Experimental early-life febrile seizures cause a sustained increase in excitatory neurotransmission in newborn dentate granule cells.

Prolonged febrile seizures (FS) are a risk factor for the development of hippocampal-associated temporal lobe epilepsy. The dentate gyrus is the major gateway to the hippocampal network and one of the sites in the brain where neurogenesis continues postnatally. Previously, we found that experimental FS increase the survival rate and structural integration of newborn dentate granule cells (DGCs). In addition, mature post-FS born DGCs express an altered receptor panel. Here, we aimed to study if these molecular and structural changes are accompanied by an altered cellular functioning. Experimental FS were induced by hyperthermia in 10-days-old Sprague-Dawley rats. Proliferating progenitor cells were labeled the next day by injecting green fluorescent protein expressing retroviral particles bilaterally in the dentate gyri. Eight weeks later, spontaneous excitatory and inhibitory postsynaptic events (sEPSCs and sIPSCs, respectively) were recorded from labeled DGCs using the whole-cell patch-clamp technique. Experimental FS resulted in a robust decrease of the inter event interval (p < .0001) and a small decrease of the amplitude of sEPSCs (p < .001). Collectively the spontaneous excitatory charge transfer increased (p < .01). Experimental FS also slightly increased the frequency of sIPSCs (p < .05), while the amplitude of these events decreased strongly (p < .0001). The net inhibitory charge transfer remained unchanged. Experimental, early-life FS have a long-term effect on post-FS born DGCs, as they display an increased spontaneous excitatory input when matured. It remains to be established if this presents a mechanism for FS-induced epileptogenesis.

Experimental febrile seizures increase dendritic complexity of newborn dentate granule cells.

OBJECTIVE: Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG. METHODS: Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology. RESULTS: At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks. SIGNIFICANCE: Experimental FS increase dendritic complexity and the number of mushroom-type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied.

Proteomic Alterations in B Lymphocytes of Sensitized Mice in a Model of Chemical-Induced Asthma.

INTRODUCTION AND AIM: The role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the "sensitized signature" of B lymphocytes comparing TDI-sensitized mice with control mice. RESULTS: Sixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses. CONCLUSION: This study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.

Neuropeptides as targets for the development of anticonvulsant drugs.

Epilepsy is a common neurological disorder characterized by recurrent seizures. These seizures are due to abnormal excessive and synchronous neuronal activity in the brain caused by a disruption of the delicate balance between excitation and inhibition. Neuropeptides can contribute to such misbalance by modulating the effect of classical excitatory and inhibitory neurotransmitters. In this review, we discuss 21 different neuropeptides that have been linked to seizure disorders. These neuropeptides show an aberrant expression and/or release in animal seizure models and/or epilepsy patients. Many of these endogenous peptides, like adrenocorticotropic hormone, angiotensin, cholecystokinin, cortistatin, dynorphin, galanin, ghrelin, neuropeptide Y, neurotensin, somatostatin, and thyrotropin-releasing hormone, are able to suppress seizures in the brain. Other neuropeptides, such as arginine-vasopressine peptide, corticotropin-releasing hormone, enkephalin, ß-endorphin, pituitary adenylate cyclase-activating polypeptide, and tachykinins have proconvulsive properties. For oxytocin and melanin-concentrating hormone both pro- and anticonvulsive effects have been reported, and this seems to be dose or time dependent. All these neuropeptides and their receptors are interesting targets for the development of new antiepileptic drugs. Other neuropeptides such as nesfatin-1 and vasoactive intestinal peptide have been less studied in this field; however, as nesfatin-1 levels change over the course of epilepsy, this can be considered as an interesting marker to diagnose patients who have suffered a recent epileptic seizure.

Origin and functional diversification of an amphibian defense peptide arsenal.

The skin secretion of many amphibians contains an arsenal of bioactive molecules, including hormone-like peptides (HLPs) acting as defense toxins against predators, and antimicrobial peptides (AMPs) providing protection against infectious microorganisms. Several amphibian taxa seem to have independently acquired the genes to produce skin-secreted peptide arsenals, but it remains unknown how these originated from a non-defensive ancestral gene and evolved diverse defense functions against predators and pathogens. We conducted transcriptome, genome, peptidome and phylogenetic analyses to chart the full gene repertoire underlying the defense peptide arsenal of the frog Silurana tropicalis and reconstruct its evolutionary history. Our study uncovers a cluster of 13 transcriptionally active genes, together encoding up to 19 peptides, including diverse HLP homologues and AMPs. This gene cluster arose from a duplicated gastrointestinal hormone gene that attained a HLP-like defense function after major remodeling of its promoter region. Instead, new defense functions, including antimicrobial activity, arose by mutation of the precursor proteins, resulting in the proteolytic processing of secondary peptides alongside the original ones. Although gene duplication did not trigger functional innovation, it may have subsequently facilitated the convergent loss of the original function in multiple gene lineages (subfunctionalization), completing their transformation from HLP gene to AMP gene. The processing of multiple peptides from a single precursor entails a mechanism through which peptide-encoding genes may establish new functions without the need for gene duplication to avoid adaptive conflicts with older ones.

Proteome changes in auricular lymph nodes and serum after dermal sensitization to toluene diisocyanate in mice.

Some reactive chemicals, such as diisocyanates, are capable of initiating an allergic response, which can lead to occupational asthma after a latency period. Clinical symptoms such as cough, wheezing, and dyspnea occur only late, making it difficult to intervene at an early stage. So far, most studies using proteomics in lung research have focused on comparisons of healthy versus diseased subjects. Here, using 2D-DIGE, we explored proteome changes in the local draining lymph nodes and serum of mice dermally sensitized once or twice with toluene-2,4-diisocyanate (TDI) before asthma is induced. In the lymph nodes, we found 38 and 58 differentially expressed proteins after one and two treatments, respectively, between TDI-treated and vehicle-treated mice. In serum, seven and 16 differentially expressed proteins were detected after one and two treatments, respectively. We identified 80-85% of the differentially expressed proteins by MS. Among them, lymphocyte-specific protein-1, coronin 1a, and hemopexin were verified by Western blotting or ELISA in an independent group of mice. This study revealed alterations in the proteomes early during sensitization in a mouse model before the onset of chemical-induced asthma. If validated in humans, these changes could lead to earlier diagnosis of TDI-exposed workers.

Purification, molecular cloning and functional characterization of HelaTx1 (Heterometrus laoticus): the first member of a new κ-KTX subfamily.

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 µM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.

Molecular diversity of the telson and venom components from Pandinus cavimanus (Scorpionidae Latreille 1802): transcriptome, venomics and function.

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7-30 µg and a paralysis or lethal effect was observed at 30 µg of venom (death at 1.5 µg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 µg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage-gated Na(+) and K(+) channels. Some fractions block K(+) currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC-MS/MS and MALDI-TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom.

Conservation of fruitless' role as master regulator of male courtship behaviour from cockroaches to flies.

In Drosophila melanogaster, male courtship behaviour is regulated by the fruitless gene. In D. melanogaster, fruitless encodes a set of putative transcription factors that are sex-specifically spliced. Male-specific variants are necessary and sufficient to elicit male courtship behaviour. Fruitless sequences have been reported in other insect species, but there are no data available on their functional role. In the present work, we cloned and sequenced fruitless in males of the German cockroach, Blattella germanica, and we studied its expression in male brain and testes. B. germanica fruitless encodes a 350-amino acid protein with BTB and Zinc finger domains typical of fruitless sequences. Upon RNAi-mediated knockdown of fruitless in B. germanica, males no longer exhibit courtship behaviour, thus implying that fruitless is necessary for male sexual behaviour in our cockroach model. This suggests that the role of fruitless as master regulator of male sexual behaviour has been conserved along insect evolution, at least from cockroaches to flies.

Neuropeptide biology in Drosophila.

Drosophila melanogaster is since decades the most important invertebrate model. With the publishing of the genome sequence, Drosophila also became a pioneer in (neuro)peptide research. Neuropeptides represent a major group of signaling molecules that outnumber all other types of neurotransmitters/modulators and hormones. By means of bioinformatics 119 (neuro)peptide precursor genes have been predicted from the Drosophila genome. Using the neuropeptidomics technology 46 neuropeptides derived from 19 of these precursors could be biochemically characterized. At the cellular level, neuropeptides usually exert their action by binding to membrane receptors, many of which belong to the family of G-protein coupled receptors or GPCRs. Such receptors are the major target for many contemporary drugs. In this chapter, we will describe the identification, localization and functional characterization of neuropeptide-receptor pairs in Drosophila melanogaster.

Proteome analysis of multiple compartments in a mouse model of chemical-induced asthma.

Occupational asthma is the principal cause of work-related respiratory disease in the industrial world. Toluene-2,4-diisocyanate (TDI) is one of the most common respiratory sensitizers leading to occupational asthma. Using a mouse model of chemical-induced asthma, we explored proteome changes in multiple compartments of mice sensitized and challenged with TDI or acetone-olive oil (AOO; vehicle). Airway reactivity to methacholine and a bronchoalveolar lavage (BAL) cell count was assessed in treated and control mice, 1 day after challenge. Subsequently, two-dimensional differential gel electrophoresis (2D-DIGE) was performed on auricular lymph nodes, BAL, and serum comparing TDI-treated and vehicle-treated control mice. The differentially expressed proteins were identified by mass spectrometry and pathway analysis was performed. TDI-treated mice exhibit increased airway reactivity (2.6-fold increase) and a neutrophilic inflammation in the BAL fluid, compared to control mice. 2D-DIGE showed 53, 210, and 40 differentially expressed proteins in the auricular lymph nodes, BAL, and serum of TDI-treated versus vehicle-treated mice, respectively. Several of the identified proteins could be linked with inflammation, neutrophil chemotaxis, and/or oxidative stress. Physiologic and immunologic readouts of the asthmatic phenotype, such as inflammation, were confirmed in three compartments by several of the differentially expressed proteins via 2D-DIGE and computerized pathway analysis.

Identical skin toxins by convergent molecular adaptation in frogs.

The Tree of Life is rife with adaptive convergences at all scales and biological levels of complexity. However, natural selection is not likely to result in the independent evolution of identical gene products. Here we report such a striking example of evolutionary convergence in the toxic skin secretions of two distantly related frog lineages. Caeruleins are important decapeptides in pharmacological and clinical research [1] and are commonly believed to represent a single evolutionary class of peptides [2-4]. Instead, our phylogenetic analyses combining transcriptome and genome data reveal that independently evolved precursor genes encode identical caeruleins in Xenopus and Litoria frogs. The former arose by duplication from the cholecystokinin (cck) gene, whereas the latter was derived from the gastrin gene. These hormone genes that are involved in many physiological processes diverged early in vertebrate evolution, after a segmental duplication during the Cambrian period. Besides implicating convergent mutations of the peptide-encoding sequence, recurrent caerulein origins entail parallel shifts of expression from the gut-brain axis to skin secretory glands. These results highlight extreme structural convergence in anciently diverged genes as an evolutionary mechanism through which recurrent adaptation is attained across large phylogenetic distances.

Approaches to identify endogenous peptides in the soil nematode Caenorhabditis elegans.

The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past without the genomic information was challenging. Since the rise of many genome-sequencing projects and the significant boost of mass spectrometry instrumentation, many hyphenated techniques can be used to explore the "peptidome" of individual species, organs, or even cell cultures. The peptidomic approach aims to identify endogenously present (neuro)peptides by using liquid chromatography and mass spectrometry in a high-throughput way. Here we outline the basic procedures for the maintenance of C. elegans nematodes and describe in detail the peptide extraction procedures. Two peptidomics strategies (off-line HPLC-MALDI-TOF MS and on-line 2D-nanoLC-Q-TOF MS/MS) and the necessary instrumentation are described.

Bioinformatic approaches to the identification of novel neuropeptide precursors.

With the entire genome sequence of several animals now available, it is becoming possible to identify in silico all putative peptides and their precursors in an organism. In this chapter we describe a searching algorithm that can be used to scan the genome for predicted proteins with the structural hallmarks of (neuro)peptide precursors. We also describe how to use search strings such as the presence of a glycine residue as a putative amidation site, dibasic cleavage sites, the presence of a signal peptide, and specific peptide motifs to improve a standard BLAST search and make it suitable for searching (neuro)peptides in EST data. We briefly explain how bioinformatic tools and in silico predicted peptide precursor sequences can aid experimental peptide identification with mass spectrometry.

Peptidomic survey of the locust neuroendocrine system.

Neuropeptides are important controlling agents in animal physiology. In order to understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. We here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, we analysed the brain, suboesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e. pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosaemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.

Unraveling the protective effect of a Drosophila phosphatidylethanolamine-binding protein upon bacterial infection by means of proteomics.

This study addresses the biological function of CG18594, a Drosophila melanogaster phosphatidylethanolamine-binding protein (PEBP) that we named PEBP1, by combining fly genetics, survival experiments and differential proteomics. We demonstrate that transgenic flies overexpressing PEBP1 are highly protected against bacterial infection due to the release of immunity-related proteins in their hemolymph. Apart from proteins that have been reported earlier to participate in insect immunity, we also identify proteins involved in metabolism and signaling, and, in addition, twelve (hypothetical) proteins with unknown function. This is the first report demonstrating an immune function for a Drosophila PEBP protein.

Identification of new members of the (short) neuropeptide F family in locusts and Caenorhabditis elegans.

Both the long and short neuropeptides F (NPF) represent important families of invertebrate neuropeptides that have been implicated in the regulation of reproduction and feeding behavior. In the present study, two short NPFs (SNRSPS(L/I)R(L/I)RFamide and SPS(L/I)R(L/I)RFamide) were de novo sequenced by mass spectrometry in two major pest insects, the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. They are two of the most widespread peptides in the locust neuroendocrine system. A peptide that was previously reported to accelerate egg development in S. gregaria is shown to represent a truncated form of long NPF. This peptide is most likely derived by a novel processing mechanism involving cleavage at RY. In addition, an NPF peptide from the nematode Caenorhabditis elegans was isolated and sequenced by tandem mass spectrometry.

APRP, the second peptide encoded by the adipokinetic hormone gene(s), is highly conserved in evolution: a role in control of ecdysteroidogenesis?

Since the early days of cloning the first adipokinetic hormone (AKH) gene, researchers recognized that this gene also codes for a joining region and for a second peptide called adipokinetic hormone precursor related peptide (APRP). In species with more than one AKH gene, such as locusts, APRPs can form both homodimers and heterodimers. Database analysis showed that APRPs might belong to the ancient family of growth hormone releasing factor but they still are functionally orphan. We investigated whether some of the APRP forms play a role in control of reproduction or/and growth via stimulation of ecdysteroidogenesis.

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans.

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC(50)=18nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.

Cloning of neuropeptide-like precursor 1 in the gray flesh fly and peptide identification and expression.

The neuropeptide-like precursor 1 (NPLP1) was first identified in a peptidomics experiment on Drosophila melanogaster. Limited data on this novel neuropeptide precursor suggest a role in the regulation of ecdysis in holometabolous larvae. In this study, we characterized the NPLP1 precursor in the gray flesh fly, Neobellieria bullata, which is an excellent model for physiological assays and hence to discover the role of the NPLP1 peptides. Antisera against three of the D. melanogaster NPLP1 neuropeptides stained an identical set of neurons in the central nervous system of N. bullata compared to D. melanogaster. A novel approach was applied to identify the N. bullata NPLP1 orthologs. Using a combination of affinity chromatography, mass spectrometry, cDNA cloning and RACE experiments, we obtained almost the complete coding sequence of the NPLP1 mRNA. Three encoded NPLP1 peptides were identified in central nervous system extracts by mass spectrometry. Neither doses of 25-250pmol of synthetic Neb-MGYamide and Neb-PQNamide peptides, nor the NPLP1 antisera did affect the speed of retraction, contraction and tanning in the pupariation bioassay.