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In vitro cytotoxicity, cell cycle arrest, and antioxidation studies of ruthenium(II) complex [Ru(dmb)2(AHPIP)](ClO4)2
Neurotherapeutics (2014) 11:319–323
DOI 10.1007/s13311-014-0261-6
REVIEW
Molecular Biomarkers of Epileptogenesis
Katarzyna Lukasiuk & Albert J. Becker
Published online: 25 February 2014
# The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Epileptogenesis, a process leading to a reduced
threshold for seizures after transient brain insults, as well as
the mechanisms underlying the propensity to generate spontaneous epileptic seizures, are highly dynamic processes. Biomarkers—objective measures of biological processes—would
be excellent tools for monitoring epileptogenesis and the
dynamics of increased seizure propensity, as well as the potential to interfere, for example pharmacologically, with these
key pathological aspects of epilepsy. Molecular biomarkers
have revolutionized therapies, as well as response prediction
and monitoring of therapies in other biomedical fields. However, high-impact molecular biomarkers are still not available
in the context of epilepsy. Several factors, such as the large
heterogeneity of epileptic syndromes and their underlying
pathological patterns, as well as the limited availability of
tissue samples, represent a particular challenge to the development of molecular biomarkers in epileptogenesis and epilepsy. However, substantial technical progress has been made
recently with respect to biomarker characterization and monitoring by large throughput analysis on the genomic, mRNA,
and proteomic levels, starting from minute amounts of brain
tissue or body fluids, for example cerebrospinal fluid, blood,
serum, or plasma. Given the substantial cellular- and networklevel functional pathophysiology involved in epilepsy, it may
be beneficial in the future to combine molecular analysis with
other methods, such as imaging and electrophysiological
biomarkers.
K. Lukasiuk (*)
The Nencki Institute of Experimental Biology, Polish Academy of
Sciences, 3 Pasteur Street, 02 093 Warsaw, Poland
e-mail: k.lukasiuk@nencki.gov.pl
A. J. Becker
Department of Neuropathology, University of Bonn Medical Center,
Bonn, Germany
e-mail: Albert_Becker@uni-bonn.de
Key Words Cerebrospinal fluid . epilepsy . plasma .
seizures . traumatic brain injury
The Biomarker Concept
Biomarkers are defined as “measures” of disease processes, that
is, factors that can be objectively determined and interpreted as
indicators of pathogenic processes, such as those related to
epileptogenesis and ictogenesis [1]. In the context of
epileptogenesis, molecular biomarkers would be particularly
helpful in identifying patients with an increased propensity of
developing chronic spontaneous seizures after an epileptogenic
insult. Furthermore, molecular biomarkers reflecting the presence, type, and severity of neuropathologically damaged tissue
with epileptogenic potential should be of great value [2]. Considering the transition from epileptogenesis to a spontaneous
epileptic condition and the semiological manifestation of an
associated increased seizure propensity, molecular biomarkers
that reflect dynamic changes in the seizure threshold will be of
significant importance. It should be noted that potential molecular biomarkers for epilepsy development may also be suitable for
monitoring or predicting therapy responses, for example by
interventions retarding or even stopping epileptogenic processes,
or raising the seizure threshold in the transition stage to chronic
epilepsy, that is, the late stages of epilepsy development. Finally,
biomarkers may become surrogate markers for spontaneous
seizures, that is, measures used in therapeutic trials as a substitute
for clinically meaningful end point, and through this eliminating
the need to wait for spontaneous seizures to occur [2].
Epileptogenesis after a transient insult to the brain is accompanied by pathogenic processes that may serve as the source of
potential biomarkers. These processes include reactive
astrogliosis, the presence of activated microglia cells and leukocytes, blood–brain barrier (BBB) dysfunction, neuronal cell loss
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Lukasiuk and Becker
In recent years, great progress has been made in using biomarkers to predict disease outcome, as well as therapy response in other central nervous system disorders, in particular
in neuro-oncology [6]. This is particularly true for chemotherapy with procarbazine/CCNU/vincristine in patients with anaplastic oligodendrogliomas with 1p/19q co-deletion. Recent
data from elderly patients with glioblastoma multiforme show
the relevance of O(6)-methylguanine methyltransferase promoter methylation to the outcome of Temozolomid therapy.
However, developing biomarkers in the context of epilepsy
is clearly substantially more complex. There are two main
differences that we will consider here. First, in the context of
potential epileptogenesis, after a transient insult to the brain,
putative molecular biomarkers would need to be able to predict the future development of a disease that is not yet
emergent at the time point of biomarker assessment. Second,
and representing a major difference from neuro-oncology,
molecular biomarkers for epilepsy will need to reflect the
complex dynamics of seizure propensity. They could also
constitute surrogate markers that demonstrate the potential to
pharmacologically antagonize or retard epileptogenesis, thus
avoiding epilepsy surgery for the patient. These considerations clearly show that although brain tissue from patients
undergoing epilepsy surgery in the pharmaco-resistant stage
should be used for the development of molecular biomarkers,
such molecules may be particularly useful if they can be
analyzed in the blood and/or CSF.
The field of neuro-oncology is somewhat ahead of the field
of epileptogenesis in the consideration of blood-based biomarkers [7]. Similar to the function of molecular biomarkers
for brain tumors, a transient dysfunction of the BBB may
participate in the appearance of blood or serum biomarkers
for epileptogenesis. In CSF, molecular correlates of cellular
damage and inflammation associated with epileptogenesis or
epileptic seizures, or the consequences of tissue damage and
reorganization may be particularly pronounced if the affected
anatomical structures are close to the ventricles. An ideal
marker would be extremely sensitive and highly specific.
However, the cellular composition of affected brain tissue,
variability of blood and CSF marker half-life, and the dynamics of BBB integrity may substantially affect molecular biomarker levels in the blood and CSF. Similar to tumor markers,
molecular biomarkers for epilepsy will not be perfect and
perhaps do not need to be. It will be necessary to interpret
molecular biomarkers applied to epilepsy with caution. Theoretically, several classes of molecules should be considered as potential molecular biomarkers, including cellular components derived from epileptogenic lesions,
such as messenger RNAs, microRNAs, and alterations
in DNA methylation reflecting pathology in the central
nervous system. Proteins that reflect inflammation or
neurodegeneration in an epileptic lesion should also be
considered as potential molecular biomarkers. In the
following discussion, we will present what has been
discovered so far with respect to molecular biomarkers
in the context of epileptogenesis and the transition to a
spontaneous epileptic condition.
Fig. 1 Schematic overview of key alterations during epileptogenesis.
The key pathological elements of epileptogenesis are summarized.
Epileptogenesis can emerge from different, potentially epileptogenic,
insults. A plethora of mechanisms and potential biomarkers contribute
to the conversion from a normal to a chronic epileptic brain structure.
Notably, during epileptogenesis, occasional seizures can occur, but do not
necessarily reflect the transition to the stage of chronic recurrent seizure
activity (modified from [28]). BBB = blood–brain barrier
and neurogenesis, axonal regeneration, and—on the cellular
level—altered expression and distribution of neurotransmitter
receptors and ion channels [3, 4]. In the transition to the spontaneous seizure stage, the targeting of the receptors and channels
may be particularly suited to modifying the epileptic threshold.
For the development of molecular biomarkers, focal epilepsies may represent a rather promising setting compared with
other central nervous disorders, in which native disease tissue is
rarely available. A significant number of patients with focal
epilepsies develop pharmacoresistance to antiepileptic drugs. In
many of these patients, surgical removal of the epileptogenic
focus results in seizure control [2, 5]. The complementary availability of brain tissue from focal epilepsy patients undergoing
surgery, and cerebrospinal fluid (CSF) and/or peripheral blood
from different time points relative to the time point of neurosurgical intervention would be particularly valuable. If
blood and CSF reflect pathogenic mechanisms in epileptogenic brain tissue, they would be well suited to the
discovery of molecular biomarkers. A complementary
approach may be the characterization and assessment of
potential biomarkers in animal models of focal epilepsy
and their potential translation to human patients (Fig. 1).
Requirements of Biomarkers in the Epileptogenesis
Context
Biomarkers in Epilepsy
Identification of Molecular Biomarkers
for Epileptogenesis
Despite the growing knowledge of the molecular events occurring in the brain during epileptogenesis, there are currently
no validated biomarkers that would allow the reliable prediction of increased likelihood of epilepsy development. Ideal
biomarkers should not only be specific and sensitive, but also
easily accessible. In the case of epileptogenesis, accessing the
brain tissue of patients at risk is typically not feasible. Brain
tissue is sometimes available from patients with traumatic
brain injury (TBI) requiring surgical intervention, or from
biopsies taken for diagnostic purposes, but such circumstances are rare, and direct evaluation of brain tissue is not
included in the typical diagnostic process. Therefore, molecular biomarkers of epileptogenesis should either be based on
brain imaging or derived from peripheral tissues.
As recently proposed by Engel et al. [2], the search for
molecular biomarkers of epileptogenesis should start with
proof-of-concept studies in a properly chosen animal model.
Ideally, such a model should allow tissue sampling at different
stages of epileptogenesis and a comparison between animals
developing epilepsy to those that do not. The outcome measure should be occurrence of spontaneous seizures based on
video electroencephalography monitoring. Next, a potential
biomarker should be validated in different epilepsy models,
and its sensitivity and specificity should be determined. Only
then, the predictive value of the candidate biomarker should
be tested in an appropriate group of patients for translation.
There is no identified candidate biomarker that would fulfill
the above described criteria yet.
Only a few reports in the literature propose candidate
biomarkers together with outcome measures such as appearance of spontaneous seizures or their frequency. Although
correlation with seizure frequency does not imply that a candidate molecule will become a biomarker, such studies are
also included in this review as they may point to interesting
mechanisms for epileptogenesis biomarker discovery. The
majority of these studies were carried out based on experimental models of epilepsy. Despite the very preliminary state
of this research, some ideas presented here may be promising
and constitute the starting point for further studies.
A recent and intriguing approach complementary to the discovery of imaging biomarkers is the evaluation of brain metabolites associated with changes in brain metabolism during
epileptogenesis. Glucose metabolism studies by positron emission tomography in both the pilocarpine and kainic acid models
of epilepsy revealed hypometabolism in several brain areas
during the latency phase and after the onset of recurrent seizures
[8–10]. Interestingly, the level of hypometabolism in the entorhinal cortex has been shown to be correlated with the development
of recurrent seizures [10]. Additionally, in the lateral fluid percussion model, glucose metabolism parameters detected in the
321
ipsilateral hippocampus with positron emission tomography
1 week, 1 month, and 3 months after TBI were able to predict
the development of epilepsy [11].
Metabolites other than glucose have been tested as potential
imaging biomarkers. It has been shown that the level of myoinositol, a metabolite linked to astrocyte activation, is elevated
in the hippocampus during the latency phase in the pilocarpine
and kainate models of epilepsy [12, 13], and that this increase
can be detected with IH-magnetic resonance spectroscopy. Although the level of myo-inositol reflects the extent of neuronal
damage and neurodegeneration, it does not correlate with spontaneous recurrent seizures rat pilocarpine model [12]. Levels of
glutathione, synthesized mainly in astrocytes, decrease early
after status epilepticus (SE) but subsequently increase gradually
[12, 14]. Interestingly, glutathione levels in the hippocampus,
measured with IH-MRS during epileptogenesis, correlate negatively with neuronal cell loss and with the frequency of recurrent seizures observed in the chronic epileptic stage [12]. In
animal models, levels of lactate transiently increase during
epileptogenesis; however, there is no correlation with the frequency of seizures later on [12, 15]. The level of another brain
cell-derived metabolite, N-acetyl-aspartate, is decreased early
following SE and in the latency phase, possibly reflecting
neuronal loss and/or changed neuronal metabolism [12, 15].
The fact that blood, serum, and plasma are easily accessible
makes them very attractive sources of biomarkers. To date, only
levels of inflammatory proteins in the circulating blood have
been proposed as potential markers of epileptogenesis and have
been studied in experimental insult models. The rationale was
that their presence in blood may result from either “spillover” of
cerebral inflammatory molecules or even cells, or be due to
peripheral inflammation. Plasma levels of inflammatory proteins
C-reactive protein, interleukin 1-beta, and interleukin 6 were
studied in the angular bundle stimulation model of the temporal
lobe epilepsy, but no change was detected acutely after SE,
neither during the latency phase nor in chronic epilepsy [16].
Potential Biomarkers in TBI
TBI may result in the development of epilepsy. Although there is
no approved biomarker of TBI in clinical use, several candidate
molecular biomarkers exist. These have been evaluated at acute
insult stages to examine their correlation with the level of damage
and to predict functional outcomes in terms of mortality, secondary pathologies, and neurological parameters (reviewed in [1, 17,
18]).
Candidate biomarkers of TBI include serum or CSF levels of
protein leaking from the brain, which may represent a distinct
correlate of brain damage. Intriguing candidates for such biomarkers, detected in trauma patients, as well as in animal models,
are S100 calcium binding protein B (S100B), neuron-specific
enolase, glial fibrillary acidic protein, ubiquitin carboxyl-terminal
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Lukasiuk and Becker
hydrolase L1, myelin basic protein, and tau [1, 17, 18]. In
addition to dynamic protein concentrations, levels of metabolites,
including cyclic adenosine monophosphate, lactate, pyruvate,
glycerol, glutamate, norepinephrine, homovanillic acid,
hydroxyindolacetic acid, and N-acetylaspartate, as well as products of lipid peroxidation, such as F2-isoprostane, are altered
after TBI and correlate with injury severity [1, 17]. Current
efforts to identify new candidate protein biomarkers in the CSF
of TBI patients apply proteomics methods [19].
In the studies described above, candidate TBI biomarkers
that would include the development of epilepsy as an outcome
measure are lacking.
Potential Biomarkers in SE
SE is a risk factor for epilepsy development in humans. The
majority of animal models of epileptogenesis use chemically- or
electrically-induced SE as an epileptogenic insult. Molecular
changes in the brain induced by SE are well described [3, 20],
and several peripheral biomarkers have already been suggested
(Table 1).
The CSF levels of several proteins, including glial fibrillary
acidic protein and ubiquitin carboxyl-terminal hydrolase L1,
have been shown to be elevated acutely after SE [21]. An SEinduced increase in the serum level of neuron-specific enolase
has been observed in humans and in animal models [22, 23].
The level of prolactin in plasma and serum has also been
shown to transiently increase after SE in animals and humans
[24, 25].
Profiling of the blood transcriptome early after kainic acidinduced SE in rats has shown that the levels of several messenger RNAs are dynamically affected [26]. Additionally,
changes in blood microRNA profiles were found after kainic
acid-induced SE in rats [27]. These changes, however, have
not been validated, and their functional implications for the
development of epilepsy are not clear.
The prognostic value of these biomarkers for epilepsy
development has not been studied. Moreover, the specificity
of some of these changes for SE is questionable because
similar alterations may also occur after seizures or TBI.
Conclusions and Perspectives
Pattern Combination
Several features of epileptogenesis hamper the discovery of
biomarkers. First, epileptogenesis is a dynamic process. Several measures—imaging, and histological and molecular data—indicate that pathological changes occur in the brain sequentially and in parallel, including neurodegeneration, inflammation, and functional and structural neuronal plasticity.
Time lines for each of these phenomena overlap and may
differ depending on etiology, environmental factors, or patient
genetic susceptibility. As different mechanisms predominate
at distinct phases of epileptogenesis, different biomarkers will
presumably be necessary to predict the development of seizures over the course of epileptogenesis. Furthermore, an
important issue in acquired epilepsies will be the specificity
of the candidate biomarker. Particularly frequent causes of
epilepsy, including traffic accidents and military injuries, often
involve multi-organ injuries. It will be challenging to identify
biomarkers with enough specificity to identify ongoing
epileptogenesis in such cases.
Taking into account the complexity of epileptogenesis, potential panels of comprehensive, complementary biomarkers (imaging, electroencephalography, molecular) evaluated at different
stages (i.e., different time points after the precipitating injury)
may be required to provide reasonable predictive value.
Table 1 Summary of potential biomarkers for epileptogenesis in cerebrospinal fluid (CSF) and blood serum
Molecule
Biomaterial
Remarks [reference]
S100B
NSE
Serum/CSF
Serum
Levels correlate with injury severity after trauma and have predictive value on neurological outcome [1, 17, 18]
Levels correlate with neurologic outcome after trauma and reflect neuronal damage after SE [1, 17, 18, 23, 24]
GFAP
Serum/CSF
UCHL1
MBP
Tau
miR-9
CSF
Serum
Serum/CSF
Serum/brain
Levels correlate with injury severity after trauma and have predictive value on neurologic outcome; increase in
CSF correlates with epileptogenesis stage in the kainic acid SE model [1, 17, 18, 22]
Increase in CSF correlates with epileptogenesis in the kainic acid model [22]
Released in the course of brain trauma; predictive value for epileptogenesis to be determined [1, 17, 18]
Released in the course of brain trauma; predictive value for epileptogenesis to be determined [1, 17, 18]
Increased after traumatic injury; predictive value for epileptogenesis to be determined [20]
Prolactin
CSF, serum
Transiently increased in serum of SE animal model in early epileptogenesis; serum levels in SE patients not
increased; predictive value for epileptogenesis to be determined [25, 26]
S100B = S100 calcium binding protein B; NSE = neuron-specific enolase; GFAP = glial fibrillary acidic protein; UCHL1 = ubiquitin carboxyl-terminal
hydrolase L1; MBP = myelin basic protein; miR-9 = microRNA-9; SE = status epilepticus
Biomarkers in Epilepsy
Translation of Biomarkers Into Therapeutic Approaches
Given the basic concept of biomarkers in epileptogenesis and the
decreased seizure threshold prediction at the transition from
epileptogenesis to the chronic seizure stage, it may be expected
that molecular biomarkers that are either up- or down-regulated,
or altered, for example by phosphorylation, at this stage not only
correlate with these features, but also play a putative functional
role in these processes. It would be ideal to detect biomarkers that
correlate with specific pathologic aspects of epilepsy as they may
also give insights into therapeutic options. New avenues for
epilepsy biomarker discovery and therapy development may
result from studies on the manipulation of gene expression in
animal models of insult-induced epileptogenesis employing, for
example, small interfering RNA-mediated antagonism of abundant gene transcription in downstream pathways or the compensatory overexpression of certain molecules, which are reduced in
epilepsy.
Acknowledgments AJB’s work is supported by EpiTarget,
EuroEpinomics and DFG (SFB 1089, KFO 177), the Else KrönerFresenius and German Israeli Foundations, and BonFor. KL’s work is
supported by Ministry of Science and Higher Education grant DNP/
N119/ESF-EuroEPINOMICS/2012 and EpiTarget.
Required Author Forms Disclosure forms provided by the authors are
available with the online version of this article.
Disclosure The authors declare that they have no conflicts of interest
related to this manuscript.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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