A hydrophobic groove in secretagogin allows for alternate interactions with SNAP-25 and syntaxin-4 in endocrine tissues.

Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and ß-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.

Pseudouridine-Modifying Enzymes SapB and SapH Control Entry into the Pseudouridimycin Biosynthetic Pathway.

Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5'-aminopseudouridine and formamidinylated, N-hydroxylated Gly-Gln dipeptide moieties to allow Watson-Crick base pairing and to mimic protein-ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (KM = 34 µM) over uridine (KM = 901 µM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5'-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5'-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (KM = 181 µM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.

Adverse effects of Δ9-tetrahydrocannabinol on neuronal bioenergetics during postnatal development.

Ongoing societal changes in views on the medical and recreational roles of cannabis increased the use of concentrated plant extracts with a Δ9-tetrahydrocannabinol (THC) content of more than 90%. Even though prenatal THC exposure is widely considered adverse for neuronal development, equivalent experimental data for young age cohorts are largely lacking. Here, we administered plant-derived THC (1 or 5 mg/kg) to mice daily during P5-P16 and P5-P35 and monitored its effects on hippocampal neuronal survival and specification by high-resolution imaging and iTRAQ proteomics, respectively. We found that THC indiscriminately affects pyramidal cells and both cannabinoid receptor 1+ (CB1R)+ and CB1R- interneurons by P16. THC particularly disrupted the expression of mitochondrial proteins (complexes I-IV), a change that had persisted even 4 months after the end of drug exposure. This was reflected by a THC-induced loss of membrane integrity occluding mitochondrial respiration and could be partially or completely rescued by pH stabilization, antioxidants, bypassed glycolysis, and targeting either mitochondrial soluble adenylyl cyclase or the mitochondrial voltage-dependent anion channel. Overall, THC exposure during infancy induces significant and long-lasting reorganization of neuronal circuits through mechanisms that, in large part, render cellular bioenergetics insufficient to sustain key developmental processes in otherwise healthy neurons.

Acute molecular effects of pressure-controlled intermittent coronary sinus occlusion in patients with advanced heart failure.

AIMS: Cardiac repair has steered clinical attention and remains an unmet need, because available regenerative therapies lack robust mechanistic evidence. Pressure-controlled intermittent coronary sinus occlusion (PICSO), known to induce angiogenetic and vasoactive molecules as well as to reduce regional ischemia, may activate endogenous regenerative processes in failing myocardium. We aimed to investigate the effects of PICSO in patients with advanced heart failure undergoing cardiac resynchronization therapy. METHODS AND RESULTS: Eight out of 32 patients were treated with PICSO, and the remainder served as controls. After electrode testing including left ventricular leads, PICSO was performed for 20 min. To test immediate molecular responses, in both patient groups, coronary venous blood samples were taken at baseline and after 20 min, the time required for the intervention. Sera were tested for microRNAs and growth factors. To test the ability of up-regulated soluble factors on cell proliferation and expression of transcription factors [e.g. Krüppel-like factor 4 (KLF-4)], sera were co-cultured with human cardiomyocytes and fibroblasts. As compared with controls, significant differential expression (differences between pre-values and post-values in relation to both patient cohorts) of microRNA patterns associated with cardiac development was observed with PICSO. Importantly, miR-143 (P < 0.048) and miR-145 (P < 0,047) increased, both targeting a network of transcription factors (including KLF-4) that promote differentiation and repress proliferation of vascular smooth muscle cells. Additionally, an increase of miR-19b (P < 0.019) known to alleviate endothelial cell apoptosis was found, whereas disadvantageous miR-320b (P < 0.023) suspect to impair expression of c-myc, normally provoking cell cycle re-entry in post-mitotic myocytes and miR-25 (P < 0.023), decreased, a target of anti-miR application to improve contractility in the failing heart. Co-cultured post-PICSO sera significantly increased cellular proliferation both in fibroblasts (P < 0.001) and adult cardiomycytes (P < 0.004) sampled from a transplant recipient as compared with controls. Adult cardiomyocytes showed a seven-fold increase of the transcription factor KLF-4 protein when co-cultured with treated sera as compared with controls. CONCLUSIONS: Here, we show for the first time that PICSO, a trans-coronary sinus catheter intervention, is associated with an increase in morphogens secreted into cardiac veins, normally present during cardiac development, and a significant induction of cell proliferation. Present findings support the notion that epigenetic modifications, that is, haemodynamic stimuli on venous vascular cells, may reverse myocardial deterioration. Further investigations are needed to decipher the maze of complex interacting molecular pathways in failing myocardium and the potential role of PICSO to reinitiate developmental processes to prevent further myocardial decay eventually reaching clinical significance.

Diversity matters: combinatorial information coding by GABAA receptor subunits during spatial learning and its allosteric modulation.

In the hippocampus, GABA inhibition tunes network oscillations and shapes synchronous activity during spatial learning and memory coding. Once released from the presynapse, GABA primarily binds to ionotropic GABAA receptors (GABAARs), which are heteropentamers combinatorially assembled from nineteen known subunits to induce Cl- currents postsynaptically. Dissecting GABAAR subtype specificities in neurobiology is daunting because of differences in their developmental dynamics, regional distribution and subcellular compartmentalization. Here, we review recent data to show that the combination of single-cell mRNA-seq and neuroanatomy can reveal unprecedented cell-type and network-specificity of GABAAR subunits and limit the permutation in subunit configurations, thus rationalizing GABAAR physiology and pharmacology. By comparing RNA-seq data on principal cells and interneurons we discuss a tight match between GABAAR subunit allocation, diversity in the origins of GABA inputs and operational rules at synaptic and extrasynaptic sites. We propose that coincident analysis of all GABAAR subunits, particularly in relation to specific behaviors, could overcome existing pitfalls of the genetic and pharmacological manipulation of single subunits. By using α1 and α5 GABAAR subunits, we single out hippocampal spatial learning as a process in which, despite the many studies available to date, neither consensus nor causality exists with regards to GABAAR subtype requirements, curtailing a unifying concept on postsynaptic coding of GABA signals. Finally, we address the modulation of GABAAR subunits by dopamine and endocannabinoids through receptor heteromerization, cross-modulation of signal transduction and allostery. In sum, data in this review infer that multiparametric computation gains momentum to improve knowledge on GABAARs function in cognition and neuropsychiatric illnesses.

Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for ß cell specification.

OBJECTIVE: Specification of endocrine cell lineages in the developing pancreas relies on extrinsic signals from non-pancreatic tissues, which initiate a cell-autonomous sequence of transcription factor activation and repression switches. The steps in this pathway share reliance on activity-dependent Ca2+ signals. However, the mechanisms by which phasic Ca2+ surges become converted into a dynamic, cell-state-specific and physiologically meaningful code made up by transcription factors constellations remain essentially unknown. METHODS: We used high-resolution histochemistry to explore the coincident expression of secretagogin and transcription factors driving ß cell differentiation. Secretagogin promoter activity was tested in response to genetically manipulating Pax6 and Pax4 expression. Secretagogin null mice were produced with their pancreatic islets morphologically and functionally characterized during fetal development. A proteomic approach was utilized to identify the Ca2+-dependent interaction of secretagogin with subunits of the 26S proteasome and verified in vitro by focusing on Pdx1 retention. RESULTS: Here, we show that secretagogin, a Ca2+ sensor protein that controls α and ß cell turnover in adult, is in fact expressed in endocrine pancreas from the inception of lineage segregation in a Pax4-and Pax6-dependent fashion. By genetically and pharmacologically manipulating secretagogin expression and interactome engagement in vitro, we find secretagogin to gate excitation-driven Ca2+ signals for ß cell differentiation and insulin production. Accordingly, secretagogin-/- fetuses retain a non-committed pool of endocrine progenitors that co-express both insulin and glucagon. We identify the Ca2+-dependent interaction of secretagogin with subunits of the 26S proteasome complex to prevent Pdx1 degradation through proteasome inactivation. This coincides with retained Nkx6.1, Pax4 and insulin transcription in prospective ß cells. CONCLUSIONS: In sum, secretagogin scales the temporal availability of a Ca2+-dependent transcription factor network to define ß cell identity.

A TRPV1-to-secretagogin regulatory axis controls pancreatic ß-cell survival by modulating protein turnover.

Ca2+-sensor proteins are generally implicated in insulin release through SNARE interactions. Here, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single-cell RNA-seq reveals retained expression of the TRP family members in ß-cells from diabetic donors. Amongst these, pharmacological probing identifies Ca2+-permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s fails to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock-out(-/-) mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin-/- mice is due to ß-cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin-dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1-to-secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of ß-cells.

Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration.

The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. Here we demonstrate the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin, a Ca2+-sensor protein, to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrate that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identify a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

Formation of GABAA receptor complexes containing α1 and α5 subunits is paralleling a multiple T-maze learning task in mice.

There is limited information on the role of GABA type A receptors (GABAARs) containing α1, α5 and γ2 subunits in learning and memory. Here, we assessed the possible role of such receptors in spatial learning using the multiple T-maze (MTM) paradigm. C57BL/6J mice were trained in the MTM which induced elevated levels of α1 and α5 subunit-containing hippocampal GABAAR complexes. Moreover, spatial learning evoked a significant increase in the colocalization of α1 and α5 subunits in both, CA1 and dentate gyrus regions of the hippocampus suggesting the formation of complexes containing both subunits. Additionally, the presence of α1, α5 and γ2 subunits in high molecular weight GABAARs was detected and significant correlation in the level of α1-containing complexes with those containing α5 and γ2 subunits was demonstrated. Accordingly, α1 deficiency led to decreased levels of γ2 subunit-containing complexes, however, had no effect on α5-containing ones. On the other hand, α1 knockout mice showed impaired performance in the MTM correlating with increased levels of α5 subunit-containing GABAARs in comparison to trained floxed control animals which quickly learned the task. Taken together, these results suggest that α1, α5 and γ2-containing hippocampal GABAAR complexes play an essential role in spatial learning and memory in which targeted disruption of the α1 subunit produces profound deficits.

Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord.

Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k(+) neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2(+) and/or calretinin(+). Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.

The differential hippocampal phosphoproteome of Apodemus sylvaticus paralleling spatial memory retrieval in the Barnes maze.

Protein phosphorylation is a well-known and well-documented mechanism in memory processes. Although a large series of protein kinases involved in memory processes have been reported, information on phosphoproteins is limited. It was therefore the aim of the study to determine a partial and differential phosphoproteome along with the corresponding network in hippocampus of a wild caught mouse strain with excellent performance in several paradigms of spatial memory. Apodemus sylvaticus mice were trained in the Barnes maze, a non-invasive test system for spatial memory and untrained mice served as controls. Animals were sacrificed 6h following memory retrieval, hippocampi were taken, proteins extracted and in-solution digestion was carried out with subsequent iTRAQ double labelling. Phosphopeptides were enriched by a TiO2-based method and semi-quantified using two fragmentation principles on the LTQ-orbitrap Velos. In hippocampi of trained animals phosphopeptide levels representing signalling, neuronal, synaptosomal, cytoskeletal and metabolism proteins were at least twofold reduced or increased. Furthermore, a network revealing a link to pathways of ubiquitination, the androgen receptor, small GTPase Rab5 and MAPK signaling as well as synucleins was constructed. This work is relevant for interpretation of previous work and the design of future studies on protein phosphorylation in spatial memory.