Balloon cells promote immune system activation in focal cortical dysplasia type 2b.

AIMS: Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro-epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. METHODS: The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well-characterised FCD 2 cohort. RESULTS: Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T-lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. CONCLUSION: We conclude that, next to mutation-driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b.

Homing of allogeneic nestin-positive hair follicle-associated pluripotent stem cells after maternal transplantation in experimental model of cortical dysplasia.

An embryo has the capability to accept allo- or xeno-geneic cells, which probably makes it an ideal candidate for stem cell transplantation of various cerebral cortex abnormalities, such as cortical dysplasia. The aim of this study was to determine hair follicle-associated pluripotent (HAP) stem cells homing into various organs of mother and fetus. Cells were obtained, analyzed for immunophenotypic features, and then labelled with CM-Dil; nestin(+)HAP stem cells or media phosphate-buffered saline (PBS) were intravenously delivered on day 16 of gestation in BALB/c mice, which intraperitoneally received methylazoxymethanol (MAM) one day in advance, and homing was assessed at 24 h after cell injection. Flow cytometry and immunocytochemistry manifested positive expression of nestin in HAP stem cells. For both mother and fetus, brain, lungs, liver, and spleen were the host organs for cell implants. For the brain, the figure was considerably higher in fetus, 4.05 ± 0.5% (p ≤ 0.05 vs. mother). MAM-injected mice had a downward trend for SDF-1α and CXCR4 (p ≤ 0.05 vs. control), but HAP stem cells group showed an upward trend for CXCR4 (p ≤ 0.05 vs. MAM). We conclude the HAP stem cells show homing potential in experimental cortical dysplasia, which may permit these cells to be a target in future work on prenatal therapy of neural disorders.

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.

Inflammation in epilepsy: clinical observations.

Over the past decade, an increasing number of observations indicate that activation of inflammatory processes occurs in variety of focal epilepsies. Understanding the feature and consequences of neuroinflammation, including the contribution to development and perpetuation of seizures, as well as to mood or cognitive dysfunction, is a major requisite for delineating its role in epilepsy. The present article discusses the most recent observations supporting the involvement of the inflammatory response in human focal epilepsy. It also evaluates emerging evidence concerning the possibility to identify epilepsy-associated inflammatory biomarkers in cerebrospinal fluid and serum, as well as the potential application of neuroimaging approaches to study the inflammatory reactions in chronic epilepsy patients in vivo, aiming to improve the recognition of appropriate patient populations who might benefit from antiinflammatory or immunomodulatory therapies.

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.

Doublecortin expression in focal cortical dysplasia in epilepsy.

PURPOSE: Doublecortin (DCX) is a microtubule-associated protein with regulatory roles in radial and tangential migration of neurons during cortical development. In normal adult cortex there is restricted expression, and DCX is widely used as a marker of neurogenesis. Imperfect corticogenesis is thought to underpin many focal cortical pathologies in epilepsy surgical series, including focal cortical dysplasia (FCD). The aim was to study DCX expression patterns in such lesions compared to normal developing and mature cortex. METHOD: Cases of FCD types Ia (13) and IIb (4), pediatric hippocampal sclerosis (HS) (5), temporal lobe sclerosis (5), glioneuronal tumors (5), gray matter heterotopia (3), and control tissues (16) from a wide age range [20 gestational weeks (GW) to 85 years] were studied using immunohistochemistry to DCX. RESULTS: In controls and all epilepsy cases, perinuclear labeling of small round cells (SRCs) and satellite perineuronal cells was observed in both postmortem and surgical tissues. In FCD Ia up to the age of 4 years, prominent DCX-positive (DCX(+)), immature cells were present along the junction of layers I and II, with processes extending into the molecular layer. These cell types were not a significant feature in other pathologies, which showed multipolar DCX(+) cells or labeling of dysmorphic cells throughout the cortex. DISCUSSION: Persistent cellular DCX expression is confirmed in normal adult cortex. Characteristic expression patterns in layer II of FCD Ia could indicate delayed or abnormal cortical maturation rather than ongoing cytogenesis. This could be indicative of enhanced local cortical plasticity as well as a potential diagnostic feature of this type of pathology.

Doublecortin-like (DCL) expression in focal cortical dysplasia and cortical tubers.

PURPOSE: Focal cortical dysplasia type IIB (FCD IIB) and cortical tubers of patients with tuberous sclerosis complex (TSC) are malformations of cortical development that are frequently associated with intractable epilepsy. Their histopathologic and molecular features suggest developmental abnormalities during the early stages of cortical development, which may involve neural progenitor cells. The aim of our study was to define the expression and cell-specific distribution of doublecortin-like (DCL), a protein critically involved in neuronal division and radial migration during early corticogenesis, in both FCD and TSC. METHODS: DCL was studied in epilepsy surgery cases with FCD IIB (n = 8) and TSC (cortical tubers; n = 8) using immunocytochemistry, confocal analysis, and Western blotting. RESULTS: Autopsy and surgical control neocortical specimens were characterized by modest DCL immunoreactivity (IR) throughout all cortical layers, but DCL IR was not detectable in the white matter. Balloon cells (BCs) in FCD and giant cells (GCs) in TSC expressed strong DCL IR. Most of the large dysplastic neurons (DNs) were positive for DCL in both FCD and TSC. Coexpression of DCL with the neural progenitor or neuroblast markers nestin, GFAPdelta, and doublecortin was observed in both FCD and TSC specimens. The increased DCL expression within the dysplastic cortex, compared to control cortex, was confirmed by Western blot analysis. DISCUSSION: The prominent postnatal expression of DCL by BCs/GCs and DNs in FCD and TSC supports an important role for this microtubule associated protein, also during early human cortical development, which could be relevant to the pathogenesis of these developmental glioneuronal malformations.

Ki-67 immunoreactivity in type II malformations of cortical development.

Malformations of cortical development (MCD) are a well-recognized cause of chronic epilepsy. Most MCD are defined by the neuronal component of the lesion and are considered to be relatively "static" lesions. Twenty-one cases of MCD type II (Taylor type cortical dysplasia) were retrospectively evaluated with Ki-67 antibody looking for evidence of cell proliferation and evaluating populations of cells that may be proliferating. Resections were from 13 males and 8 females who ranged in age at the time of surgery from 6 weeks to 57 years (mean 8.6 y) and who had a duration of seizures before surgery of 1.5 months to 34.4 years (mean 6.5 y). Dysmorphic neurons were observed in all cases and balloon cells in 18/21 (86%) cases. Ki-67 labeling indices ranged from 0.2% to 4.9% (mean 2.0%). Coimmunolabeling with Ki-67 and antibodies to glial fibrillary acidic protein, CD68, and CD45RB showed that the majority of the Ki-67-positive cells were astrocytic (glial fibrillary acidic protein positive). In 9/21 cases (43%), endothelial cell staining with Ki-67 was also observed. These results suggest that low rates of cell proliferation are observable in type II MCD. The proliferating cells appear to be primarily astrocytic and endothelial in nature and suggest that these lesions are not static. Dysmorphic neurons and balloon cells in MCD were not observed to stain with Ki-67.