Jan A. Gorter

Global Ischemia Induces Downregulation of Glur2 mRNA and Increases AMPA Receptor-Mediated Ca2+ Influx in Hippocampal CA1 Neurons of Gerbil

Transient, severe forebrain or global ischemia leads to delayed cell death of pyramidal neurons in the hippocampal CA1. The precise molecular mechanisms underlying neuronal cell death after global ischemia are as yet unknown. Glutamate receptor-mediated Ca2+ influx is thought to play a critical role in this cell death. In situ hybridization revealed that the expression of mRNA encoding GluR2 (the subunit that limits Ca2+ permeability of AMPA-type glutamate receptors) was markedly and specifically reduced in gerbil CA1 pyramidal neurons after global ischemia but before the onset of neurodegeneration. To determine whether the change in GluR2 expression is functionally significant, we examined the AMPA receptor-mediated rise in cytoplasmic free Ca2+ level ([Ca2+]i) in individual CA1 pyramidal neurons by optical imaging with the Ca2+indicator dye fura-2 and by intracellular recording. Seventy-two hours after ischemia, CA1 neurons that retained the ability to fire action potentials exhibited a greatly enhanced AMPA-elicited rise in [Ca2+]i. Basal [Ca2+]i in these neurons was unchanged. These findings provide evidence for Ca2+entry directly through AMPA receptors in pyramidal neurons destined to die. Downregulation of GluR2 gene expression and an increase in Ca2+ influx through AMPA receptors in response to endogenous glutamate are likely to contribute to the delayed neuronal death after global ischemia.

Progression of spontaneous seizures after status epilepticus is associated with mossy fibre sprouting and extensive bilateral loss of hilar parvalbumin and somatostatin‐immunoreactive neurons

The development of spontaneous limbic seizures was investigated in a rat model in which electrical tetanic stimulation of the angular bundle was applied for up to 90 min. This stimulation produced behavioural and electrographic seizures that led to a status epilepticus (SE) in most rats (71%). Long‐term EEG monitoring showed that the majority of the rats (67%) that underwent SE, displayed a progressive increase of seizure activity once the first seizure was recorded after a latent period of about 1 week. The other SE rats (33%) did not show this progression of seizure activity. We investigated whether these different patterns of evolution of spontaneous seizures could be related to differences in cellular or structural changes in the hippocampus. This was the case regarding the following changes. (i) Cell loss in the hilar region: in progressive SE rats this was extensive and bilateral whereas in nonprogressive SE rats it was mainly unilateral. (ii) Parvalbumin and somatostatin‐immunoreactive neurons: in the hilar region these were almost completely eliminated in progressive SE rats but were still largely present unilaterally in nonprogressive SE rats. (iii) Mossy fibre sprouting: in progressive SE rats, extensive mossy fibre sprouting was prominent in the inner molecular layer. In nonprogressive SE rats, mossy fibre sprouting was also present but less prominent than in progressive SE rats. Although mossy fibre sprouting has been proposed to be a prerequisite for chronic seizure activity in experimental temporal lobe epilepsy, the extent of hilar cell death also appears to be an important factor that differentiates between whether or not seizure progression will occur.

Potential new antiepileptogenic targets indicated by microarray analysis in a rat model for temporal lobe epilepsy.

To get insight into the mechanisms that may lead to progression of temporal lobe epilepsy, we investigated gene expression during epileptogenesis in the rat. RNA was obtained from three different brain regions [CA3, entorhinal cortex (EC), and cerebellum (CB)] at three different time points after electrically induced status epilepticus (SE): acute phase [group D (1 d)], latent period [group W (1 week)], and chronic epileptic period [group M (3–4 months)]. A group that was stimulated but that had not experienced SE and later epilepsy was also included (group nS). Gene expression analysis was performed using the Affymetrix Gene Chip System (RAE230A). We used GENMAPP and Gene Ontology to identify global biological trends in gene expression data. The immune response was the most prominent process changed during all three phases of epileptogenesis. Synaptic transmission was a downregulated process during the acute and latent phases. GABA receptor subunits involved in tonic inhibition were persistently downregulated. These changes were observed mostly in both CA3 and EC but not in CB. Rats that were stimulated but that did not develop spontaneous seizures later on had also some changes in gene expression, but this was not reflected in a significant change of a biological process. These data suggest that the targeting of specific genes that are involved in these biological processes may be a promising strategy to slow down or prevent the progression of epilepsy. Especially genes related to the immune response, such as complement factors, interleukins, and genes related to prostaglandin synthesis and coagulation pathway may be interesting targets.

Upregulation of metabotropic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy

Reactive gliosis is a prominent morphological feature of mesial temporal lobe epilepsy. Because astrocytes express glutamate receptors, we examined changes in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and transforming growth factor (TGF)‐β in glial cells of the hippocampal regions in an experimental rat model of spontaneous seizures. Rats that exhibited behavioural status epilepticus (SE) directly after 1 h of electrical angular bundle stimulation, displayed chronic spontaneous seizures after a latent period of 1–2 weeks as observed using continuous electrographic monitoring. SE resulted in hypertrophy of astrocytes and microglia activation throughout the hippocampus as revealed by immunolabelling studies. A dramatic, seizure intensity‐dependent increase in vimentin immunoreactivity (a marker for reactive astrocytes) was revealed in CA3 and hilar regions where prominent neuronal loss occurs. Increased vimentin labelling was first apparent 24 h after onset of SE and persisted up to 3 months. mGluR2/3 and mGluR5 protein expression increased markedly in glial cells of CA3 and hilus by 1 week after SE, and persisted up to 3 months after SE. Double immunolabelling of brain sections with vimentin confirmed co‐localization with glial fibrillary acidic protein (GFAP), mGluR2/3 and mGluR5 in reactive astrocytes. TGF‐β, a cytokine implicated in mGluR3‐mediated neuroprotection, was also upregulated during the first 3 weeks after SE throughout the hippocampus. This study demonstrates seizure‐induced upregulation of two mGluR subtypes in reactive astrocytes, which − together with the increased production of TGF‐β − may represent a novel mechanism for modulation of glial function and for changes in glial‐neuronal communication in the course of epileptogenesis.

Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins.

We examined the regulation of glutamate transporter protein expression after stimulation with selective metabotropic glutamate receptor (mGluR) agonists in cultured human glial cells. mGluR3 and mGluR5 are expressed in human astrocytes and in human glioma cells in vivo as well as in vitro, as shown by either RT‐PCR or western blot analysis. The selective group I agonist (S)‐3,5‐dihydroxyphenylglycine produced a significant down‐regulation of both GLAST and GLT‐1 protein expression in astrocytes cultured in the presence of growth factors. This condition mimics the morphology of reactive glial cells in vivo including an increased expression of mGluR5 protein (observed in pathological conditions). In contrast, (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine, a selective agonist of group II metabotropic glutamate receptors, positively modulates the expression of GLAST and GLT‐1 proteins. A similar opposite effect of (S)‐3,5‐dihydroxyphenylglycine and (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine was observed for the expression of EAAT3 protein in U373 glioblastoma cell line. Selective group I and II antagonists prevented these effects. Pharmacological inhibition of mitogen‐activated protein kinase and phosphatidylinositol‐3‐K pathways reduces the induction of GLT‐1 observed in response to the group II metabotropic glutamate receptor agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine. Thus, mGluR3 and mGluR5 can critically and differentially modulate the expression of glutamate transporters and may represent interesting pharmacological targets to regulate the extracellular levels of glutamate in pathological conditions.

Expression and Cellular Distribution of Multidrug Resistance–related Proteins in the Hippocampus of Patients with Mesial Temporal Lobe Epilepsy

Summary:  Purpose: This study investigated the cellular distribution of different multidrug resistance (MDR)‐related proteins such as P‐glycoprotein (P‐gp), the multidrug resistance–associated proteins (MRP) 1 and 2, and the major vault protein (MVP) in normal and sclerotic hippocampus of patients with medically refractory mesial temporal lobe epilepsy (MTLE).

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-1β signaling. Downstream signaling in the IL-1β 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-1β and blocked by IL-1β 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-1β 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.

Expression pattern of miR‐146a, an inflammation‐associated microRNA, in experimental and human temporal lobe epilepsy

Increasing evidence supports the involvement of inflammatory and immune processes in temporal lobe epilepsy (TLE). MicroRNAs (miRNA) represent small regulatory RNA molecules that have been shown to act as negative regulators of gene expression controlling different biological processes, including immune‐system homeostasis and function. We investigated the expression and cellular distribution of miRNA‐146a (miR‐146a) in a rat model of TLE as well as in human TLE. miR‐146a analysis in rat hippocampus was performed by polymerase chain reaction and immunocytochemistry at 1 week and 3–4 months after induction of status epilepticus (SE). Prominent upregulation of miR‐146a activation was evident at 1 week after SE and persisted in the chronic phase. The miR‐146a expression was confirmed to be present in reactive astrocytes. In human TLE with hippocampal sclerosis, increased astroglial expression of miR‐146a was observed mainly in regions where neuronal cell loss and reactive gliosis occurred. The increased and persistent expression of miR‐146a in reactive astrocytes supports the possible involvement of miRNAs in the modulation of the astroglial inflammatory response occurring in TLE and provides a target for future studies aimed at developing strategies against pro‐epileptogenic inflammatory signalling.

Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors.

The cell-specific distribution of multidrug resistance extrusion pumps was studied in developmental glioneuronal lesions, including focal cortical dysplasia (15 cases) and ganglioglioma (15 cases) from patients with medically intractable epilepsy. Lesional, perilesional, as well as normal brain regions were examined for the expression of the multidrug resistance gene 1 encoded P-glycoprotein (P-gp) and the multidrug resistance-associated protein 1 (MRP1) by immunocytochemistry. In normal brain MRP1 expression was below detection, whereas P-gp staining was present only in blood vessels. MRP1 and P-gp immunoreactivity was observed in dysplastic neurons of 11/15 cases of focal cortical dysplasia, as well as in the neuronal component of 14/15 ganglioglioma. Glial cells with astrocytic morphology within the lesion showed multidrug-resistant protein immunoreactivity (P-gp>MRP1). Moderate to strong MRP1 and P-gp immunoreactivity was observed in a population of large ballooned neuroglial cells. P-gp appeared to be most frequently expressed in glial fibrillary acidic protein-positive balloon cells (glial type), whereas MRP1 was more frequently expressed in microtubule-associated protein 2-positive balloon cells (neuronal type). In both types of lesions strong P-gp immunoreactivity was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal cells compared with normal cortex. The predominant intralesional cell-specific distribution of multidrug transporter proteins supports the hypothesis of a constitutive overexpression as common mechanism underlying the intrinsic pharmaco-resistance to antiepileptic drugs of both malformative and neoplastic glioneuronal developmental lesions.

Inhibition of the multidrug transporter P-glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats

Summary:  Purpose: Overexpression of multidrug transporters such as P‐glycoprotein (P‐gp) may play a significant role in pharmacoresistance, by preventing antiepileptic drugs (AEDs) from reaching their targets in the brain. Until now, many studies have described increased P‐gp expression in epileptic tissue or have shown that several AEDs act as substrates for P‐gp. However, definitive proof showing the functional involvement of P‐gp in pharmacoresistance is still lacking. Here we tested whether P‐gp contributes to pharmacoresistance to phenytoin (PHT) by using a specific P‐gp inhibitor in a model of spontaneous seizures in rats.