FMRFamid-rokon neuropeptidek és receptoraik hatásmechanizmusának genetikai analízise Drosophila melanogaster-ben = Insect FMRFamide-related neuropeptides (FaRPs) and receptors: genetic analysis of their mechanism of action in Drosophila melanogaster

Genetikai módszerekkel tanulmányoztuk ecetmuslica (Drosophila melanogaster) modellben az FMRF-rokon (FARP) és azok receptorait. (1) Intragénikus deléciós mutánsokat izoláltunk a DMS-R1 and FMRFa génekben. (2) Kettős-szálú RNS (RNAi) segítségével csendesítettük a peptid- és receptor géneket. (3) Gal4 driver-eket készítettünk az FMRFa gén 5’ régiójának fragmentjeivel, majd FMRFa peptiderg neuronokat elimináltunk. Az RNAi tesztben először az Act5C-Gal4 drivert alkalmaztuk, amely az FMRFa, DMS, DMS-R1, DSK-R1 és DSK-R2 gének esetében letalitást eredményezett. A pánneurális elav-Gal4 driverrel viszont minden kombináció életképes volt. Homozigóta életképesek az FMRFa és DMS-R1 deléciós mutánsok is. Az eredmények magyarázata az „off-target” hatás.. A mutáns kombinációknak az adult viselkedésre gyakorolt hatását stressz-indukált mozgási tesztben vizsgáltuk. A mozgási aktivitást szignifikánsan csökkentették a deléciós mutánsok és az RNAi kombinációk, valamint bizonyos FMRF-specifikus neuronok ablációja. | We accomplished a genetic analysis in Drosophila melanogaster of the FMRF-related group of peptides (FARPs) comprising (DMS the dFMRFamide, dromyosuppressin) and drosulfakinin (DSK) peptides, and their specific receptors (FP, DMS-R1 and -R2, DSK-R1 and –R2). We used three approaches: (i) isolating intragenic deletions by P transposon remobilization in the DMS-R1 and FMRFa genes, (ii) gene silencing by double-stranded inhibitory RNA (RNAi) and (iii) constructing new Gal4 drivers (RS- 8, -11 ans -17) with 5’ regulatory fragments of the FMRFa gene, and using them to ablate specific FMRFa peptidergic neurons. In the RNAi tests, when using the Act5C-Gal4 driver expressing continuously in all the tissues, silencing the FMRFa, DMS, DMS-R1, DSK-R1 and DSK-R2 genes resulted in lethality, while with the pan-neuronal elav-Gal4 driver all the combinations remained alive. Similarly, the FMRFa and DMS-R1 deletion mutants are homozygous viable. The RNAi-induced lethality is likely explained by off-target effect, i.e. silencing non-specific genes which have sections in their DNA sequence similar to that of the target gene. In order to compare the effects of the above mutant combinations on adult behavior, we examined the startle-induced locomotor activity of flies by measuring the mean velocity of movement (MVM) following repeated air-puffs. In general, the MVM was decreased by the deletion mutants, the elav-Gal4-induced RNAi knockdown and the ablation of some FMRF-specific neurons

Coordinated RNA-Seq and peptidomics identify neuropeptides and G-protein coupled receptors (GPCRs) in the large pine weevil Hylobius abietis, a major forestry pest

Hylobius abietis (Linnaeus), or large pine weevil (Coleoptera, Curculionidae), is a pest of European coniferous forests. In order to gain understanding of the functional physiology of this species, we have assembled a de novo transcriptome of H. abietis, from sequence data obtained by Next Generation Sequencing. In particular, we have identified genes encoding neuropeptides, peptide hormones and their putative G-protein coupled receptors (GPCRs) to gain insights into neuropeptide-modulated processes. The transcriptome was assembled de novo from pooled paired-end, sequence reads obtained from RNA from whole adults, gut and central nervous system tissue samples. Data analysis was performed on the transcripts obtained from the assembly including, annotation, gene ontology and functional assignment as well as transcriptome completeness assessment and KEGG pathway analysis. Pipelines were created using Bioinformatics tools and techniques for prediction and identification of neuropeptides and neuropeptide receptors. Peptidomic analysis was also carried out using a combination of MALDI-TOF as well as Q-Exactive Orbitrap mass spectrometry to confirm the identified neuropeptide. 41 putative neuropeptide families were identified in H. abietis, including Adipokinetic hormone (AKH), CAPA and DH31. Neuropeptide F, which has not been yet identified in the model beetle T. castaneum, was identified. Additionally, 24 putative neuropeptide and 9 leucine-rich repeat containing G protein coupled receptor-encoding transcripts were determined using both alignment as well as non-alignment methods. This information, submitted to the NCBI sequence read archive repository (SRA accession: SRP133355), can now be used to inform understanding of neuropeptide-modulated physiology and behaviour in H. abietis; and to develop specific neuropeptide-based tools for H. abietis control

Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of HbrKrrPdmrCasrGrh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a ‘switching factor’. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, we have isolated a novel allele of svp. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage.This work was supported by the Swedish Research Council, by the Swedish Strategic Research Foundation, by the Knut and Alice Wallenberg foundation, by the Swedish Brain Foundation, by the Swedish Cancer Foundation, by the Swedish Royal Academy of Sciences [S.T.], and by a grant from the Spanish Ministerio de Ciencia e Innovación [BFU-2008-04683-C02-02 to L.T.]

Lineage-unrelated neurons generated in different temporal windows and expressing different combinatorial codes can converge in the activation of the same terminal differentiation gene

It is becoming increasingly clear that the activation of specific terminal differentiation genes during neural development is critically dependent upon the establishment of unique combinatorial transcription factor codes within distinct neural cell subtypes. However, it is still unclear to which extent these codes are shared by lineage-unrelated neurons expressing the same terminal differentiation genes. Additionally, it is not known if the activation of a specific terminal differentiation gene is restricted to cells born at a particular developmental time point. Here, we utilize the terminal differentiation gene FMRFa which is expressed by the Ap4 and SE2 neurons in the Drosophila ventral nerve cord, to explore these issues in depth. We find that the Ap4 and SE2 neurons are generated by different neural progenitors and use different combinatorial codes to activate FMRFa expression. Additionally, we find that the Ap4 and SE2 neurons are generated in different temporal gene expression windows. Extending the investigation to include a second Drosophila terminal differentiation gene, Leucokinin, we find similar results, suggesting that neurons generated by different progenitors might commonly use different transcription factor codes to activate the same terminal differentiation gene. Furthermore, these results imply that the activation of a particular terminal differentiation gene in temporally unrestricted.This work was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (BFU-2008- 04683-C02-02 to L.T.)

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions

Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated by RF-amide-related peptides through the G{alpha}q/11 pathway

MrgA1 and MrgC11 belong to a recently identified family of orphan G-protein coupled receptors, called mrgs (mas-related genes). They are only expressed in a specific subset of sensory neurons that are known to detect painful stimuli. However, the precise physiological function of Mrg receptors and their underlying mechanisms of signal transduction are not known. We therefore have screened a series of neuropeptides against human embryonic kidney (HEK) 293 cells that stably express either MrgA1 or MrgC11 to identify ligands and/or agonists. MrgA1- or MrgC11-specific agonists stimulated dose-dependent increases in intracellular free Ca2+ in a pertussis toxin-insensitive manner, but failed to alter basal or forskolin-stimulated levels of intracellular cAMP. Furthermore, studies using embryonic fibroblasts derived from various G{alpha} protein knockout mice demonstrated that both the MrgA1 and MrgC11 receptors are coupled to the G{alpha}q/11 signaling pathway. Screening of neuropeptides identified surrogate agonists, most of these peptides included a common C-terminal -RF(Y)G or -RF(Y) amide motif. Structure-function studies suggest that endogenous ligands of Mrg receptors are likely to be RF(Y)G and/or RF(Y) amide-related peptides and that postprocessing of these peptides may serve to determine Mrg receptor-ligand specificity. The differences in ligand specificity also suggest functional diversity amongst the Mrg receptors

Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability

Excitatory synapses in the CNS are highly dynamic structures that can show activity- dependent remodeling and stabilization in response to learning and memory. Synapses are enveloped with intricate processes of astrocytes known as perisynaptic astrocytic processes (PAPs). PAPs are motile structures displaying rapid actin-dependent movements and are characterized by Ca²⁺ elevations in response to neuronal activity. Despite a debated implication in synaptic plasticity, the role of both Ca²⁺ events in astrocytes and PAP morphological dynamics remain unclear.Results In the hippocampus, we found that PAPs show extensive structural plasticity that is regulated by synaptic activity through astrocytic metabotropic glutamate receptors and intracellular calcium signaling. Synaptic activation that induces long-term potentiation caused a transient PAP motility increase leading to an enhanced astrocytic coverage of the synapse. Selective activation of calcium signals in individual PAPs using exogenous metabotropic receptor expression and two-photon uncaging reproduced these effects and enhanced spine stability. In vivo imaging in the somatosensory cortex of adult mice revealed that increased neuronal activity through whisker stimulation similarly elevates PAP movement. This in vivo PAP motility correlated with spine coverage and was predictive of spine stability.Conclusions This study identifies a novel bidirectional interaction between synapses and astrocytes, in which synaptic activity and synaptic potentiation regulate PAP structural plasticity, which in turn determines the fate of the synapse. This mechanism may represent an important contribution of astrocytes to learning and memory processes

Neuromoduláció sejtszintű mechanizmusainak vizsgálata ideg-izom és ideg-mirigy kapcsolatokban. = Cellular mechanisms of the neuromodulation in neuromuscular and neuroglandular junctions.

Az éticsiga nyálmirigy jó modell az ideg-izom, ideg-mirigy szinapszis és a szekréció molekuláris mechanizmusának a tanulmányozásában. A mirigyben négyféle sejttípust azonosítottunk és megállapítottuk, hogy azok száma, eloszlása az aktív és inaktív állatokban eltérő és az ingerületáttevődés nem-szinaptikus úton valósul meg. A varikozitásokban lévő vezikulákból dopamin, szerotonin és neuropeptidek szabadulnak fel. A mirigysejtek közötti gap-junction ill. deszmoszóma kapcsolatok biztosítják a szerv szinkron működését. Leírtuk a mirigysejtek passzív és aktív elektromos tulajdonságait, és a feszültségfüggő ion-konduktanciákat. Jellemeztük a transzmitter receptorokat, amelyek heteromér formában vannak jelen, ily módon egymás hatását antagonizálják. Eredményeink ujabb adatokkal szolgálnak a puhatestüek 5HT receptorainak osztályozásához. A transzmitterhatást számos neuropeptid modulálja, amely csak a puhatestüekben fordul elő, de a PACAP is, melynek jelenlétét és szerepét puhatestűekben kimutattuk. Egy endogén neuropeptid szignalizációs útvonalát vizsgáltuk és megállapítottuk, hogy G-protein kapcsolt és amelyben mindkét disszociált alegység részt vesz és a deszenzitizációt egy protein kináz enzim végzi. A fiziológiai ingerek hatására a cisztikus sejtek száma megnőtt, melyek kivétel nélkül a programozott sejthalál morfológiai és molekuláris jeleit mutatták. Megállapítottuk, hogy a nyálfelszabadulás fiziológiai útja a mitochondrium-kaszpáz mediált útvonal. | The snail salivary gland is a suitable model in studying neuro-muscular, neuro-glandular synapse and the molecular mechanism of salivation. For cell types were identified in the gland and it was found, that distribution and number of different cell types were different in active and inactive snails. Vesicles in varicosities liberate dopamine, serotonin and number of neuropeptides. Gap-junction and desmosome-like structures ensure the synchronization of gland cells. Passive and active electrical properties of cells were examined and voltage-dependent ion currents were characterized. It was found that receptors occur in heterometric form, they bind both DA and 5HT and thereby antagonizing its effect. Our results contribute to the characterization and classification of 5HT-receptors in mollusc. The transmitter effect is modulated by peptides present only in molluscs, and by PACAP occurrence and distribution of which is described in mollusc. Signalling pathway of the neuropeptide (MIP) was studied and found that it is G-protein coupled and both subunits are active and the desensitization of the receptor is performed by receptor kinase. Physiological stimuli such as transmitters or nerve stimulation increased the number of cystic cells. These cells every time revealed a morphological and molecular signs of the programmed cell death. It is concluded, that the mitochondrial-caspase pathway of the programmed cell death is a physiological way of the saliva release

The regenerative nidi of the locust midgut as a model to study epithelial cell differentiation from stem cells

A better knowledge of the regulatory mechanisms involved in stem cell proliferation and/or differentiation could reveal new methods for the treatment of some diseases. Most previous studies in the field of stem cell biology have been carried out on cultured isolated cells. In the case of adult tissue stem cells, mesenchymal bone marrow derived cells have been most widely studied, while the undifferentiated stem cells present in the epithelial tissues are less known. In order to advance further our understanding of epithelial tissue stem cells, new in vivo models are required. The present study focuses on the dynamics of a new and simple model of intestinal epithelial regeneration found in the midgut of the migratory locust, Locusta migratoria (Linnaeus 1758). The locust midgut consists of three cell types: columnar cells, endocrine cells and undifferentiated regenerative clustered cells. The undifferentiated epithelial midgut cells give rise to two other cell types and are located in a nest of regenerative cells known as regenerative niche. We have performed single and continuous bromodeoxyuridine (BrdU) administration experiments to study regeneration niches and their cellular dynamics. Immunocytochemistry and immunofluorescence techniques were used to detect the incorporation of BrdU into regenerative niches and the presence of FMRFamide-like immunoreactivity, as a marker for endocrine cell differentiation. Some isolated label retaining cells (LRC) were observed at the niche base 10-15 days after the final BrdU administration. We propose that these cells are the stem cells of this epithelial tissue. We also calculated the length of the cell cycle phases for a subpopulation of transit amplifying cells within the regenerative niche: G1, 2.5+/-0.5 h; S, 5.5+/-0.5 h; G2, 0.75+/-0.25 h; M, 2.5+/-0.5 h. These amplifying cells will give rise to the columnar epithelial non-endocrine lineage. The differentiation of an endocrine cell from a niche stem cell occurs less frequently and thus leads to a lower proportion of endocrine cells as compared with epithelial columnar digestive cells (up to three endocrine cells per niche). Endocrine cell commitment seems to occur very early in the differentiation process within the niche, as double-labelled BrdU and FMRF endocrine cells have never been found. The only exception is the endocrine cells located in the ampullar region of the midgut, some of which show double immunostaining after long-term chronic BrdU injection. In summary, we have characterized a new and simple animal model of epithelial stem cell regeneration that may be useful for understanding the complex biological process that drives tissue renewal from undifferentiated and uncommitted progenitor cells

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, we have used a very highly specific marker that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. Our data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2–A4. Moreover, we show that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5–A7, although they undergo apoptosis before expressing Capa. In addition, we have performed a targeted genetic screen to identify players involved in ABCA specification. We have found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, we show that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third suboesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification.This work was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (BFU-2008-04683-C02-02 to L.T.)