Karin Boer

The IL-1β system in epilepsy-associated malformations of cortical development

Focal cortical dysplasia (FCD) and glioneuronal tumors (GNT) are recognized causes of chronic intractable epilepsy. The cellular mechanism(s) underlying their epileptogenicity remain largely unknown. Compelling evidence in experimental models of seizures indicates an important role of interleukin (IL)-1beta in the mechanisms of hyperexcitability leading to the occurrence of seizures. We immunocytochemically investigated the brain expression and cellular distribution pattern of IL-1beta, IL-1 receptor (IL-1R) types I and II and IL-1R antagonist (IL-1Ra) in FCD and GNT specimens, and we correlate these parameters with the clinical history of epilepsy in patients with medically intractable seizures. In normal control cortex, and in perilesional regions with histologically normal cortex, IL-1beta, IL-1Rs and IL-1Ra expression was undetectable. In all FCD and GNT specimens, IL-1beta and its signalling receptor IL-1RI were highly expressed by more than 30% of neurons and glia whereas the decoy receptor IL-RII and IL-Ra were expressed to a lesser extent by approximately 10% and 20% of cells, respectively. These findings show a high expression of IL-1beta and its functional receptor (IL-1RI) in FCD and GNT specimens together with a relative paucity of mechanisms (IL-1RII and IL-1Ra) apt to inactivate IL-1beta actions. Moreover, the number of IL-1beta- and IL-1RI-positive neurons was positively correlated with the frequency of seizures, whereas the number of IL-1Ra-positive neurons and astroglial cells was negatively correlated with the duration of epilepsy prior to surgery. The expression of IL-1beta family members in these developmental lesions may contribute to their intrinsic and high epileptogenicity, thus possibly representing a novel target for antiepileptic strategies.

Inflammatory processes in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex

Cortical tubers and subependymal giant cell tumors (SGCT) are two major cerebral lesions associated with tuberous sclerosis complex (TSC). In the present study, we investigated immunocytochemically the inflammatory cell components and the induction of two major pro-inflammatory pathways (the interleukin (IL)-1beta and complement pathways) in tubers and SGCT resected from TSC patients. All lesions were characterized by the prominent presence of microglial cells expressing class II-antigens (HLA-DR) and, to a lesser extent, the presence of CD68-positive macrophages. We also observed perivascular and parenchymal T lymphocytes (CD3(+)) with a predominance of CD8(+) T-cytotoxic/suppressor lymphoid cells. Activated microglia and reactive astrocytes expressed IL-1beta and its signaling receptor IL-1RI, as well as components of the complement cascade, such as C1q, C3c and C3d. Albumin extravasation, with uptake in astrocytes, was observed in both tubers and SGCT, suggesting that alterations in blood brain barrier permeability are associated with inflammation in TSC-associated lesions. Our findings demonstrate a persistent and complex activation of inflammatory pathways in cortical tubers and SGCT.

Differential expression patterns of chloride transporters, Na+-K+-2Cl−-cotransporter and K+-Cl−-cotransporter, in epilepsy-associated malformations of cortical development

Malformations of cortical development are recognized causes of chronic medically intractable epilepsy. An increasing number of observations suggests an important role for cation-chloride co-transporters (CCTs) in controlling neuronal function. Deregulation of their expression may contribute to the mechanisms of hyperexcitability that lead to seizures. In the present study the expression and cell-specific distribution of Na+-K+-2Cl--cotransporter (NKCC1) and K+-Cl--cotransporter (KCC2) were studied immunocytochemically in different developmental lesions, including focal cortical dysplasia (FCD) type IIB (n=9), hemimegalencephaly (HMEG, n=6) and ganglioglioma (GG, n=9) from patients with medically intractable epilepsy and in age-matched controls. In normal control adult cortex, NKCC1 displayed low neuronal and glial expression levels. In contrast KCC2 showed strong and diffuse neuropil staining. Notable glial immunoreactivity (IR) was not found for KCC2. NKCC1 was highly expressed in the majority of FCD, HMEG and GG specimens. NKCC1 IR was observed in neurons of different size, including large dysplastic neurons, in balloon cells (in FCD and HMEG cases) and in glial cells with astrocytic morphology. The immunoreactivity pattern of KCC2 in FCD, HMEG and GG specimens was characterized by less neuropil staining and more intrasomatic IR compared with control. KCC2 IR was observed in neurons of different size, including large dysplastic neurons, but not in balloon cells or in glial cells with astrocytic morphology. Double-labeling experiments confirmed the differential cellular distribution of the two CCTs and their expression in GABA(A) receptor (alpha1 subunit)-positive dysplastic neurons. The cellular distribution of CCTs, with high expression of NKCC1 in dysplastic neurons and altered subcellular distribution of KCC2 resembles that of immature cortex and suggests a possible contribution of CCTs to the high epileptogenicity of malformations of cortical development.

Evidence of activated microglia in focal cortical dysplasia

Focal cortical dysplasia (FCD), which is caused by malformations of cortical development, is known to be a major cause of intractable epilepsy. Cortical laminar disorganization and the presence of abnormal neuronal and astroglial cell types are histological characteristics of FCD. Though, little information is known about the microglia/macrophage cell system in FCD and its possible contribution to the high epileptogenesis of this disorder. In the present study, the distribution of cells of the microglia/macrophage lineage was studied in 20 specimens of FCD (type II) by immunocytochemistry for CD68 and human HLA-DR. A significant number of microglial cells and macrophages were observed within the dysplastic cortex. The mean number of CD68- and HLA-DR-positive cells was significantly higher in FCD specimens than in normal-appearing control cortex obtained at autopsy. HLA-DR-positive cells, which represent activated microglia, were localized around blood vessels and also clustered around dysplastic neuronal cells. The density of these activated HLA-DR-positive microglial cells correlated with the duration of epilepsy, as well as with the frequency of seizures prior to surgical resection. CD68-positive macrophages were mainly located around vessels and the number of these cells did not correlate with seizure frequency, neither with the duration of symptoms prior to surgical resection. In conclusion, our findings demonstrate a specific and persistent increase in the numerical density of HLA-DR-positive activated microglia within the dysplastic region, supporting the contribution of the inflammatory response and proinflammatory molecules to the epileptogenicity of FCD.

Clinicopathological and immunohistochemical findings in an autopsy case of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder caused by mutations in either the TSC1 or TSC2 genes and characterized by developmental brain abnormalities. In the present study we discuss the neuropathological findings of a 32‐year‐old patient with a germ‐line mutation in the TSC2 gene. Post mortem MRI combined with histology and immunocytochemical analysis was applied to demonstrate widespread anatomical abnormalities of gray and white matter structure. TSC brain lesions were analyzed for loss of heterozygosity (LOH) on chromosome 16p13. The neuropathological supratentorial abnormalities were represented by multiple subependymal nodules (SENs) and cortical tubers. In addition to cerebral cortical lesions, cerebellar lesions and hippocampal sclerosis were also observed. LOH was not found in the cortical tubers and SENs of this patient. Immunocytochemical analysis of the TSC brain lesions confirmed the cell‐specific activation of the mTOR pathway in cortical tubers, SENs and cerebellum, as well as differential cellular localization of hamartin and tuberin, the TSC1 and TSC2 gene products. Examination of the pathological brain regions revealed activated microglial cells and disruption of blood‐brain barrier permeability. Predominant intralesional cell‐specific distribution was also detected for the multidrug transporter protein P‐gp, possibly explaining the mechanisms underlying the pharmacoresistance to antiepileptic drugs. Autopsy findings confirm the complexity of the brain abnormalities encountered in TSC patients and proved useful in clarifying certain aspects of the pathogenesis, epileptogenesis and pharmacoresistance of TSC lesions.

Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy.

Purpose:u2002 We previously showed that gene expression of synaptic vesicle protein 2A (SV2A), the binding site for the antiepileptic drug levetiracetam, is reduced during epileptogenesis in the rat. Since absence of SV2A has been associated with increased epileptogenicity, changes in expression of SV2A could have consequences for the progression of epilepsy. Therefore we investigated hippocampal SV2A protein expression of temporal lobe epilepsy (TLE) patients and in rats during epileptogenesis and in the chronic epileptic phase.

GENE EXPRESSION PROFILE ANALYSIS OF EPILEPSY-ASSOCIATED GANGLIOGLIOMAS

Gangliogliomas (GG) constitute the most frequent tumor entity in young patients undergoing surgery for intractable epilepsy. The histological composition of GG, with the presence of dysplastic neurons, corroborates their maldevelopmental origin. However, their histogenesis, the pathogenetic relationship with other developmental lesions, and the molecular alterations underlying the epileptogenicity of these tumors remain largely unknown. We performed gene expression analysis using the Affymetrix Gene Chip System (U133 plus 2.0 array). We used GENMAPP and the Gene Ontology database to identify global trends in gene expression data. Our analysis has identified various interesting genes and processes that are differentially expressed in GG when compared with normal tissue. The immune and inflammatory responses were the most prominent processes expressed in GG. Several genes involved in the complement pathway displayed a high level of expression compared with control expression levels. Higher expression was also observed for genes involved in cell adhesion, extracellular matrix and proliferation processes. We observed differential expression of genes as cyclin D1 and cyclin-dependent kinases, essential for neuronal cell cycle regulation and differentiation. Synaptic transmission, including GABA receptor signaling was an under-expressed process compared with control tissue. These data provide some suggestions for the molecular pathogenesis of GG. Furthermore, they indicate possible targets that may be investigated in order to dissect the mechanisms of epileptogenesis and possibly counteract the epileptogenic process in these developmental lesions.

Inhibitory networks in epilepsy-associated gangliogliomas and in the perilesional epileptic cortex

Developmental glioneuronal lesions, such as gangliogliomas (GG) are increasingly recognized causes of chronic pharmaco-resistant epilepsy. It has been postulated that chronic epilepsy in patients with malformations of cortical development is associated with dysfunction of the inhibitory GABA-ergic system. We aimed to identify the subtypes of interneurons present within GG specimens and the expression and cellular distribution patterns of GABA receptors (GABAR) and GABA transporter 1 (GAT1). The expression of the various components of the GABA-ergic system were also analyzed in the perilesional cortex. We investigated the expression of parvalbumin, calbindin, calretinin, GABA(A)R (a1 subunit)(,) GABA(B) (R1 and R2) and GAT-1 using immunocytochemistry in 30 specimens of GG obtained during epilepsy surgery, including 10 cases with sufficient amount of perilesional cortex. Immunocytochemistry for calbindin (CB), calretinin (CR) and parvalbumin (PV) demonstrate the presence of inhibitory neurons of different subtypes within the GG specimens. Calcium-binding protein-positive interneurons represent a small fraction of the total neuronal population. Both GABA(A)R and GABA(B)R (R1 and R2) subtypes were detected within the neuronal component of GG specimens. In addition, GABA(B)R2 immunoreactivity (IR) was observed in glial cells. GG specimens displayed also expression of GAT-1 IR. Compared to normal cortex, the density of PV- and CB-immunoreactive interneurons was reduced in the perilesional cortex of GG patients, whereas CR-labeling was similar to that observed in normal cortex. GAT-1 IR was also significantly reduced in the perilesional specimens. The cellular distribution of components of the GABA-ergic system in GG, together with the perilesional changes suggest that alterations of the GABA-ergic system may contribute to the complex abnormal functional network of these highly epileptogenic developmental lesions.

Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsy-associated lesional pathologies.

Aims: Glial fibrillary acidic protein (GFAP)‐δ is a novel isoform that differs in its C‐terminal sequence from other GFAP isoforms. Previous studies suggest restriction of expression to the subpial layer, subventricular zone and the subgranular zone astrocytes, with an absence in pathological conditions causing reactive gliosis. GFAP‐δ is speculated to have roles in regulation of astrocyte size and motility and a subpopulation of GFAP‐δ‐positive glia may be multipotent stem cells. The aim of this study was to investigate its expression in common causes of lesion‐related refractory epilepsy. Methods: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD) type IIB, cortical tuberous sclerosis (TSC) lesions, gangliogliomas, grey matter heterotopias and hemimegalencephaly from a wide age range of patients using both surgical and post mortem tissue specimens were studied. Results: GFAP‐δ expression was observed in CA4 and CA1 astrocytes in HS with less frequent labelling in the granule cell layer, even where granule cell dispersion was present. No significant labelling was noted in the subiculum in HS cases or in any subfields in non‐HS epilepsy cases. Balloon cells in FCDIIB and hemimegalencephaly, giant cells in TSC and the astrocytic component of gangliogliomas showed immunoreactivity, colocalizing with conventional GFAP. No neuronal expression for GFAP‐δ was seen in any of the pathologies. Quantitative analysis in 10 FCDIIB and five TSC cases revealed greater numbers of GFAP‐δ‐positive balloon cells than conventional GFAP. There was no GFAP‐δ expression within nodular heterotopia. Conclusions: GFAP‐δ expression patterns in HS overall appears to mirror regional reactive gliosis. It is a useful marker for the demonstration of balloon cells in FCD and TSC, which may be relevant to their abnormal size and localization. The lack of GFAP‐δ within heterotopia supports their composition from cells destined for deeper cortical layers.

GFAPdelta in radial glia and subventricular zone progenitors in the developing human cortex.

A subpopulation of glial fibrillary acidic protein (GFAP)-expressing cells located along the length of the lateral ventricles in the subventricular zone (SVZ) have been identified as the multipotent neural stem cells of the adult mammalian brain. We have previously found that, in the adult human brain, a splice variant of GFAP, termed GFAPδ, was expressed specifically in these cells. To investigate whether GFAPδ is also present in the precursors of SVZ astrocytes during development and whether GFAPδ could play a role in the developmental process, we analyzed GFAPδ expression in the normal developing human cortex and in the cortex of foetuses with the migration disorder lissencephaly type II. We demonstrated for the first time that GFAPδ is specifically expressed in radial glia and SVZ neural progenitors during human brain development. Expression of GFAPδ in radial glia starts at around 13 weeks of pregnancy and disappears before birth. GFAPδ is continuously expressed in the SVZ progenitors at later gestational ages and in the postnatal brain. Co-localization with Ki67 proved that these GFAPδ-expressing cells are able to proliferate. Furthermore, we showed that the expression pattern of GFAPδ was disturbed in lissencephaly type II. Overall, these results suggest that the adult SVZ is indeed a remnant of the foetal SVZ, which develops from radial glia. Furthermore, we provide evidence that GFAPδ can distinguish resting astrocytes from proliferating SVZ progenitors.