Marina de Wit

Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE may involve defects in the post-transcriptional regulation of gene expression. MicroRNAs (miRNAs) are non-coding RNAs that control the expression of genes at the post-transcriptional level. Here, we performed a genome-wide miRNA profiling study to examine whether miRNA-mediated mechanisms are affected in human mTLE. miRNA profiles of the hippocampus of autopsy control patients and two mTLE patient groups were compared. This revealed segregated miRNA signatures for the three different patient groups and 165 miRNAs with up- or down-regulated expression in mTLE. miRNA in situ hybridization detected cell type-specific changes in miRNA expression and an abnormal nuclear localization of select miRNAs in neurons and glial cells of mTLE patients. Of several cellular processes implicated in mTLE, the immune response was most prominently targeted by deregulated miRNAs. Enhanced expression of inflammatory mediators was paralleled by a reduction in miRNAs that were found to target the 3′-untranslated regions of these genes in reporter assays. miR-221 and miR-222 were shown to regulate endogenous ICAM1 expression and were selectively co-expressed with ICAM1 in astrocytes in mTLE patients. Our findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins thereby further facilitating the immune response. This mechanism may have broad implications given the central role of astrocytes and the immune system in human neurological disease. Overall, this work extends the current concepts of human mTLE pathogenesis to the level of miRNA-mediated gene regulation.

Possible role of the innate immunity in temporal lobe epilepsy.

Purpose: Temporal lobe epilepsy (TLE) is a multifactorial disease often involving the hippocampus. So far the etiology of the disease has remained elusive. In some pharmacoresistant TLE patients the hippocampus is surgically resected as treatment. To investigate the involvement of the immune system in human TLE, we performed large‐scale gene expression profiling on this human hippocampal tissue.

Depolarization-induced phosphorylation of the protein kinase C substrate B 50 (GAP 43) in rat cortical synaptosomes

Abstract: We studied the molecular events underlying K+‐induced phosphorylation of the neuron‐specific protein kinase C substrate B‐50. Rat cortical synaptosomes were prelabelled with 32P‐labelled orthophosphate. B‐50 phosphorylation was measured by an immunoprecipitation assay. In this system, various phorbol esters, as well as a synthetic diacylglycerol derivative, enhance B‐50 phosphorylation. K+ depolarization induces a transient enhancement of B‐50 phosphorylation, which is totally dependent on extracellular Ca2+. Also, the application of the Ca2+ ionophore A23187 induces B‐50 phosphorylation, but the magnitude and kinetics of A23187‐induced B‐50 phosphorylation differ from those induced by depolarization. The protein kinase inhibitors 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7), N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), and staurosporine antagonize K+‐ as well as PDB‐induced B‐50 phosphorylation, whereas trifluoperazine and calmidazolium are ineffective under both conditions. We suggest that elevation of the intracellular Ca2+ level after depolarization is a trigger for activation of protein kinase C, which subsequently phosphorylates its substrate B‐50. This sequence of events could be of importance for the mechanism of depolarization‐induced transmitter release.

N‐Terminal‐Specific Anti‐B‐50 (GAP‐43) Antibodies Inhibit Ca2+‐Induced Noradrenaline Release, B‐50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B‐50 (GAP‐43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin‐binding domain of B‐50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B‐50 into streptolysin O‐permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N‐terminal residues 39–43 of rat B‐50 dose‐dependently inhibited Ca2+‐induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C‐terminal‐directed (residues 132–213) anti‐B‐50 antibodies were without effect in the same dose range. NM2 inhibited PKC‐mediated B‐50 phosphorylation at Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32P‐B‐50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B‐50 in the absence of Ca2+. Similar concentrations of NM6 did not affect B‐50 phosphorylation or dephosphorylation or B‐50/calmodulin binding. We conclude that the N‐terminal residues 39–43 of the rat B‐50 protein play an important role in the process of Ca2+‐induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca2+‐ and phosphorylation‐dependent fashion.

Evidence for the Binding of Calmodulin to Endogenous B‐50 (GAP‐43) in Native Synaptosomal Plasma Membranes

Abstract: The neuron‐specific protein B‐50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B‐50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B‐50/CaM binding, we used the cross‐linker disuccimidyl suberate (DSS) to form a covalent B‐50/CaM complex, which is stable on SDS‐PAGE. Upon addition of DSS, purified B‐50 and calmodulin form a 70‐kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti‐B‐50 and anti‐CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70‐kDa B‐50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B‐50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B‐50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.

Interspecies Trait Genetics Reveals Association of Adcy8 with Mouse Avoidance Behavior and a Human Mood Disorder

BACKGROUND Identifying susceptibility genes for endophenotypes by studying analogous behaviors across species is an important strategy for understanding the pathophysiology underlying psychiatric disorders. This approach provides novel biological pathways plus validated animal models critical for selective drug development. One such endophenotype is avoidance behavior. METHODS In the present study, novel automated registration methods for longitudinal behavioral assessment in home cages are used to screen a panel of recently generated mouse chromosome substitution strains that are very powerful in quantitative trait loci (QTL) detection of complex traits. In this way, we identified chromosomes regulating avoidance behavior (increased sheltering preference) independent of motor activity levels (horizontal distance moved). Genetic information from the mouse QTL-interval was integrated with that from the homologous human linkage region for a mood disorder. RESULTS We genetically mapped a QTL for avoidance behavior on mouse chromosome 15, homologous with a human genome region (8q24) linked to bipolar disorder. Integrating the syntenic mouse QTL-interval with genotypes of 1868 BPD cases versus 14,311 control subjects revealed two associated genes (ADCY8 and KCNQ3). Adenylyl cyclase 8 (Adcy8) was differentially expressed in specific brain regions of mouse strains that differ in avoidance behavior levels. Finally, we showed that chronic infusion of the human mood stabilizer carbamazepine (that acts via adenylyl cyclase activity) significantly reduced mouse avoidance behavior, providing a further link between human mood disorders and this mouse home cage behavior. CONCLUSIONS Our data suggest that Adcy8 might encode a translational behavioral endophenotype of bipolar disorder.

Studies on the Role of B‐50 (GAP‐43) in the Mechanism of Ca2+‐Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B‐50, protein kinase C (PKC), and PKC‐mediated B‐50 phosphorylation in the mechanism of Ca2+‐induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin‐O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP‐dependent manner as a result of an elevation of the free Ca2+ concentration from 10−8 to 10−5M Ca2+ The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca2+‐induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca2+‐induced NA release was inhibited by either N‐ or C‐terminal‐directed anti‐B‐50 antibodies, confirming a role of B‐50 in the process of exocytosis. In addition, both anti‐B‐50 antibodies inhibited PKC‐mediated B‐50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC‐mediated B‐50 phosphorylation in Ca2+‐induced NA release. PKC pseudosubstrate PKC19‐36, which inhibited B‐50 phosphorylation (IC50 value, 10−5M), failed to inhibit Ca2+‐induced NA release, even when added before the Ca2+ trigger. Similar results were obtained with PKC inhibitor H‐7, whereas polymyxin B inhibited B‐50 phosphorylation as well as Ca2+‐induced NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC‐mediated B‐50 phosphorylation is initiated at a slightly higher Ca2+ concentration than NA release. Moreover, phorbol ester‐induced PKC down‐regulation was not paralleled by a decrease in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Finally, the Ca2+‐ and phorbol ester‐induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B‐50 is involved in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Evidence is presented challenging a role of PKC‐mediated B‐50 phosphorylation in the mechanism of NA exocytosis after Ca2+ influx. An involvement of PKC or PKC‐mediated B‐50 phosphorylation before the Ca2+ trigger is not ruled out. We suggest that the degree of B‐50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca2+ influx resulting in exocytosis of NA.

Protein expression profiling of inflammatory mediators in human temporal lobe epilepsy reveals co-activation of multiple chemokines and cytokines

Mesial temporal lobe epilepsy (mTLE) is a chronic and often treatment-refractory brain disorder characterized by recurrent seizures originating from the hippocampus. The pathogenic mechanisms underlying mTLE remain largely unknown. Recent clinical and experimental evidence supports a role of various inflammatory mediators in mTLE. Here, we performed protein expression profiling of 40 inflammatory mediators in surgical resection material from mTLE patients with and without hippocampal sclerosis, and autopsy controls using a multiplex bead-based immunoassay. In mTLE patients we identified 21 upregulated inflammatory mediators, including 10 cytokines and 7 chemokines. Many of these upregulated mediators have not previously been implicated in mTLE (for example, CCL22, IL-7 and IL-25). Comparing the three patient groups, two main hippocampal expression patterns could be distinguished, pattern I (for example, IL-10 and IL-25) showing increased expression in mTLE + HS patients compared to mTLE-HS and controls, and pattern II (for example, CCL4 and IL-7) showing increased expression in both mTLE groups compared to controls. Upregulation of a subset of inflammatory mediators (for example, IL-25 and IL-7) could not only be detected in the hippocampus of mTLE patients, but also in the neocortex. Principle component analysis was used to cluster the inflammatory mediators into several components. Follow-up analyses of the identified components revealed that the three patient groups could be discriminated based on their unique expression profiles. Immunocytochemistry showed that IL-25 IR (pattern I) and CCL4 IR (pattern II) were localized in astrocytes and microglia, whereas IL-25 IR was also detected in neurons. Our data shows co-activation of multiple inflammatory mediators in hippocampus and neocortex of mTLE patients, indicating activation of multiple pro- and anti-epileptogenic immune pathways in this disease.

Evidence for a relationship between B 50 (GAP 43) and [3H]noradrenaline release in rat brain synaptosomes

Phosphorylation of the neuron-specific substrate of protein kinase C (PKC), B-50 (GAP-43), was studied parallel with noradrenaline release in rat brain synaptosomes. Both could be evoked by treating the synaptosomes with high K+ or veratridine. Phorbol 12,13-dibutyrate enhanced depolarization-induced B-50 phosphorylation and noradrenaline release. To investigate the involvement of PKC-mediated B-50 phosphorylation in noradrenaline release, we applied a variety of kinase inhibitors. Prior to measuring the effects of these inhibitors in intact synaptosomes, we determined their effectivity and specificity in a membrane phosphorylation assay. H-7 most specifically inhibited PKC-dependent phosphorylation, whereas calmidazolium inhibited calmodulin-dependent phosphorylation. Polymyxin B affected both protein kinase systems. Only polymyxin B effectively inhibited noradrenaline release in the intact synaptosomes. We conclude that PKC as well as calmodulin-dependent processes are important for the release event. Data are discussed in view of the presumed function of B-50 as a calmodulin-binding protein.

Hippocampal distribution of vesicular glutamate transporter 1 in patients with temporal lobe epilepsy.

Purpose:  Vesicular glutamate transporters (VGLUTs) are responsible for loading synaptic vesicles with glutamate, determining the phenotype of glutamatergic neurons, and have been implicated in the regulation of quantal size and presynaptic plasticity. We analyzed VGLUT subtype expression in normal human hippocampus and tested the hypothesis that alterations in VGLUT expression may contribute to long‐term changes in glutamatergic transmission reported in patients with temporal lobe epilepsy (TLE).