Astrocyte immune responses in epilepsy.

Astrocytes, the major glial cell type of the central nervous system (CNS), are known to play a major role in the regulation of the immune/inflammatory response in several human CNS diseases. In epilepsy-associated pathologies, the presence of astrogliosis has stimulated extensive research focused on the role of reactive astrocytes in the pathophysiological processes that underlie the development of epilepsy. In brain tissue from patients with epilepsy, astrocytes undergo significant changes in their physiological properties, including the activation of inflammatory pathways. Accumulating experimental evidence suggests that proinflammatory molecules can alter glio-neuronal communications contributing to the generation of seizures and seizure-related neuronal damage. In particular, both in vitro and in vivo data point to the role of astrocytes as both major source and target of epileptogenic inflammatory signaling. In this context, understanding the astroglial inflammatory response occurring in epileptic brain tissue may provide new strategies for targeting astrocyte-mediated epileptogenesis. This article reviews current evidence regarding the role of astrocytes in the regulation of the innate immune responses in epilepsy. Both clinical observations in drug-resistant human epilepsies and experimental findings in clinically relevant models will be discussed and elaborated, highlighting specific inflammatory pathways (such as interleukin-1ß/toll-like receptor 4) that could be potential targets for antiepileptic, disease-modifying therapeutic strategies.

Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 ß.

OBJECTIVE: Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K⁺ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1ß (IL-1ß) on Kir4.1 mRNA and protein expression. METHODS: We investigated Kir4.1 (Kcnj10) and IL-1ß mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64), comparing the expression in tumor patients with (n = 38) and without epilepsy (n = 26). RESULTS: Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1ß mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1ß. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1ß and the cytoplasmic translocation of high mobility group box 1 (HMGB1). CONCLUSIONS: Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the inflammatory cytokine IL-1ß.

Overexpression of ADK in human astrocytic tumors and peritumoral tissue is related to tumor-associated epilepsy.

PURPOSE: Adenosine kinase (ADK), a largely astrocyte-based metabolic enzyme, regulates adenosine homeostasis in the brain. Overexpression of ADK decreases extracellular adenosine and consequently leads to seizures. We hypothesized that dysfunction in the metabolism of tumor astrocytes is related to changes in ADK expression and that those changes might be associated with the development of epilepsy in patients with tumors. METHODS: We compared ADK expression and cellular distribution in surgically removed tumor tissue (n = 45) and peritumoral cortex (n = 20) of patients with glial and glioneuronal tumors to normal control tissue obtained at autopsy (n = 11). In addition, we compared ADK expression in tumor patients with and without epilepsy. To investigate ADK expression, we used immunohistochemistry and Western blot analysis. ADK activity measurement was performed in surgical specimens of astrocytomas World Health Organization (WHO) grade III (n = 3), peritumoral cortex (n = 3), and nonepileptic cortex (n = 3). KEY FINDINGS: Immunohistochemistry predominantly showed cytoplasmic labeling in tumors and peritumoral tissue containing infiltrating tumor cells. ADK immunoreactivity was significantly stronger in tumor and peritumoral tissue compared to normal white matter and normal cortex, especially in astrocytoma WHO grade III, as confirmed by Western blot analysis and ADK activity measurements. Importantly, we found a significantly higher expression of ADK in the peritumoral infiltrated tissue of patients with epilepsy than in patients without epilepsy. SIGNIFICANCE: These results suggest a dysregulation of ADK in astrocytic brain tumors. Moreover, the upregulation of ADK observed in peritumoral infiltrated tissue of glioma patients with epilepsy supports the role of this enzyme in tumor-associated epilepsy.

Activation of Toll-like receptor, RAGE and HMGB1 signalling in malformations of cortical development.

Recent evidence in experimental models of seizures and in temporal lobe epilepsy support an important role of high-mobility group box 1 and toll-like receptor 4 signalling in the mechanisms of hyperexcitability leading to the development and perpetuation of seizures. In this study, we investigated the expression and cellular distribution of toll-like receptors 2 and 4, and of the receptor for advanced glycation end products, and their endogenous ligand high-mobility group box 1, in epilepsy associated with focal malformations of cortical development. Immunohistochemistry showed increased expression of toll-like receptors 2 and 4 and receptor for advanced glycation end products in reactive glial cells in focal cortical dysplasia, cortical tubers from patients with the tuberous sclerosis complex and in gangliogliomas. Toll-like receptor 2 was predominantly detected in cells of the microglia/macrophage lineage and in balloon cells in focal cortical dysplasia, and giant cells in tuberous sclerosis complex. The toll-like receptor 4 and receptor for advanced glycation end products were expressed in astrocytes, as well as in dysplastic neurons. Real-time quantitative polymerase chain reaction confirmed the increased receptors messenger RNA level in all pathological series. These receptors were not detected in control cortex specimens. In control cortex, high-mobility group box 1 was ubiquitously detected in nuclei of glial and neuronal cells. In pathological specimens, protein staining was instead detected in the cytoplasm of reactive astrocytes or in tumour astrocytes, as well as in activated microglia, predictive of its release from glial cells. In vitro experiments in human astrocyte cultures showed that nuclear to cytoplasmic translocation of high-mobility group box 1 was induced by interleukin-1ß. Our findings provide novel evidence of intrinsic activation of these pro-inflammatory signalling pathways in focal malformations of cortical development, which could contribute to the high epileptogenicity of these developmental lesions.

Ontogenetic modifications of neuronal excitability during brain maturation: developmental changes of neurotransmitter receptors.

The development of the human brain depends on a precisely orchestrated cascade of events, including proliferation, migration and maturation of neural progenitor cells. Different mechanisms coordinate these stages to reach a normal structural organization, producing appropriate excitatory and inhibitory networks. Here, we will briefly review the developmental changes of glutamate (Glu) and γ-aminobutyric acid (GABA) receptors, with particular attention to the development of the human brain. We will also briefly discuss recent evidence on the involvement of the endocannabinoid signaling in the regulation of neuronal excitability during early brain development..

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-1ß 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.

Induction of sodium channel Na(x) (SCN7A) expression in rat and human hippocampus in temporal lobe epilepsy.

PURPOSE: In a recent large-scale gene-expression study in a rat model of temporal lobe epilepsy (TLE) a persistent up-regulation in the expression of the SCN7A gene was revealed. The SCN7A gene encodes an atypical sodium channel (Na(x) ), which is involved in osmoregulation via a sensing mechanism for the extracellular sodium concentration. Herein we investigated the expression and cellular distribution of SCN7A mRNA and protein in normal and epileptic rat and human hippocampus. METHODS: SCN7A/Na(x) expression analysis was performed by polymerase chain reaction (PCR), immunocytochemistry, and western blot analysis. RESULTS: Increased expression of SCN7A/Na(x) mRNA/protein was observed during epileptogenesis and in the chronic epileptic phase in the post-status epilepticus (SE) model of TLE. The up-regulation was confirmed in human hippocampal tissue resected from pharmacoresistant patients with hippocampal sclerosis (HS). In both epileptic rat and human hippocampus, increased Na(x) expression was observed in neurons and reactive astrocytes compared to control tissue. CONCLUSIONS: The increased and persistent expression of SCN7A/Na(x) in the epileptic rat and human hippocampus supports the possible involvement of this channel in the complex reorganization occurring within the hippocampus during the epileptogenic process in TLE. Further studies are needed for a complete understanding of the functional role of SCN7A in epilepsy.

Evaluation of the innate and adaptive immunity in type I and type II focal cortical dysplasias.

PURPOSE: Induction of inflammatory pathways has been reported in epileptic patients with focal malformations of cortical development. In the present study we examined the innate and adaptive immune responses in focal cortical dysplasia (FCD) with different histopathologic and pathogenetic features. METHODS: The inflammatory cell components and the induction of major proinflammatory pathways and molecules [complement pathway, interleukin (IL)-1ß, and chemokine monocyte chemotactic protein-1 (MCP1)] was investigated in surgical specimens of sporadic type IA and type IIB FCD using immunocytochemical methods. RESULTS: FCD II but not FCD I cases exhibit activation of the mammalian target of rapamycin (mTOR) cascade with strong neuronal expression of the phosphorylated isoform of S6 protein. Microglia reactivity was increased in all lesions (FCD I and II) compared to control tissue; however, the number of HLA-DR-positive cells was significantly higher in FCD II than in FCD I. In FCD II specimens we also observed perivascular and parenchymal T lymphocytes (CD3(+) ), with a predominance of CD8(+) T-cytotoxic/suppressor lymphocytes, as well as a few dendritic cells. Expression of components of the complement cascade, IL-1ß, and MCP1 was prominent in FCD II cases. DISCUSSION: Our findings indicate a prominent activation of both innate and adaptive immunity, with involvement of different inflammatory pathways in FCD II cases, supporting the possible involvement of inflammation in the epileptogenesis of these lesions, as well as the notion that FCD II is pathologically distinct from FCD I.

Defective microglial development in the hippocampus of Cx3cr1 deficient mice.

Microglial cells participate in brain development and influence neuronal loss and synaptic maturation. Fractalkine is an important neuronal chemokine whose expression increases during development and that can influence microglia function via the fractalkine receptor, CX3CR1. Mice lacking Cx3cr1 show a variety of neuronal defects thought to be the result of deficient microglia function. Activation of CX3CR1 is important for the proper migration of microglia to sites of injury and into the brain during development. However, little is known about how fractalkine modulates microglial properties during development. Here we examined microglial morphology, response to ATP, and K(+) current properties in acute brain slices from Cx3cr1 knockout mice across postnatal hippocampal development. We found that fractalkine signaling is necessary for the development of several morphological and physiological features of microglia. Specifically, we found that the occurrence of an outward rectifying K(+) current, typical of activated microglia, that peaked during the second and third postnatal week, was reduced in Cx3cr1 knockout mice. Fractalkine signaling also influenced microglial morphology and ability to extend processes in response to ATP following its focal application to the slice. Our results reveal the developmental profile of several morphological and physiological properties of microglia and demonstrate that these processes are modulated by fractalkine signaling.

The inflammatory molecules IL-1ß and HMGB1 can rapidly enhance focal seizure generation in a brain slice model of temporal lobe epilepsy.

Epilepsy is a neurological disorder characterized by a hyperexcitable brain tissue and unpredictable seizures, i.e., aberrant firing discharges in large neuronal populations. It is well established that proinflammatory cytokines, in addition to their canonical involvement in the immune response, have a crucial role in the mechanism of seizure generation. The purpose of the present study was to investigate the role of interleukin-1ß (IL-1ß) and high mobility group B1 (HMGB1) in the generation of seizure-like discharges using two models of focal epilepsy in a rat entorhinal cortex slice preparation. Seizure like-discharges were evoked by either slice perfusion with low Mg(2+) and picrotoxin or with a double NMDA local stimulation in the presence of the proconvulsant 4-amino-pyridine. The effects of IL-1ß or HMGB1 were evaluated by monitoring seizure discharge generation through laser scanning microscope imaging of Ca(2+) signals from neurons and astrocytes. In the picrotoxin model, we revealed that both cytokines increased the mean frequency of spontaneous ictal-like discharges, whereas only IL-1ß reduced the latency and prolonged the duration of the first ictal-like event. In the second model, a single NMDA pulse, per se ineffective, became successful when it was performed after IL-ß or HMGB1 local applications. These findings demonstrate that both IL-1ß and HMGB1 can rapidly lower focal ictal event threshold and strengthen the possibility that targeting these inflammatory pathways may represent an effective therapeutic strategy to prevent seizures.

Cerebellar axon/myelin loss, angiogenic sprouting, and neuronal increase of vascular endothelial growth factor in a preterm infant with kernicterus.

We performed histologic and immunohistochemical analysis of cerebellar sections from a preterm infant (32 weeks 5 days) dead on the 4th day of life with the diagnosis of kernicterus and compared the results with 1 age-matched nonicteric patient. Poorer Luxol fast blue-periodic acid Schiff and Bodian-Luxol fast blue stainings as well as neurofilament expression were observed in the kernicterus case, indicating loss of axon neurites and myelin fibers. Elevated claudin-5 and cluster of differentiation 34 expression associated with increased blood vessel density suggests bilirubin-induced angiogenic sprouting. Upregulation of vascular endothelial growth factor and its receptor 2 was observed in nucleus dentatus and Purkinje neurons. Although upregulation of multidrug resistance-associated protein 1 was increased in cerebellar neurons, it was not able to prevent bilirubin-induced neurotoxicity. These data add new insights into the pathophysiology of kernicterus, revealing vascular endothelial growth factor and its receptor 2, as well as angiogenic sprouting, as new players in neurologic damage by unconjugated bilirubin.

MicroRNA-146a: a key regulator of astrocyte-mediated inflammatory response.

Increasing evidence supports the involvement of microRNAs (miRNA) in the regulation of inflammation in human neurological disorders. In the present study we investigated the role of miR-146a, a key regulator of the innate immune response, in the modulation of astrocyte-mediated inflammation. Using Taqman PCR and in situ hybridization, we studied the expression of miR-146a in epilepsy-associated glioneuronal lesions which are characterized by prominent activation of the innate immune response. In addition, cultured human astrocytes were used to study the regulation of miR-146a expression in response to proinflammatory cytokines. qPCR and western blot were used to evaluate the effects of overexpression or knockdown of miR-146a on IL-1ß signaling. Downstream signaling in the IL-1ß pathway, as well as the expression of IL-6 and COX-2 were evaluated by western blot and ELISA. Release several cytokines was evaluated using a human magnetic multiplex cytokine assay on a Luminex® 100™/200™ platform. Increased expression of miR-146a was observed in glioneuronal lesions by Taqman PCR. MiR-146a expression in human glial cell cultures was strongly induced by IL-1ß and blocked by IL-1ß receptor antagonist. Modulation of miR-146a expression by transfection of astrocytes with anti-miR146a or mimic, regulated the mRNA expression levels of downstream targets of miR-146a (IRAK-1, IRAK-2 and TRAF-6) and the expression of IRAK-1 protein. In addition, the expression of IL-6 and COX-2 upon IL-1ß stimulation was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. Modulation of miR-146a expression affected also the release of several cytokines such as IL-6 and TNF-α. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation and suggests that this miR may represent a novel target for therapeutic strategies.