Jasper J. Anink

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-1β. 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.

TOLL-LIKE RECEPTOR SIGNALING IN AMYOTROPHIC LATERAL SCLEROSIS SPINAL CORD TISSUE

Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the receptor for advanced glycation endproducts (RAGE) in the regulation of both innate and adaptive immunity in different pathologies associated with neuroinflammation. In the present study we investigated the expression and cellular distribution of TLR2, TLR4, RAGE and their endogenous ligand high mobility group box 1 (HMGB1) in the spinal cord of control (n=6) and sporadic ALS (n=12) patients. The immunohistochemical analysis of TLR2, TLR4 and RAGE showed increased expression in reactive glial cells in both gray (ventral horn) and white matter of ALS spinal cord. TLR2 was predominantly detected in cells of the microglia/macrophage lineage, whereas the TLR4 and RAGE was strongly expressed in astrocytes. Real-time quantitative PCR analysis confirmed the increased expression of both TLR2 and TLR4 and HMGB1 mRNA level in ALS patients. In ALS spinal cord, HMGB1 signal is increased in the cytoplasm of reactive glia, indicating a possible release of this molecule from glial cells. Our findings show increased expression of TLR2, TLR4, RAGE and HMGB1 in reactive glia in human ALS spinal cord, suggesting activation of the TLR/RAGE signaling pathways. The activation of these pathways may contribute to the progression of inflammation, resulting in motor neuron injury. In this context, future studies, using animal models, will be important to achieve a better understanding of these signaling pathways in ALS in view of the development of new therapeutic strategies.

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.

Fetal brain lesions in tuberous sclerosis complex: TORC1 activation and inflammation

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or TSC2 genes and characterized by developmental brain abnormalities. We defined the spectrum of brain abnormalities in fetal TSC brain ranging from 23 to 38 gestational weeks. We hypothesized (i) prenatal activation of the target‐of‐rapamycin complex 1 (TORC1) signaling pathway; and (ii) activation of inflammatory pathways in fetal brain lesions. Immunocytochemical analysis of cortical tubers, as well as subependymal lesions in all cases confirmed the cell‐associated activation of the TORC1 signaling pathway in both the cortical tubers and subependymal lesions (including a congenital subependymal giant cell astrocytoma) with expression of pS6, p4EBP1 and c‐myc proteins, as well as of p70 S6 kinase 1. The lesions contained macrophages and T‐lymphocytes; giant cells within the lesions expressed inflammatory response markers including major histocompatibility complex class I and II, Toll‐like receptors (TLR) 2 and 4 and receptor for advanced glycation end products (RAGE). These observations indicate that brain malformations in TSC are likely a consequence of increased TORC1 activation during embryonic brain development. We also provide evidence supporting the possible immunogenicity of giant cells and the early activation of inflammatory pathways in TSC brain.

BRAF V600E Mutation Is Associated with mTOR Signaling Activation in Glioneuronal Tumors

BRAF V600E mutations have been recently reported in glioneuronal tumors (GNTs). To evaluate the expression of the BRAF V600E mutated protein and its association with activation of the mammalian target of rapamycin (mTOR) pathway, immunophenotype and clinical characteristics in GNTs, we investigated a cohort of 174 GNTs. The presence of BRAF V600E mutations was detected by direct DNA sequencing and BRAF V600E immunohistochemical detection. Expression of BRAF‐mutated protein was detected in 38/93 (40.8%) gangliogliomas (GGs), 2/4 (50%) desmoplastic infantile gangliogliomas (DIGs) and 23/77 (29.8%) dysembryoplastic neuroepithelial tumors (DNTs) by immunohistochemistry. In both GGs and DNTs, the presence of BRAF V600E mutation was significantly associated with the expression of CD34, phosphorylated ribosomal S6 protein (pS6; marker of mTOR pathway activation) in dysplastic neurons and synaptophysin (P < 0.05). In GGs, the presence of lymphocytic cuffs was more frequent in BRAF‐mutated cases (31 vs. 15.8%; P = 0.001). The expression of both BRAF V600E and pS6 was associated with a worse postoperative seizure outcome in GNT (P < 0.001). Immunohistochemical detection of BRAF V600E‐mutated protein may be valuable in the diagnostic evaluation of these glioneuronal lesions and the observed association with mTOR activation may aid in the development of targeted treatment involving specific pathogenic pathways.

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.

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations.

mTOR Hyperactivation in down syndrome hippocampus appears early during development.

The mammalian target of rapamycin (mTOR) signaling pathway is a key developmental pathway involved in mechanisms underlying cellular aging and neurodegeneration. We hypothesized that its deregulation may occur during early brain development in patients with Down syndrome (DS). The expression patterns and cellular distribution of components of mTOR signaling (phosphorylated S6, phosphorylated S6 kinase, phosphorylated eukaryotic initiation factor 4E binding protein 1, and phosphorylated mTOR) were investigated in developing hippocampi from controls and patients with DS and from adults with DS and Alzheimer disease-associated pathology using immunocytochemistry. In control hippocampi, only phosphorylated S6 was detected prenatally (19-41 gestational weeks); it became undetectable 2 months postnatally. Increased expression of phosphorylated S6, phosphorylated S6 kinase, phosphorylated eukaryotic initiation factor 4E binding protein 1, and phosphorylated mTOR was observed in DS hippocampus compared with controls. Phosphorylated S6 and phosphorylated S6 kinase were detected prenatally and persisted throughout postnatal development. Prominent expression of mTOR components was observed in pyramidal neurons with granulovacuolar degeneration and in neurons containing neurofibrillary tangles in the hippocampi of DS subjects with Alzheimer disease pathology. These findings suggest that a dysregulated mTOR pathway may contribute to both early hippocampal developmental abnormalities and hippocampal functional impairment developing before neurodegeneration. Moreover, the expression patterns of mTOR components in adult DS hippocampus support its association with Alzheimer disease-related histopathologic changes.

Cell injury and premature neurodegeneration in focal malformations of cortical development.

Several lines of evidence suggest that cell injury may occur in malformations of cortical development associated with epilepsy. Moreover, recent studies support the link between neurodevelopmental and neurodegenerative mechanisms. We evaluated a series of focal cortical dysplasia (FCD, n = 26; type I and II) and tuberous sclerosis complex (TSC, n = 6) cases. Sections were processed for terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate nick‐end labeling (TUNEL) labeling and immunohistochemistry using markers for the evaluation of apoptosis signaling pathways and neurodegeneration‐related proteins/pathways. In both FCD II and TSC specimens, we observed significant increases in both TUNEL‐positive and caspase–3‐positive cells compared with controls and FCD I. Expression of β‐amyloid precursor protein was observed in neuronal soma and processes in FCD II and TSC. In these specimens, we also observed an abnormal expression of death receptor‐6. Immunoreactivity for phosphorylated tau was only found in older patients with FCD II and TSC. In these cases, prominent nuclear/cytoplasmic p62 immunoreactivity was detected in both dysmorphic neurons and balloon/giant cells. Our data provide evidence of complex, but similar, mechanisms of cell injury in focal malformations of cortical development associated with mammalian target of rapamycin pathway hyperactivation, with prominent induction of apoptosis‐signaling pathways and premature activation of mechanisms of neurodegeneration.

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

ObjectiveDecreased 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-1β (IL-1β) on Kir4.1 mRNA and protein expression.MethodsWe investigated Kir4.1 (Kcnj10) and IL-1β 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).ResultsTwenty-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-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. 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-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1).ConclusionsTaken 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-1β.