Neuronal microRNAs safeguard ER Ca2+ homeostasis and attenuate the unfolded protein response upon stress.

Ca2+ is a critical mediator of neurotransmitter release, synaptic plasticity, and gene expression, but also excitotoxicity. Ca2+ signaling and homeostasis are coordinated by an intricate network of channels, pumps, and calcium-binding proteins, which must be rapidly regulated at all expression levels. Τhe role of neuronal miRNAs in regulating ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs) was investigated to understand the underlying mechanisms that modulate ER Ca2+ release. RyRs and IP3Rs are critical in mounting and propagating cytosolic Ca2+ signals by functionally linking the ER Ca2+ content, while excessive ER Ca2+ release via these receptors is central to the pathophysiology of a wide range of neurological diseases. Herein, two brain-restricted microRNAs, miR-124-3p and miR-153-3p, were found to bind to RyR1-3 and IP3R3 3'UTRs, and suppress their expression at both the mRNA and protein level. Ca2+ imaging studies revealed that overexpression of these miRNAs reduced ER Ca2+ release upon RyR/IP3R activation, but had no effect on [Ca2+]i under resting conditions. Interestingly, treatments that cause excessive ER Ca2+ release decreased expression of these miRNAs and increased expression of their target ER Ca2+ channels, indicating interdependence of miRNAs, RyRs, and IP3Rs in Ca2+ homeostasis. Furthermore, by maintaining the ER Ca2+ content, miR-124 and miR-153 reduced cytosolic Ca2+ overload and preserved protein-folding capacity by attenuating PERK signaling. Overall, this study shows that miR-124-3p and miR-153-3p fine-tune ER Ca2+ homeostasis and alleviate ER stress responses.

Neuronal microRNAs modulate TREK two-pore domain K+ channel expression and current density.

The TREK family of leak potassium channels has been found to play critical roles in nociception, sensitivity to general anaesthetics, neuroprotection, and memory. The three members of the family, TREK1, TREK2 and TRAAK establish the resting potential and modify the duration, frequency and amplitude of action potentials. Despite their apparent importance, the repertoire of regulatory interactions utilized by cells to control their expression is poorly understood. Herein, the contribution of miRNAs in the regulation of their post-transcriptional gene expression has been examined. Using different assays, miR-124 and to a lesser extent miR-128 and miR-183 were found to reduce TREK1 and TREK2 levels through specific binding to their 3'UTRs. In contrast, miR-9 which was predicted to bind to TRAAK 3'UTR, did not alter its expression. Expression of miR-124, miR-128 and miR-183 was found to mirror that of Trek1 and Trek2 mRNAs during brain development. Moreover, application of proinflammatory mediators in dorsal root ganglion (DRG) neurons revealed an inverse correlation between miR-124 and Trek1 and Trek2 mRNA expression. Voltage clamp recordings of TREK2-mediated currents showed that miR-124 reduced the sensitivity of TREK2-expressing cells to non-aversive warmth stimulation. Overall, these findings reveal a significant regulatory mechanism by which TREK1 and TREK2 expression and hence activity are controlled in neurons and uncover new druggable targets for analgesia and neuroprotection.Abbreviations: microRNA: miRNA; UTR: untranslated region; K2p channels: two-pore domain K+channels; DRG: dorsal root ganglion; CNS: central nervous system; FBS: fetal bovine serum; TuD: Tough Decoy; TREK: tandem P-domain weak inward rectifying K+ (TWIK)-related K+ channel 1; TRAAK: TWIK-related arachidonic acid K+.

Evaluation of Antifouling Potential and Ecotoxicity of Secondary Metabolites Derived from Red Algae of the Genus Laurencia.

Red algae of the genus Laurencia are known to biosynthesize and secrete an immense variety of secondary metabolites possessing a spectrum of biological activities against bacteria, invertebrates and mammalian cell lines. Following a rigorous cross-species screening process, herein we report the antifouling potential of 25 secondary metabolites derived from species of the genus Laurencia, as well as the thorough evaluation of the ecotoxicity of selected metabolites against non-target marine arthropods and vertebrate cell lines. A number of these secondary metabolites exhibited potent antifouling activity and performed well in all screening tests. Our results show that perforenol (9) possesses similar antifouling activity with that already described for bromosphaerol, which is used herein as a benchmark.

Refining a steroidogenic model: an analysis of RNA-seq datasets from insect prothoracic glands.

BACKGROUND: The prothoracic gland (PG), the principal steroidogenic organ of insects, has been proposed as a model for steroid hormone biosynthesis and regulation. RESULTS: To validate the robustness of the model, we present an analysis of accumulated transcriptomic data from PGs of two model species, Drosophila melanogaster and Bombyx mori. We identify that the common core components of the model in both species are encoded by nine genes. Five of these are Halloween genes whose expression differs substantially between the PGs of these species. CONCLUSIONS: We conclude that the PGs can be a model for steroid hormone synthesis and regulation within the context of mitochondrial cholesterol transport and steroid biosynthesis but beyond these core mechanisms, gene expression in insect PGs is too diverse to fit in a context-specific model and should be analysed within a species-specific framework.

Should I stay or should I go? The settlement-inducing protein complex guides barnacle settlement decisions.

Reproduction in barnacles relies on chemical cues that guide their gregarious settlement. These cues have been pinned down to several sources of settlement pheromones, one of which is a protein termed settlement-inducing protein complex (SIPC), a large glycoprotein acting as a pheromone to induce larval settlement and as an adhesive in surface exploration by the cyprids. Settlement assays in laboratory conditions with Amphibalanus (=Balanus) amphitrite cyprids in the presence of SIPC showed that cyprids exhibit settlement preference behaviour at lower concentrations of SIPC [half maximal effective concentration (EC50)=3.73 nmol l-1] and settlement avoidance behaviour at higher concentrations (EC50=101 nmol l-1). By using truncated fragments of SIPC in settlement assays, we identify that domains at the N-terminus of SIPC transduce settlement preference cues that mask the settlement avoidance cues transduced by domains at its C-terminus. Removing the N-terminal 600 amino acids from SIPC resulted in truncated fragments that transduced only settlement avoidance cues to the cyprids. From the sexual reproduction point of view, this bimodal response of barnacles to SIPC suggests that barnacles will settle gregariously when conspecific cues are sparse but will not settle if conspecific cues inform of overcrowding that will increase reproductive competition and diminish their reproductive chances.

Probing the settlement signals of Amphibalanus amphitrite.

To achieve their reproductive potential, barnacles combine tactile exploration of surface structural properties and integration of cellular signals originating from their antennular sensory setae within a developmentally defined, temporally narrow window of settlement opportunity. Behavioural assays with cyprids coupled with biometric analysis of scanning electron microscopy-acquired images in the presence of specific chemical compounds were used to investigate how settlement on a substratum is altered in response to the presence of these compounds. It is shown that impeding tactile exploration, altering cellular signalling and/or inducing malformations of anatomical features of the antennular sensory setae can disrupt the settlement behaviour of the model barnacle species Amphibalanus amphitrite. It is concluded that surface exploration by the cyprids relies on mechanical and nociception-related and calcium-mediated signals while a protein kinase C signalling cascade controls the timely metamorphosis of the cyprids to sessile juveniles.

IP3 receptors: some lessons from DT40 cells.

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+-induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R. DT40 cells are a pre-B-lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40-knockout cells with both alleles of each of the three IP3R genes disrupted provide the only null-background for analysis of homogenous recombinant IP3R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP3R behavior. Patch-clamp recording from the nuclear envelope and Ca2+ release from intracellular stores loaded with a low-affinity Ca2+ indicator address the mechanisms leading to activation of IP(3)R. We show that IP3 causes intracellular IP3R to cluster and re-tune their responses to IP3 and Ca2+, better equipping them to mediate regenerative Ca2+ signals. Finally, we show that DT40 cells reliably count very few IP3R into the plasma membrane, where they mediate about half the Ca2+ entry evoked by the B-cell antigen receptor.

"Hit" to lead optimization and chemoinformatic studies for a new series of Autotaxin inhibitors.

Robust experimental evidence has highlighted the role of Autotaxin (ATX)/Lysophosphatidic acid (LPA) axis not only in the pathogenesis of chronic inflammatory conditions and especially in fibroproliferative diseases but also in several types of cancer. As a result, different series of substrate-, lipid-based and small-molecule ATX inhibitors have been identified thus far by both academia and pharma. The "crowning achievement" of these drug discovery campaigns was the development and entry of the first-in-class ATX inhibitor (ziritaxestat, GLPG-1690) in advanced clinical trials against idiopathic pulmonary fibrosis. Herein, the potency optimization efforts of a new series of Autotaxin inhibitors, namely 2-substituted-2,6-dihydro-4H-thieno[3,4-c]pyrazol-1-substituted amide, is described using a previously identified novel chemical scaffold as a "hit". The mode of inhibition of the most promising ATX inhibitors was investigated, while their cellular activity, aqueous solubility and cytotoxicity were evaluated. Our pharmacological results were corroborated by chemoinformatic tools (molecular docking and molecular dynamics simulations) deployed, to provide insight into the binding mechanism of the synthesized inhibitors to ATX.

Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3) that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP(3)R, we demonstrate that high concentrations of cAMP similarly sensitize all IP(3)R isoforms to IP(3) by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP(3) binding to IP(3)R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP(3). In single channel recordings from excised nuclear patches of cells expressing IP(3)R2, cAMP alone had no effect, but it increased the open probability of IP(3)R2 activated by a submaximal concentration of IP(3) alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP(3)R subtypes to IP(3). For IP(3)R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP(3). Using stably expressed short hairpin RNA to reduce expression of the G-protein, G alpha(s), we demonstrate that attenuation of AC activity by loss of G alpha(s) more substantially reduces sensitization of IP(3)R by PTH than does comparable direct inhibition of AC. This suggests that G alpha(s) may also specifically associate with each AC x IP(3)R complex. We conclude that all three subtypes of IP(3)R are regulated by cAMP independent of PKA. In HEK cells, where IP(3)R2 selectively associates with AC6, G alpha(s) also associates with the AC x IP(3)R signaling junction.

A missense mutation in Fgfr1 causes ear and skull defects in hush puppy mice.

The hush puppy mouse mutant has been shown previously to have skull and outer, middle, and inner ear defects, and an increase in hearing threshold. The fibroblast growth factor receptor 1 (Fgfr1) gene is located in the region of chromosome 8 containing the mutation. Sequencing of the gene in hush puppy heterozygotes revealed a missense mutation in the kinase domain of the protein (W691R). Homozygotes were found to die during development, at approximately embryonic day 8.5, and displayed a phenotype similar to null mutants. Reverse transcription PCR indicated a decrease in Fgfr1 transcript in heterozygotes and homozygotes. Generation of a construct containing the mutation allowed the function of the mutated receptor to be studied. Immunocytochemistry showed that the mutant receptor protein was present at the cell membrane, suggesting normal expression and trafficking. Measurements of changes in intracellular calcium concentration showed that the mutated receptor could not activate the IP(3) pathway, in contrast to the wild-type receptor, nor could it initiate activation of the Ras/MAP kinase pathway. Thus, the hush puppy mutation in fibroblast growth factor receptor 1 appears to cause a loss of receptor function. The mutant protein appears to have a dominant negative effect, which could be due to it dimerising with the wild-type protein and inhibiting its activity, thus further reducing the levels of functional protein. A dominant modifier, Mhspy, which reduces the effect of the hush puppy mutation on pinna and stapes development, has been mapped to the distal end of chromosome 7 and may show imprinting.

Dataset and validation of the approaches to study skills inventory for students.

There exists a vast amount of research on how students, in higher education, approach their studying and learning. Such research resulted in a multitude of questionnaires and tools to capture the way students perform in higher education institutions. One of these tools is the Approaches to Study Skills Inventory for Students (ASSIST) that was developed in the '80 s and '90 s. This inventory broadly classifies students, as approaching their study, in a deep, a strategic and/or a surface manner. Although it has gone through rigorous validation in many studies, there exist no publicly available dataset of the results of these studies and so the raw datasets cannot be re-used or integrated with other similar datasets. Here, we report and make publicly available the raw data of an 8-year longitudinal survey using this inventory in a cohort study of 1181 students from a department of a higher education institution. We validated our dataset using reliability analyses that confirmed, and compared well, with the results of previous studies.

Activation of IP(3) receptors by synthetic bisphosphate ligands.

Ca(2+) release by d-myo-inositol 1,4,5-trisphosphate receptors (IP(3)Rs) is widely considered to require the vicinal 4,5-bisphosphate motif of IP(3), with P-5 and P-4 engaging the alpha and beta domains of the binding site; using synthesis and mutagenesis we show that the adenine of synthetic glyconucleotides, through an interaction with Arg504, can replace the interaction of either P-1 or P-5 with the alpha-domain producing, respectively, the most potent bisphosphate agonist yet synthesised and the first agonist of IP(3)R without a vicinal bisphosphate motif; this will stimulate new approaches to IP(3)R ligand design.

Protein kinase A and C are "Gatekeepers" of capacitative Ca2+ entry in the prothoracic gland cells of the silkworm, Bombyx mori.

Application of protein kinases A and C inhibitors to the prothoracic glands cells of the silkworm, Bombyx mori, resulted in slow and gradual increases in intracellular Ca(2+) ([Ca(2+)](i)). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic gland cells of B. mori suggests that these increases of [Ca(2+)](i) are mediated neither by voltage-gated Ca(2+) channels nor by intracellular Ca(2+) stores. Rather they result from slow Ca(2+) leak from plasma membrane Ca(2+) channels that are sensitive to agents that inhibit capacitative Ca(2+) entry and are abolished in the absence of extracellular Ca(2+). Okadaic acid, an inhibitor of PP1 and PP2A phosphatases, blocked the increase in [Ca(2+)](i) produced by the inhibitors of protein kinase A and C. The combined results indicate that the capacitative Ca(2+) entry channels in prothoracic gland cells of B. mori are probably modulated by protein kinases A and C.

A fragment of the alarmin prothymosin α as a novel biomarker in murine models of bacteria-induced sepsis.

Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.

Antibodies to inositol 1,4,5-triphosphate receptor 1 in patients with cerebellar disease.

OBJECTIVE: To describe newly identified autoantibodies associated with cerebellar disorders. DESIGN/METHODS: We first screened the sera of 15 patients with cerebellar ataxia, without any known associated autoantibodies, with immunocytochemistry on mouse brain. After characterization and validation of a newly identified antibody, 85 additional patients with suspected autoimmune cerebellar disease were screened using a cell-based assay. RESULTS: Immunoglobulin G from one of the first 15 patients demonstrated a distinct staining pattern on Purkinje neurons. This autoantibody, as characterized further by immunoprecipitation and mass spectrometry, was binding inositol 1,4,5-triphosphate receptor 1 (IP3R1), an intracellular channel that mediates the release of Ca2+ from intracellular stores. Anti-IP3R1 specificity was then validated with a cell-based assay. On this basis, screening of 85 other patients with cerebellar disease revealed 2 additional IP3R1-positive patients. All 3 patients presented with cerebellar ataxia; the first was eventually diagnosed with primary progressive multiple sclerosis, the second had a homozygous CAG insertion at the gene TBP, and the third was thought to have a neurodegenerative disease. CONCLUSIONS: We independently identified an autoantibody against IP3R1, a protein highly expressed in Purkinje neurons, confirming an earlier report. Because a mouse knockout model for IP3R1 exhibits ataxia and epilepsy, this autoantibody may have a functional role. The heterogeneity of the antibody-positive patients suggests that this antibody may either have a direct involvement in disease pathogenesis or it is a surrogate marker secondary to cerebellar injury. Anti-IP3R1 antibodies should be further explored in various ataxic and epileptic syndromes as they may denote a marker of response to immunotherapies.

Synthesis of adenophostin A analogues conjugating an aromatic group at the 5'-position as potent IP3 receptor ligands.

Previous structure-activity relationship studies of adenophostin A, a potent IP(3) receptor agonist, led us to design the novel adenophostin A analogues 5a-c, conjugating an aromatic group at the 5'-position to develop useful IP(3) receptor ligands. The common key intermediate, a D-ribosyl alpha-D-glucoside 10alpha, was stereoselectively synthesized by a glycosidation with the 1-sulfinylglucoside donor 11, which was conformationally restricted by a 3,4-O-cyclic diketal protecting group. After introduction of an aromatic group at the 5-position of the ribose moiety, an adenine base was stereoselectively introduced at the anomeric beta-position to form 7a-c, where the tetra-O-i-butyryl donors 9a-c were significantly more effective than the corresponding O-acetyl donor. Thus, the target compounds 5a-c were synthesized via phosphorylation of the 2', 3' ', and 4' '-hydroxyls. The potencies of compounds 5a-c for Ca(2+) release were shown to be indistinguishable from that of adenophostin A, indicating that bulky substitutions at the 5'-position of adenophostin A are well-tolerated in the receptor binding. This biological activity of 5a-c can be rationalized by molecular modeling using the ligand binding domain of the IP(3) receptor.

A systematic study of C-glucoside trisphosphates as myo-inositol trisphosphate receptor ligands. Synthesis of beta-C-glucoside trisphosphates based on the conformational restriction strategy.

Beta-C-glucoside trisphosphates having a C2 side chain (3,7-anhydro-2-deoxy-D-glycero-D-gulo-octitol 1,5,6-trisphosphate, 11) and a C3 side chain (4,8-anhydro-2,3-dideoxy-D-glycero-D-gulo-nonanitol 1,6,7-trisphosphate, 12) were designed as structurally simplified analogues of a potent D-myo-inositol 1,4,5-trisphosphate (IP3) receptor ligand, adenophostin A. Construction of the beta-C-glucosidic structure, which was the key to their synthesis, was achieved by two different methods based on the conformational restriction strategy: (1) radical cyclization with a temporary connecting silicon tether and (2) silane reduction of glyconolactols having an anomeric allyl substituent. Using these methods, the target beta-C-glycoside trisphosphates 11 and 12 were successfully synthesized. A structure-activity relationship was established on a series of C-glucoside trisphosphates, including the previously synthesized related compounds, which were a C-glycosidic analogue 3 of adenophostin A, its uracil congener 5, alpha-C-glucoside trisphosphates 7-9 having a C1, C2, or C3 side chain, and the beta-C-glucoside trisphosphates 10-12 having a C1, C2, or C3 side chain. The O-glycosidic linkage of adenophostin A and its analogues proved to be replaced by the chemically and biologically more stable C-glycosidic linkage. The alpha-C2-glucoside trisphosphate 8 stimulates Ca2+ release with a potency similar to that of IP3 in spite of its simplified structure, indicating a better fit to the receptor than the beta-C-glucoside trisphosphates and also the alpha-congeners having a shorter or longer C1 side chain, which was supported by molecular modeling using the ligand binding domain of the IP3 receptor.

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.

Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects.

Rapid functional assays of recombinant IP3 receptors.

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).