Ca-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 β-cells from diabetic donors. Amongst these, pharmacological probing identifies Ca-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 β-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 β-cells.

Background: Endocannabinoids can affect pancreatic ␤ cell physiology. Results: Anandamide and 2-arachidonoylglycerol binding to CB 1 receptors induces focal adhesion kinase phosphorylation, which is a prerequisite of insulin release. Conclusion: Focal adhesion kinase activation downstream from CB 1 receptors couples cytoskeletal reorganization to insulin release. Significance: Identifies the molecular blueprint of 2-arachidonoylglycerol signaling in the endocrine pancreas, and outlines a kinase activation cascade linking endocannabinoid signals to insulin release.

Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB 1 cannabinoid receptor (CB 1 R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB 1 Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB 1 R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB 1 R −/− islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.A nandamide (AEA) and 2-arachydonoylglycerol (2-AG), major endocannabinoids (eCBs), are involved in the regulation of energy homeostasis through coordinated actions in peripheral organs (adipose tissue, liver, and pancreas) and brain (hypothalamus, ventral striatum) (1). eCB signals are particularly significant to coordinate the regulated release of insulin and glucagon from mature pancreatic islets (2-6). Genetic evidence from CB 1 cannabinoid receptor −/− (CB 1 R −/− ) mice supports these findings because CB 1 R −/− mice are lean, resistant to high fat diet-induced obesity and diabetes (4, 7-9). Whether eCBs impact the formation of the endocrine pancreas and predispose it to long-lasting changes in hormone release postnatally remains unknown.Because eCBs broadly affect cell proliferation, fate, motility, and differentiation (e.g., in sperm, hematopoietic and T cells, and neurons) (10-13), it is likely that they play a role in the cellular organization of developing pancreatic islets, possibly by affecting the spatial segregation of α and β cells. A contribution of eCBs to cell diversification and positioning in the developing pancreas is supported by the temporal control of their levels in fetal tissues (14) and circulation (15). Moreover, α and β cells in mature pancreatic islets express the molecular machinery for eCB metabolism together with CB 1 Rs and transient receptor potential cation channels, particularly subfamily V member 1 (TRPV1) (2,16,17). Understanding these developmental processes is also relevant to po...

Edited by Varda RotterKeywords: p53 p73 Protoporphyrin IX Photodynamic therapy Fluorescent band shift Fluorescence polarization a b s t r a c tThe p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53-or p73-dependent cancer cell death.

Stress‐induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin‐releasing hormone‐releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal‐regulated kinase 1 and tyrosine hydroxylase with the Ca2+‐sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress‐induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate‐limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long‐lasting cortical excitability following acute stress.

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 Ca 2+ -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.human fetus | calcium-binding protein | cell motility | restorative strategy | olfactory system T he subventricular zone of adult rodents and its human fetal equivalent (1) generate neuroblasts that migrate through the rostral migratory stream (RMS) to supply the olfactory bulb with new neurons (2). Tangential neuroblast migration in the RMS is regulated by various environmental factors including growth factors (3-10), ephrins (11), and cell adhesion-related cues (12)(13)(14). Many of these regulatory events are executed through direct intercellular interactions: Chain-migrating neuroblasts move in contact with each other (15), and neuron-glia communication shapes the journey of newborn cells toward the olfactory bulb (16,17). In particular, astrocytes can vector neuroblast movement by secreting netrin (18) and VGEF (19,20) and regulate the speed of migration by releasing GABA (21, 22) while actively reconfiguring their own network in response to neuroblast-derived signals through the Slit/Robo machinery (16).Brain extracellular matrix is a juxtacellular scaffold that defines the microenvironmental arrangements surrounding each cell. Extracellular matrix components were earlier implicated in neuroblast migration: Tenascin-R can mediate activity-dependent neuroblast recruitment, (23) and hyaluronan and its receptor, Rhamm, driving hyaluronan-mediated motility, are selectively expressed in the adult RMS (24). Notably, members of the matrix metalloproteinase (MMP) family are recognized as critical for restructuring the extracellular matrix by cleaving all its components (25, 26). However, MMP activity and efficacy in neurogenic niches were studied chiefly in the early postnatal nervous system (12), during axonal growth, and upon cancer cell invasion (27-29) with pathologically increased proliferative capacity. In the RMS, MMP inhibitors reduce the rate of neuroblast migration in young mice, but chain migration remains unaffected in the ...

ObjectiveSpecification 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.MethodsWe used high-resolution histochemistry to explore the coincident expression of secretagogin and transcription factors driving β 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.ResultsHere, we show that secretagogin, a Ca2+ sensor protein that controls α and β 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 β 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 β cells.ConclusionsIn sum, secretagogin scales the temporal availability of a Ca2+-dependent transcription factor network to define β cell identity.

Endocannabinoids are small signaling lipids, with 2-arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB1 cannabinoid receptors (CB1Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of 2-AG signaling in DAGLα null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGLα overexpression spherically excludes cholinergic growth cones from 2-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB1R-mediated exclusion responses lasted 3 days, indicating sustained spherical 2-AG availability. Overall, these data suggest that extracellular 2-AG concentrations can be sufficient to activate CB1Rs along discrete spherical boundaries to modulate neuronal responsiveness.