Upregulation of dysbindin in temporal lobe epileptic foci of human and experimental animals

The gene encoding dystrobrevin-binding-protein-1 (dysbindin) is expressed in many areas of the central nervous system and plays a role in intracellular vesicle trafficking, synaptic vesicle trafficking, and neurotransmitter release. At a cellular level, dysbindin is thought to mediate presynaptic glutamatergic transmission. Using Western blotting and immunofluorescence, we investigated dysbindin expression in brain tissues of the patients with temporal lobe epilepsy (TLE) and rats with TLE (lithium chloride pilocarpine model) to explore its possible role in epileptogenesis. Twenty-five samples of temporal neocortex from patients undergoing surgery for drug-refractory TLE epilepsy and 10 histologically normal temporal lobes tissues from control subjects were used in our study. We also examined dysbindin expression in the hippocampus and adjacent cortex from experimental Sprague-Dawley rats. Dysbindin was expressed in the cytoplasm of neurons from epileptic specimens, and levels of dysbindin proteins were significantly increased in patients with TLE. Dysbindin was also expressed in the neurons of the hippocampus and adjacent cortex from experimental and control rats. Western blotting of rat brain tissue showed that dysbindin was upregulated gradually from 6 h after kindling. Maximal expression was seen around 2 months in chronic epileptic phase. These results demonstrated that the increased expression of dysbindin might play a role in the pathogenesis of drug-refractory TLE.     

Modulation of Dopaminergic Neuronal Differentiation from Sympathoadrenal Progenitors

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferase (DNMT). Increasing evidence suggests that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. Here, we evaluated DNA methyltransferase 1 (Dnmt1) and Dnmt3a expression in brain tissues of epileptic patients to explore their possible role in epileptogenesis. Tissue samples from temporal neocortices of 25 patients with intractable temporal lobe epilepsy (TLE) and ten histologically normal temporal lobes from control patients were used to detect Dnmt1 and Dnmt3a expression through immunohistochemistry, immunofluorescence, and Western blotting analysis. We found that both Dnmt1 and Dnmt3a expression were principally expressed in the nucleus and the cytoplasm of NeuN-positive neurons, but not in GFAP-positive astrocytes. Levels of the two DNMT proteins were significantly increased in patients with TLE. Our study suggests that DNMT1 and DNMT3a may play a role in the pathogenesis of TLE.     

Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy

We investigated the activation of the IL-1 beta system and markers of adaptive immunity in rat brain during epileptogenesis using models of temporal lobe epilepsy (TLE). The same inflammatory markers were studied in rat chronic epileptic tissue and in human TLE with hippocampal sclerosis (HS). IL-1 beta was expressed by both activated microglia and astrocytes within 4 h from the onset of status epilepticus (SE) in forebrain areas recruited in epileptic activity; however, only astrocytes sustained inflammation during epileptogenesis. Activation of the IL-1 beta system during epileptogenesis was associated with neurodegeneration and blood-brain barrier breakdown. In rat and human chronic epileptic tissue, IL-1 beta and IL-1 receptor type 1 were broadly expressed by astrocytes, microglia and neurons. Granulocytes appeared transiently in rat brain during epileptogenesis while monocytes/macrophages were present in the hippocampus from 18 h after SE onset until chronic seizures develop, and they were found also in human TLE hippocampi. In rat and human epileptic tissue, only scarce B- and T-lymphocytes and NK cells were found mainly associated with microvessels. These data show that specific inflammatory pathways are chronically activated during epileptogenesis and they persist in chronic epileptic tissue, suggesting they may contribute to the etiopathogenesis of TLE.     

Embryonic stem cell-derived neural precursor grafts for treatment of temporal lobe epilepsy

Complex partial seizures arising from mesial temporal lobe structures are a defining feature of mesial temporal lobe epilepsy (TLE). For many TLE patients, there is an initial traumatic head injury that is the precipitating cause of epilepsy. Severe TLE can be associated with neuropathological changes, including hippocampal sclerosis, neurodegeneration in the dentate gyrus, and extensive reorganization of hippocampal circuits. Learning disabilities and psychiatric conditions may also occur in patients with severe TLE for whom conventional anti-epileptic drugs are ineffective. Novel treatments are needed to limit or repair neuronal damage, particularly to hippocampus and related limbic regions in severe TLE and to suppress temporal lobe seizures. A promising therapeutic strategy may be to restore inhibition of dentate gyrus granule neurons by means of cell grafts of embryonic stem cell-derived GABAergic neuron precursors. “Proof-of-concept” studies show that human and mouse embryonic stem cell-derived neural precursors can survive, migrate, and integrate into the brains of rodents in different experimental models of TLE. In addition, studies have shown that hippocampal grafts of cell lines engineered to release GABA or other anticonvulsant molecules can suppress seizures. Furthermore, transplants of fetal GABAergic progenitors from the mouse or human brain have also been shown to suppress the development of seizures. Here, we review these relevant studies and highlight areas of future research directed toward producing embryonic stem cell-derived GABAergic interneurons for cell-based therapies for treating TLE.     

Abnormal expression of stathmin 1 in brain tissue of patients with intractable temporal lobe epilepsy and a rat model

Microtubule dynamics have been shown to contribute to neurite outgrowth, branching, and guidance. Stathmin 1 is a potent microtubule-destabilizing factor that is involved in the regulation of microtubule dynamics and plays an essential role in neurite elongation and synaptic plasticity. Here, we investigate the expression of stathmin 1 in the brain tissues of patients with intractable temporal lobe epilepsy (TLE) and experimental animals using immunohistochemistry, immunofluorescence and western blotting. We obtained 32 temporal neocortex tissue samples from patients with intractable TLE and 12 histologically normal temporal lobe tissues as controls. In addition, 48 Sprague Dawley rats were randomly divided into six groups, including one control group and five groups with epilepsy induced by lithium chloride-pilocarpine. Hippocampal and temporal lobe tissues were obtained from control and epileptic rats on Days 1, 7, 14, 30, and 60 after kindling. Stathmin 1 was mainly expressed in the neuronal membrane and cytoplasm in the human controls, and its expression levels were significantly higher in patients with intractable TLE. Moreover, stathmin 1 was also expressed in the neurons of both the control and the experimental rats. Stathmin 1 expression was decreased in the experimental animals from 1 to 14 days postseizure and then significantly increased at Days 30 and 60 compared with the control group. Many protruding neuronal processes were observed in the TLE patients and in the chronic stage epileptic rats. These data suggest that stathmin 1 may participate in the abnormal network reorganization of synapses and contribute to the pathogenesis of TLE.     

Long-term alteration of calcium homeostatic mechanisms in the pilocarpine model of temporal lobe epilepsy

The pilocarpine model of temporal lobe epilepsy is an animal model that shares many of the clinical and pathophysiological characteristics of temporal lobe or limbic epilepsy in humans. This model of acquired epilepsy produces spontaneous recurrent seizure discharges following an initial brain injury produced by pilocarpine-induced status epilepticus. Understanding the molecular mechanisms mediating these long lasting changes in neuronal excitability would provide an important insight into developing new strategies for the treatment and possible prevention of this condition. Our laboratory has been studying the role of alterations in calcium and calcium-dependent systems in mediating some of the long-term neuroplasticity changes associated with epileptogenesis. In this study, [Ca(2+)](i) imaging fluorescence microscopy was performed on CA1 hippocampal neurons acutely isolated from control and chronically epileptic animals at 1 year after the induction of epileptogenesis with two different fluorescent dyes (Fura-2 and Fura-FF) having high and low affinities for [Ca(2+)](i). The high affinity Ca(2+) indicator Fura-2 was utilized to evaluate [Ca(2+)](i) levels up to 900 nM and the low affinity indicator Fura-FF was employed for evaluating [Ca(2+)](i) levels above this range. Baseline [Ca(2+)](i) levels and the ability to restore resting [Ca(2+)](i) levels after a brief exposure to several glutamate concentrations in control and epileptic neurons were evaluated. Epileptic neurons demonstrated a statistically significantly higher baseline [Ca(2+)](i) level in comparison to age-matched control animals. This alteration in basal [Ca(2+)](i) levels persisted up to 1 year after the induction of epileptogenesis. In addition, the epileptic neurons were unable to rapidly restore [Ca(2+)](i) levels to baseline following the glutamate-induced [Ca(2+)](i) loads. These changes in Ca(2+) regulation were not produced by a single seizure and were not normalized by controlling the seizures in the epileptic animals with anticonvulsant treatment. Peak [Ca(2+)](i) levels in response to different concentrations of glutamate were the same in both epileptic and control neurons. Thus, glutamate produced the same initial [Ca(2+)](i) load in both epileptic and control neurons. Characterization of the viability of acutely isolated neurons from control and epileptic animals utilizing standard techniques to identify apoptotic or necrotic neurons demonstrated that epileptic neurons had no statistically significant difference in viability compared to age-matched controls. These results provide the first direct measurement of [Ca(2+)](i) levels in an intact model of epilepsy and indicate that epileptogenesis in this model produced long-lasting alterations in [Ca(2+)](i) homeostatic mechanisms that persist for up to 1 year after induction of epileptogenesis. These observations suggest that altered [Ca(2+)](i) homeostatic mechanisms may underlie some aspects of the epileptic phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.     

Mitochondrial complex I deficiency in the epileptic focus of patients with temporal lobe epilepsy

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE.     

Increased expression of caspase 2 in experimental and human temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is often caused by a neurodegenerative brain insult that triggers epileptogenesis, and eventually results in spontaneous seizures, i.e., epilepsy. Understanding the mechanisms of cell death is a key for designing new drug therapies for preventing the neurodegeneration associated with TLE. Here, we investigated the expression of caspase 2, a protein involved in programmed cell death, during the course of epilepsy. We investigated caspase 2 expression in hippocampal samples derived from patients operated on for drug refractory TLE. To understand the evolution of altered-caspase 2 expression during the epileptic process, we also examined caspase 2 expression and activity in the rat hippocampus after status epilepticus-induced acute damage, during epileptogenesis, and after the onset of epilepsy. Caspase 2 expression was enhanced in the hippocampal neurons in chronic TLE patients. In rats, status epilepticus-induced caspase 2 labeling paralleled the progression of neurodegeneration. Proteolytic activation and cleavage of caspase 2 was also detected in the rat brain undergoing epileptogenesis. Our data suggest that caspase 2-mediated programmed cell death participates in the seizure-induced degenerative process in experimental and human TLE. The work was supported by The Academy of Finland, The Kuopio University Foundation, The Neurology Foundation, The North-Savo Regional Fund of the Finnish Cultural Foundation, Sigrid Juselius Foundation, and The Vaajasalo Foundation.     

Neuroprotective strategies to avert seizure-induced neurodegeneration in epilepsy

Neurodegeneration in limbic circuits is a hallmark feature of chronic temporal lobe epilepsy (TLE). Studies in experimental animal models and human patients indicate that seizure-induced neuronal injury involves some active, as well as passive cell death processes. Experimental approaches that inhibit active steps in cell death programs have been shown to reduce neuronal cell death and sclerosis, but not to prevent epileptogenesis in animal models of TLE. These findings suggest that we need additional research using both animal models and brain slices from human patients to understand the pathological mechanisms underlying seizure generation. Such comparative studies will also aid in evaluating the potential therapeutic value of inhibiting cell death in seizure disorders.     

Neuroprotective Strategies to Avert Seizure-Induced Neurodegeneration in Epilepsy

Summary:  Neurodegeneration in limbic circuits is a hallmark feature of chronic temporal lobe epilepsy (TLE). Studies in experimental animal models and human patients indicate that seizure-induced neuronal injury involves some active, as well as passive cell death processes. Experimental approaches that inhibit active steps in cell death programs have been shown to reduce neuronal cell death and sclerosis, but not to prevent epileptogenesis in animal models of TLE. These findings suggest that we need additional research using both animal models and brain slices from human patients to understand the pathological mechanisms underlying seizure generation. Such comparative studies will also aid in evaluating the potential therapeutic value of inhibiting cell death in seizure disorders.     

Cannabinoid regulation of neurons in the dentate gyrus during epileptogenesis: Role of CB1R-associated proteins and downstream pathways

The hippocampal formation plays a central role in the development of temporal lobe epilepsy (TLE), a disease characterized by recurrent, unprovoked epileptic discharges. TLE is a neurologic disorder characterized by acute long-lasting seizures (i.e., abnormal electrical activity in the brain) or seizures that occur in close proximity without recovery, typically after a brain injury or status epilepticus. After status epilepticus, epileptogenic hyperexcitability develops gradually over the following months to years, resulting in the emergence of chronic, recurrent seizures. Acting as a filter or gate, the hippocampal dentate gyrus (DG) normally prevents excessive excitation from propagating through the hippocampus, and is considered a critical region in the progression of epileptogenesis in pathological conditions. Importantly, lipid-derived endogenous cannabinoids (endocannabinoids), which are produced on demand as retrograde messengers, are central regulators of neuronal activity in the DG circuit. In this review, we summarize recent findings concerning the role of the DG in controlling hyperexcitability and propose how DG regulation by cannabinoids (CBs) could provide avenues for therapeutic interventions. We also highlight possible pathways and manipulations that could be relevant for the control of hyperexcitation. The use of CB compounds to treat epilepsies is controversial, as anecdotal evidence is not always validated by clinical trials. Recent publications shed light on the importance of the DG as a region regulating incoming hippocampal excitability during epileptogenesis. We review recent findings concerning the modulation of the hippocampal DG circuitry by CBs and discuss putative underlying pathways. A better understanding of the mechanisms by which CBs exert their action during seizures may be useful to improve therapies.     

Cellular pathology of amygdala neurons in human temporal lobe epilepsy

The amygdala complex substantially contributes to the generation and propagation of focal seizures in patients suffering from temporal lobe epilepsy (TLE). A cellular substrate for increased excitability in the human amygdala, however, remains to be identified. Here, we analyzed the three-dimensional morphology of 264 neurons from different subregions of the amygdaloid complex obtained from 17 "en bloc" resected surgical specimens using intracellular Lucifer Yellow (LY) injection and confocal laser scanning microscopy. Autopsy samples from unaffected individuals ( n=3, 20 neurons) served as controls. We have identified spine-laden, spine-sparse and aspinous cells in the lateral, basal, accessory basal and granular nuclei. Semiquantitative analysis points to significant changes in neuronal soma size, number of dendrites and spine densities in specimens from epilepsy patients compared to controls. Neuronal somata in the epilepsy group were smaller compared to controls ( P<0.01), neurons had fewer first-order dendrites ( P<0.01), whereas the maximum density of spines per dendritic segment in these cells was increased in TLE patients ( P<0.01). There were also dendritic alterations such as focal constrictions or spine bifurcations. These changes were consistent between amygdaloid subregions. The dendritic morphology of amygdaloid neurons in TLE patients points to substantial changes in synaptic connectivity and would be compatible with altered neuronal circuitries operating in the epileptic human amygdala. Although the morphological alterations differ from those described in hippocampal subregions of a similar cohort of TLE patients, they appear to reflect a characteristic pathological substrate associated with seizure activity/propagation within the amygdaloid complex.     

Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy

Purpose: Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphologic feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathologic hallmark of TLE. Methods: Using immunocytochemistry and Western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). Key Findings: In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up‐regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3–4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of patients with TLE. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several proinflammatory molecules (interleukin‐1β and lipopolysaccharide). Significance: These results suggest that dysregulation of ADK in astrocytes is a common pathologic hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte‐based adenosine cycle and inflammation. Whether this interaction also can play a role in vivo needs to be further investigated.     

BACE1 elevation is associated with aberrant limbic axonal sprouting in epileptic CD1 mice

The brain is capable of remarkable synaptic reorganization following stress and injury, often using the same molecular machinery that governs neurodevelopment. This form of plasticity is crucial for restoring and maintaining network function. However, neurodegeneration and subsequent reorganization can also play a role in disease pathogenesis, as is seen in temporal lobe epilepsy and Alzheimer's disease. β-Secretase-1 (BACE1) is a protease known for cleaving β-amyloid precursor protein into β-amyloid (Aβ), a major constituent in amyloid plaques. Emerging evidence suggests that BACE1 is also involved with synaptic plasticity and nerve regeneration. Here we examined whether BACE1 immunoreactivity (IR) was altered in pilocarpine-induced epileptic CD1 mice in a manner consistent with the synaptic reorganization seen during epileptogenesis. BACE1-IR increased in the CA3 mossy fiber field and dentate inner molecular layer in pilocarpine-induced epileptic mice, relative to controls (saline-treated mice and mice 24-48 h after pilocarpine-status), and paralleled aberrant expression of neuropeptide Y. Regionally increased BACE1-IR also occurred in neuropil in hippocampal area CA1 and in subregions of the amygdala and temporal cortex in epileptic mice, colocalizing with increased IR for growth associated protein 43 (GAP43) and polysialylated-neural cell adhesion molecule (PSA-NCAM), but reduced IR for microtubule-associated protein 2 (MAP2). These findings suggest that BACE1 is involved in aberrant limbic axonal sprouting in a model of temporal lobe epilepsy, warranting further investigation into the role of BACE1 in physiological vs. pathological neuronal plasticity.     

Astrocyte calcium signaling and epilepsy

Studies performed over the last decade, in both animal models and human epilepsy, support the view that a defective K(+) buffering due to an altered expression of K(+) and aquaporin channels in astrocytes represents a possible causative factor of the pathological neuronal hyperexcitability in the epileptic brain. More recent studies, however, reappraised the role of neurons in epileptogenesis and suggested that Ca(2+)-dependent gliotransmission directly contributes to the excessive neuronal synchronization that predisposes the brain network to seizures. Significant support for this view comes from the finding that astrocytes from hyperexcitable networks respond to neuronal signals with massive Ca(2+) elevations and generate a recurrent excitatory loop with neurons that has the potential to promote a focal seizure. The specific aim of this review is on the one hand, to provide an overview of the experimental findings that hinted at a direct role of Ca(2+)-dependent gliotransmission in the generation of seizure-like discharges in models of focal epilepsy; and on the other hand, to emphasize the importance of developing new experimental tools that could help us to understand the amazing complexity of neuron-astrocyte partnership in brain disorders.     

Heat shock protein 70 expression in epilepsy suggests stress rather than protection

Although heat shock protein 70 (HSP70) has been suggested to be a stress marker or to play a protective role in brain injury, the relevance of its pathological expression in epilepsy is unclear. We investigated the expression of HSP70 in brain tissue from human temporal lobe epilepsy (TLE) patients and from kainic acid (KA)-induced seizure-related neuronal damage in vivo and in vitro. The human TLE tissue showed severe neuronal loss and gliosis in hippocampal CA3 area. The KA-induced neuronal damage was similar to pathological changes of the TLE hippocampus. An increased number of TUNEL-positive cells were observed at day 5 when compared with day 2 after seizure induction. Intense HSP70 immunofluorescence was observed in hippocampal CA3 pyramidal neurons of rat, 2 days following KA administration, which then declined in labeling by day 5. No HSP70 expression was found in Fluoro-Jade B positive dying neurons by double staining. Western blot analysis showed an increased level of p53 and Bax expression following KA treatment. In vitro, there was no apparent difference in the degree of apoptosis between HSP70 siRNA- and control empty vector-transfected primary neurons following KA treatment. Our results revealed that HSP70 was a useful indicator of stressed neurons in acute phase of epilepsy, but not associated with neuronal death, thereby suggesting that HSP70 played no role in neuroprotection during an epileptogenic state.