1.
transcription factors. One such transcription factor is Parecoxib, another NSAID, belongs to the second inducible cyclic AMP early repressor (ICER), which has generation of selective PTGS2 inhibitors. Polascheck and been suggested to play a part in epileptogenesis because it co-workers52 administered parecoxib for 18 days after suppresses kindling (repeated subthreshold stimulation pilocarpine-induced SE. Several weeks after SE, rats culminating in the occurrence of generalised seizures).63,64 underwent video-EEG monitoring to detect the Another candidate mechanism for the clustering of post-occurrence of spontaneous seizures. No reductions in injury gene expression relates to the epigenetic regulation the occurrence of epilepsy or frequency or duration of of transcription by alterations in DNA methylation or seizures were observed. However, parecoxib slightly histone modifi cations (table 4).65–78 Vol 10 February 2011
Mechanism of
Time of administration Antiepileptogenesis
of treatment
Seizure modifi cation
(>90 min), continued for 17 days (twice daily) Vol 10 February 2011
Mechanism of
Time of administration
of treatment
Seizure modifi cation
CKO=conditional knockout. mTOR=mammalian target of rapamycin (serine-threonine protein kinase). SE=status epilepticus. COX-2=cyclo-oxygenase 2. MAb=monoclonal antibody. FGF=fi broblast growth factor. BDNF=brain-derived neurotrophic factor. NTRK2=neurotrophic tyrosine kinase receptor 2. FPI=fl uid-percussion injury. TBI=traumatic brain injury. ECS=electroconvulsive shock. NKCC1=sodium-potassium-chloride co-transporter. ··=no data/not applicable. Table 3: Studies of the eff ects of various treatments on epileptogenesis induced by status epilepticus or traumatic brain injury
Interest in the role of histone acetylation as a possible From phenotype to genotype to target
therapeutic target for epileptogenesis was increased by the Epilepsy is a common comorbidity in many neurological discovery that valproate, a widely used AED, is a histone diseases caused by a wide range of genetic factors. One deacetylase (HDAC) inhibitor.79 In particular, HDAC approach to reveal novel epileptogenic mechanisms is to inhibition explains why valproate blocks seizure-induced understand why a mutation in a disease-causing gene is neurogenesis, which is one of the changes in the neuronal associated with an epilepsy phenotype in mice. This is network triggered by various epileptogenic stimuli (ie, SE, particularly interesting if the mutated gene is not directly TBI) as well as by single brief seizures.80 Valproate also associated with the expression of ligand or voltage-gated regulates the expression of several genes that regulate ion channels that regulate neuronal excitability, as is seen synaptic transmission.81 Whether epigenetic mechanisms in inherited epileptic channelopathies.
contribute to the antiepileptic eff ect of valproate is debatable Probably the most convincing evidence to support the because other HDAC inhibitors do not suppress seizures.72,73 idea of searching for novel epileptogenic mechanisms by Another question is whether valproate would prevent investigating diseases in which epilepsy is “just a acquired epileptogenesis after SE. So far, there is no comorbidity” comes from the study of tuberous sclerosis, evidence that valproate started during the latency period or which is caused by an inactivating mutation in either the after the initiation of spontaneous seizures would have any TSC1 or TSC2 gene, which encode hamartin and tuberin, eff ect on the epileptogenic process if its eff ect on the respectively. The generation of animals with conditional severity of the epileptogenic insult itself (ie, SE) is knockout of Tsc1 in astrocytes resulted in disinhibition of excluded.10,82 Another AED with known epigenetic properties the serine/threonine protein kinase mammalian target is phenobarbital, although these eff ects have been described of rapamycin (mTOR) pathway, causing structural and only in extraneuronal tissue.83 As for valproate, there is no behavioural abnormalities resembling tuberous sclerosis convincing evidence that phenobarbital would block in human beings, including the development of epileptogenic circuitry reorganisation without aff ecting the spontaneous seizures. Administration of rapamycin, an insult itself. However, it is too early to draw any conclusion mTOR inhibitor, before seizure occurrence reversed the about the antiepileptogenic potential of epigenetic hippocampal abnormalities (ie, pyramidal cell dispersion modulation, because only SE models with a very severe and astrogliosis). Moreover, epileptogenesis was initial epileptogenic insult have been tested, and the study suppressed. When treatment was started after the designs have not been tailored to address the appearance of spontaneous seizures, a positive, albeit epigenetic modulation.
less dramatic, eff ect was still observed.45 The results have Vol 10 February 2011
Experimental model
DNA methylation
Increased DNA methyltransferase-1 expression in reactive astrocytes at days 4 and 7 Decreased DNA methylation in microglia/macrophages at days 1 and 2 Increased DNA methylation at reelin promotor Histone methylation
Decreased histone H3 methylation at 6 h, 24 h, and 72 h Histone acetylation
Decreased histone H3 acetylation at 6 h and 24 h TBI (lateral fl uid percussion injury model) in rats Decreased histone H3 acetylation at 24 h; HDAC inhibition prevents decrease in H3 acetylation and reduces microglia infl ammatory response after TBI HDAC inhibition enhances learning and memory after TBI Decreased histone H3 acetylation at 6 h and 24 h; HDAC inhibition diminishes decrease in H3 acetylation and neurodegeneration, and improves recovery Valproate, but not SAHA, increases H3 and H4 acetylation, decreases neurodegeneration, and improves motor skills and cognitive functions after TBI Valproate but not trichostatin A increases seizure threshold Increased histone H4 acetylation at c-fos and BDNF promoters and decreased histone H4 acetylation after seizures Intraperitoneal pilocarpine-induced SE in rats Decreased H4 acetylation at GluR2 promoter and increase at BDNF P2 promoter after SE Intraperitoneal kainate-induced SE in mice Increased histone H4 acetylation at 0·5–6 h after SE Histone phosphorylation
Intraperitoneal pilocarpine or kainate-induced SE in mice Increased histone H3 phosphorylation Intraperitoneal kainate-induced SE in mice Increased histone H3 phosphorylation at 0·5 h after SE TBI=traumatic brain injury. CCI=controlled cortical impact. SE=status epilepticus. HDAC=histone deacetylase. SAHA=suberoylanilide hydroxamic acid. BDNF=brain-derived neurotrophic factor. GluR2=glutamate receptor 2.
Table 4: Studies of epigenetic modifi cations during epileptogenesis
been extended to acquired epilepsy models by Zeng and the generation of an epileptogenic network. Some studies colleagues,48 who showed that administration of suggest that, in murine models of Alzheimer’s disease, rapamycin at 24 h after kainate-induced SE leads to the oligomeric amyloid β might directly aff ect the voltage-development of milder epilepsy. Importantly, rapamycin gated or ligand-gated neuronal ion channels’ modulation had favourable eff ects even when started after established of neuronal excitability and axon potential fi ring.88,89 epilepsy. Huang and co-workers49 administered rapamycin Other data show that enzymes processing amyloid to rats that had spontaneous seizures after pilocarpine- precursor protein could also use sodium-channel induced SE. Rapamycin administration suppressed subunits as substrates, resulting in hyperexcitability.90 seizures, and the study also suggested that mossy-fi bre Unfortunately, preclinical trials with compounds that sprouting was diminished. After cessation of rapamycin reduce amyloid-β concentrations (ie, lithium, valproate, treatment, which itself has not been shown to have any or γ-secretase inhibitors) have not reported the eff ects of anticonvulsant eff ect,48,84,85 seizures were re-established. chronic treatments on seizures.
These studies show that, on the basis of the identifi cation In a fragile-X murine model with knockout of the Fmr1 of the epileptogenic pathway and characterisation of its gene, seizure generation seemed to be related to a role in epilepsy-associated network reorganisation, one reduction in fragile-X mental retardation protein-mediated can indeed design treatments that modify the silencing of group I metabotropic glutamate receptor epileptogenic process both in genetic and acquired (mGluR) activation-induced dendritic mRNA. This led to conditions, and even at diff erent phases of the the discovery that co-reduction in mGluR5 expression repaired most of the structural and functional Insight into novel epileptogenic mechanisms has also abnormalities in Fmr1 knockout mice, including dendritic been revealed by investigating animal models of spine density and susceptibility to audiogenic seizures.91,92 Alzheimer’s disease and fragile-X syndrome,86,87 in which Whether the use of an mGluR5 antagonist prevents the epilepsy can be a comorbidity. The pathological proteins development of the epileptogenic network/synaptic produced by mutated genes in these disease models do reorganisation in patients with fragile-X syndrome vary, and data are just emerging on their contribution to remains to be explored. Vol 10 February 2011
The use of genetic information from patients with insult alleviation (ie, reduction in the severity and neurological diseases with epilepsy as a comorbidity is an duration of SE itself by AEDs) rather than related to a exciting platform to reveal novel epileptogenic true antiepileptogenic eff ect. Consequently, even if the mechanisms. These data show that in addition to the most recent data on the eff ects of AEDs on the occurrence of diverse network alterations, for example epileptogenic process are considered,61,82,101 there is no after SE or TBI, more localised changes in dendritic spines evidence that the use of AEDs would be antiepileptogenic or the axon initial segment can also be used to locate the in adult rodents.
epileptogenic microenvironment.87,93 Fortunately, whether However, some recent data suggest that levetiracetam the drug targets revealed by these studies have an eff ect and ethosuximide could modify the epileptogenic process beyond the specifi c syndrome can be tested.
in immature animals with genetic predisposition to epilepsy, if the treatment is started before the expression Chemistry–biology interphase target-independent
of an epilepsy phenotype. Spontaneously epileptic rats discovery
(zi/zi, tm/tm double mutant) develop air-puff -induced Minozac (derived from inactive aminopyridazine) was tonic convulsions at approximately 8 weeks of age and discovered by using a molecular target-independent absence seizures by about 12 weeks. Yan and colleagues54 discovery paradigm.94 The goal was to fi nd a small administered levetiracetam during weeks 5–9, before the molecule that suppressed the increased production of occurrence of seizures. They found that the frequency proinfl ammatory cytokines in glial cultures using disease- and duration of air-puff -induced tonic seizures was relevant endpoints rather than designing a compound reduced in the levetiracetam group compared with targeting a specifi c molecular pathway. This was combined vehicle-treated rats. Also, the number and duration of with hierarchical biological screens for oral bioavailability, electrographically recorded absence seizures was reduced toxicity, brain penetrance, and stability of candidate in the levetiracetam-treated rats.
molecules before testing their effi WAG/Rij rats develop absence seizures at approximately of brain disorders. Recently, Chrzaszcz and colleagues60 3 months of age. Blumenfeld and co-workers55 started used the closed-skull midline impact model of TBI in the administration of ethosuximide at postnatal day 21 mice and administered Minozac at 3 h or 6 h after injury. in these rats. Rats in which ethosuximide was 1 week after TBI, Minozac-treated mice showed less discontinued at the age of 5 months had reduced seizure susceptibility to electroconvulsive shock-induced seizures frequency when assessed with long-term EEG at age than did sham-operated mice (table 3). Whether Minozac 5–8 months. The duration of remaining seizures was treatment prevented the long-term increase in seizure not altered compared with the vehicle group. susceptibility and occurrence of late seizures remains to Unfortunately, they did not mention whether epilepsy be explored. Whether the chemistry–biology interface had been completely prevented in any of the rats. The would provide a faster throughput approach for investigators showed that abnormalities in the SCN1A antiepileptogenesis drug discovery than hypothesis- and SCN8A sodium channels as well as in potassium/ driven “omics” approaches also remains to be seen.
sodium hyperpolarisation-activated cyclic nucleotide-gated channel 1, as assessed by immunohistochemistry, AEDs as antiepileptogenic treatments
were normalised in the ethosuximide group.
The fi rst antiepileptogenesis trial in human beings was
done more than 60 years ago.95 It attempted to prevent Proconvulsants
epileptogenesis after TBI using phenytoin. Several other Many preclinical and clinical studies have shown that
AEDs, including phenobarbital, carbamazepine, and drugs designed to prevent epileptic seizures and suppress
valproate in monotherapy or polytherapy, as well as non-
neuronal activity (ie, AEDs) do not prevent acquired AEDs such as magnesium sulphate and glucocorticoids, epileptogenesis.96,98 Recent data have provided surprising have been tested since then. These studies have failed to evidence that the administration of the proconvulsant provide evidence that the use of AEDs (or other drugs atipamezole or rimonabant could have favourable compounds) during epileptogenesis would have eff ects on antiepileptogenesis after epileptogenic brain favourable antiepileptogenic eff ects in patients.96,97 The analysis of data from experimental studies using We induced SE with electrical stimulation of the AEDs as candidate antiepileptogenic agents is amygdala and 1 week later started atipamezole treatment challenging.98 This relates to the use of SE as an with subcutaneous osmotic minipumps for 9 weeks.56 epileptogenic insult. Many studies have now shown that Atipamezole treatment had no eff ect on the proportion of the shortening of SE by AEDs favourably modifi es the rats that developed epilepsy. However, the seizure epileptogenic process.99,100 Therefore, unless the eff ect of frequency was reduced from about 8·4 to 0·7 seizures AEDs on the duration and severity of SE is carefully per day. Atipamezole-treated rats also had milder controlled and quantifi ed, it is diffi cult to determine hippocampal neurodegeneration and less intense mossy- whether the few positive eff ects on latency, seizure fi bre sprouting than did the vehicle group. This was the frequency, or seizure duration were related to the initial fi rst study to show that SE-induced epileptogenesis can Vol 10 February 2011
be favourably modifi ed by pharmacotherapy, with an None of the studies has taken into account the experimental design in which the treatment eff ect on the qualitatively or quantitatively diff erent mechanisms of severity of the epileptogenic insult was excluded and the epileptogenesis between individuals at a given time, assessment of effi cult because we lack reliable biomarkers to pinpoint the phase of epileptogenesis in individual More recently, Echegoyen and colleagues57 induced animals. Moreover, these studies have typically been epileptogenesis by lateral fl uid-percussion-induced TBI, proof-of-principle studies, which have been done in a and administered rimonabant as a single injection 2 min relatively small number of animals to show the effi after injury. The threshold for kainate-induced seizures in the whole animal group, without any attempts to
was assessed at 6 weeks after TBI. The reduction in power the study to make subgroup analyses. Therefore,
latency to kainate-induced seizures was prevented by there is a possibility of false-negative results.
rimonabant. Also, the total time spent in seizures after
kainate administration was reduced in the rimonabant Monotherapy versus polytherapy
group compared with the vehicle group. Importantly, no As the molecular and cellular studies have shown, acquired
positive eff ect was found if rimonabant was administered
epileptogenesis is regulated by multiple molecular 20 min after TBI. The same group also showed that a pathways. One could hypothesise that modulating several similarly favourable eff ect could be achieved in a pathways at the same time or sequentially would be a hyperthermia model of prolonged seizures in immature more benefi cial strategy than any single-bullet strategy. rats if the treatment was initiated 2 min after the start of An antiepileptogenic eff ect can be shown by using seizure induction.59 Dudek and colleagues58 extended the relatively specifi c treatments, such as rapamycin for studies on rimonabant to an SE model in adult animals. targeting the mTOR pathway or a specifi c monoclonal Interestingly, if rimonabant was given after the fi rst antibody to integrin α4 (table 3). However, the blockage of electrographic seizure during the kainate-induced SE (ie, epileptogenesis was not complete, and thus the polytherapy 1 min after SE onset), it had no eff ect on the proportion hypothesis remains viable. The closest approach to of rats that developed epilepsy or seizure frequency when polytherapy was made in the FGF-2–BDNF duotherapy assessed during the fi rst 10 weeks after SE.58 study, which resulted in multiple eff ects, including both Even though the compounds seem to have diff erent neurogenesis and survival of interneurons. However, the mechanisms of action (atipamezole is an antiepileptogenic eff ect was partial. Finally, both α4 MAb α -noradrenergic antagonist and rimonabant is a and FGF-2–BDNF gene therapy show effi cannabinoid receptor 1 antagonist), it remains to be systemic pilocarpine model, even though one is given found whether there is convergence in the molecular systemically (α4 MAb) and one directly to the hippocampus mechanisms or cellular location of the eff ects of these (gene therapy). This is of particular interest as recent compounds. Furthermore, whether the eff ects are model studies show that both the peripheral component of infl ammation (leucocyte stimulation) and the central cholinergic eff ect contribute to mechanisms that trigger What should antiepileptogenic treatment
SE after pilocarpine administration. Currently, no look like?
preclinical experiments have investigated whether a Diff erences across conditions and patients
combination of diff erent approaches is more favourable As mentioned earlier, there are some similarly regulated than any of the treatments alone.
genes in diff erent conditions (eg, SE and TBI) during epileptogenesis. However, even considering the bias related When to start and how long to continue
erent array platforms or other As in patients, the progression of the epileptogenic methodological issues, most analyses of epileptogenesis in process varies between the conditions and even between rodents suggest diff erences in the pattern of molecular diff erent animals with a similar epileptogenic trigger. In changes as well as in the time course and severity of the addition, the altered gene expression progresses in waves. cellular alterations between conditions, such as electrically Should this aff ect the timing of the treatment approach or chemically induced SE or TBI.102 Even the diff erent SE (fi gure)? Table 3 summarises the time of initiation of models diff er substantially. Moreover, in each condition candidate antiepileptogenic therapies in experimental there is substantial inter-animal variability. Experimental models. In all cases with a benefi cial eff ect, the treatment antiepileptogenesis studies have mostly used electrically or was initiated within 7 days after the insult, suggesting chemically induced SE as an epileptogenic trigger (table 3). that the therapeutic time window can be several days In a few reports, TBI or a genetically abnormal mTOR rather than minutes or hours, at least in SE models. In pathway serves as an epileptogenic trigger. Benefi cial eff ects most of the studies, the time window was not specifi cally have been achieved by administering rapamycin, α4 MAb, investigated. One exception was a study in a TBI model, or FGF-2–BDNF combination gene therapy (table 3). Only in which the administration of the cannabinoid antagonist rapamycin has shown effi cacy in diff erent conditions (ie, rimonabant prevented the lowering of seizure tuberous sclerosis, cortical dysplasia, and post-SE models).
susceptibility only if it was given at 2 min, but not at Vol 10 February 2011
20 min, after TBI. No similar eff ect was found in an SE model.58 Whether this indicates a true diff erence in the Search strategy and selection criteria
therapeutic time window for antiepileptogenesis between We searched all PubMed articles published up to TBI and SE models remains to be studied. Furthermore, September, 2010, with terms “epileptogenesis” and the duration of treatment has varied from a single “antiepileptogenesis”. For transcriptomics in epileptogenesis, we administration to up to 9 weeks. For clinical trials, the did searches using the following terms: “microarrays and extent of the therapeutic window is a crucial issue, as is epileptogenesis”, “transcriptome and epileptogenesis”, the question of how specifi c is the window for each “microarrays and traumatic brain injury”, and “transcriptome and traumatic brain injury”. Articles describing alterations in gene expression at timepoints longer than 4 days post-insult Conclusions
were selected. For epigenetics, a search for “epigenetic” and The molecular and cellular data on processes that underlie “epilepsy” was done. For preclinical treatment trials, only those epileptogenesis suggest a wide spectrum of treatment studies in which the therapy was initiated after the epileptogenic targets. Therefore, is it even realistic to believe that the insult were included. Articles were also identifi ed through modulation of one target pathway would be searches of the authors’ own fi les. Only articles published in antiepileptogenic, unless treating specifi c syndromes such as tuberous sclerosis? Should we focus on target selectivity versus pathophysiological process selectivity in multifactorial disorders like post-SE or post-TBI when solved will facilitate the movement from proof-of-epileptogenesis? Do “omics” provide a category of principle studies to preclinical trials. Another challenge biological mechanisms that can be set up as endpoints for is the design of compounds with acceptable bioavailability biological screens of selective molecular chemotypes? to achieve stable brain concentrations, sometimes for a In addition, how can we cross over from a proof-of- longer period of time. Forming preclinical consortia principle trial to the preclinical testing of candidate between the laboratories will make it realistic to do antiepileptogenic treatments? Many components of the randomised and blinded preclinical trials with suffi infrastructure for preclinical testing are already numbers of animals to show effi available. For example, we have a wide range of clinically endophenotypes and, thus, reduce the likelihood of relevant models and many laboratories have long-term false-negative or false-positive data. Finally, overcoming video-EEG monitoring units that can provide the the publication bias (ie, by reporting negative data) will opportunity for more representative and reliable data save resources if repetition of unnecessary studies can acquisition. However, several challenges remain to be be avoided.
faced before translating the preclinical data to the clinic Even though many questions remain, particularly and some of the problems are similar to those discussed related to translation of preclinical data to the clinic, the for stroke and amyotrophic lateral sclerosis.103,104 For recent developments in modelling, target identifi cation, example, is the prevention of the lowering of seizure and data from proof-of-principle antiepileptogenesis threshold a valid outcome measure in models, whereby preclinical studies provide encouraging signals that the only a low proportion of animals develop spontaneous prevention of the complicated process of epileptogenesis
seizures? Should the favourable eff ect be shown in is not an impossible mission, but can indeed be
more than one model to represent the diff erent favourably modifi ed.
conditions? Should we aim to identify a silver-bullet Contributors
therapy for large patient populations with heterogeneous
Both authors contributed equally to the conception, design, literature epileptogenic triggers, or accept the possibility of a search, and writing of this Review.
need for personalised treatments? Which preclinical Confl icts of interest
outcome measures show the strongest indications to We declare that we have no confl icts of interest.
move to the clinic, and eventually, to labelling a Acknowledgments
compound as antiepileptogenic? Are the eff ects on This study was supported by the Academy of Finland (AP), The Sigrid
Juselius Foundation (AP), CURE (AP), PMSE grant NN301162135 (KL), comorbidities, such as alleviation of memory and and statutory funds of the Nencki Institute (KL).
behavioural abnormalities, an extra bonus for judging References
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