The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.

18F-FDOPA PET/CT Uptake Parameters Correlate with Catecholamine Secretion in Human Pheochromocytomas.

BACKGROUND: 18F-FDOPA positron emission tomography/computed tomography (PET/CT) is a sensitive nuclear imaging technology for the diagnosis of pheochromocytomas (PHEO). However, its utility in determining predictive factors for the secretion of catecholamines remains poorly studied. METHODS: Thirty-nine histologically confirmed PHEO were included in this retrospective single-center study. Patients underwent 18F-FDOPA PET/CT before surgery, with an evaluation of several uptake parameters (standardized uptake values [SUVmax and SUVmean] and the metabolic burden [MB] calculated as follows: MB = SUVmean × tumor volume) and measurement of plasma and/or urinary metanephrine (MN), normetanephrine (NM), and chromogranin A. Thirty-five patients were screened for germline mutations in the RET, SDHx, and VHL genes. Once resected, primary cultures of 5 PHEO were used for real-time measurement of catecholamine release by carbon fiber amperometry. RESULTS: The MB of the PHEO positively correlated with 24-h urinary excretion of NM (r = 0.64, p < 0.0001), MN (r = 0.49, p = 0.002), combined MN and NM (r = 0.75, p < 0.0001), and eventually plasma free levels of NM (r = 0.55, p = 0.006). In the mutated patients (3 SDHD, 2 SDHB, 3 NF1, 1 VHL, and 3 RET), a similar correlation was observed between MB and 24-h urinary combined MN and NM (r = 0.86, p = 0.0012). For the first time, we demonstrate a positive correlation between the PHEO-to-liver SUVmax ratio and the mean number of secretory granule fusion events of the corresponding PHEO cells revealed by amperometric spikes (p = 0.01). CONCLUSION: While the 18F-FDOPA PET/CT MB of PHEO strongly correlates with the concentration of MN, amperometric recordings suggest that 18F-FDOPA uptake could be enhanced by overactivity of catecholamine exocytosis.

Dynamic Regulation of AMPAR Phosphorylation In Vivo Following Acute Behavioral Stress.

The tuning of glutamatergic transmission is an essential mechanism for neuronal communication. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ionotropic glutamate receptors that mediate fast synaptic transmission. The phosphorylation states of specific serine residues on the GluA1 and GluA2 AMPAR subunits are considered critical post-translational modifications that regulate AMPAR activity and subcellular trafficking. While behavioral stress, via stress hormones, exerts specific alterations on such glutamatergic processes, there have been conflicting data concerning the influence of stress on AMPAR phosphorylation in different brain regions, and the post-stress signaling mechanisms mediating these processes are not well delineated. Here, we examined the dynamics of phosphorylation at three AMPAR serine residues (ser831-GluA1, ser845-GluA1, and ser880-GluA2) in four brain regions [amygdala, medial prefrontal cortex (mPFC), dorsal hippocampus, and ventral hippocampus] of the rat during the hour following behavioral stress. We also tested the impact of post-stress corticosteroid receptor blockade on AMPAR phosphorylation. Both GluA1 subunit residues exhibited elevated phosphorylation after stress, yet post-stress administration of corticosteroid receptor antagonists curtailed these effects only at ser831-GluA1. In contrast, ser880-GluA2 displayed a time-dependent tendency for early decreased phosphorylation (that was selectively augmented by mifepristone treatment in the amygdala and mPFC of stressed animals) followed by increased phosphorylation later on. These findings show that the in vivo regulation of AMPAR phosphorylation after stress is a dynamic and subunit-specific process, and they provide support for the hypothesis that corticosteroid receptors have an ongoing role in the regulation of ser831-GluA1 phosphorylation during the post-stress interval.

Progesterone and nestorone promote myelin regeneration in chronic demyelinating lesions of corpus callosum and cerebral cortex.

Multiple Sclerosis affects mainly women and consists in intermittent or chronic damages to the myelin sheaths, focal inflammation, and axonal degeneration. Current therapies are limited to immunomodulators and antiinflammatory drugs, but there is no efficient treatment for stimulating the endogenous capacity of myelin repair. Progesterone and synthetic progestins have been shown in animal models of demyelination to attenuate myelin loss, reduce clinical symptoms severity, modulate inflammatory responses and partially reverse the age-dependent decline in remyelination. Moreover, progesterone has been demonstrated to promote myelin formation in organotypic cultures of cerebellar slices. In the present study, we show that progesterone and the synthetic 19-nor-progesterone derivative Nestorone® promote the repair of severe chronic demyelinating lesions induced by feeding cuprizone to female mice for up to 12 weeks. Progesterone and Nestorone increase the density of NG2(+) oligodendrocyte progenitor cells and CA II(+) mature oligodendrocytes and enhance the formation of myelin basic protein (MBP)- and proteolipid protein (PLP)-immunoreactive myelin. However, while demyelination in response to cuprizone was less marked in corpus callosum than in cerebral cortex, remyelination appeared earlier in the former. The remyelinating effect of progesterone was progesterone receptor (PR)-dependent, as it was absent in PR-knockout mice. Progesterone and Nestorone also decreased (but did not suppress) neuroinflammatory responses, specifically astrocyte and microglial cell activation. Therefore, some progestogens are promising therapeutic candidates for promoting the regeneration of myelin.

The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.

The evolutionary conserved CCR4-NOT complex functions in the cytoplasm as the main mRNA deadenylase in both constitutive mRNA turnover and regulated mRNA decay pathways. The versatility of this complex is underpinned by its modular multi-subunit organization, with distinct structural modules actuating different functions. The structure and function of all modules are known, except for that of the N-terminal module. Using different structural approaches, we obtained high-resolution data revealing the architecture of the human N-terminal module composed of CNOT1, CNOT10, and CNOT11. The structure shows how two helical domains of CNOT1 sandwich CNOT10 and CNOT11, leaving the most conserved domain of CNOT11 protruding into solvent as an antenna. We discovered that GGNBP2, a protein identified as a tumor suppressor and spermatogenic factor, is a conserved interacting partner of the CNOT11 antenna domain. Structural and biochemical analyses thus pinpoint the N-terminal CNOT1-CNOT10-CNOT11 module as a conserved protein-protein interaction platform.

A Role for the V0 Sector of the V-ATPase in Neuroexocytosis: Exogenous V0d Blocks Complexin and SNARE Interactions with V0c.

V-ATPase is an important factor in synaptic vesicle acidification and is implicated in synaptic transmission. Rotation in the extra-membranous V1 sector drives proton transfer through the membrane-embedded multi-subunit V0 sector of the V-ATPase. Intra-vesicular protons are then used to drive neurotransmitter uptake by synaptic vesicles. V0a and V0c, two membrane subunits of the V0 sector, have been shown to interact with SNARE proteins, and their photo-inactivation rapidly impairs synaptic transmission. V0d, a soluble subunit of the V0 sector strongly interacts with its membrane-embedded subunits and is crucial for the canonic proton transfer activity of the V-ATPase. Our investigations show that the loop 1.2 of V0c interacts with complexin, a major partner of the SNARE machinery and that V0d1 binding to V0c inhibits this interaction, as well as V0c association with SNARE complex. The injection of recombinant V0d1 in rat superior cervical ganglion neurons rapidly reduced neurotransmission. In chromaffin cells, V0d1 overexpression and V0c silencing modified in a comparable manner several parameters of unitary exocytotic events. Our data suggest that V0c subunit promotes exocytosis via interactions with complexin and SNAREs and that this activity can be antagonized by exogenous V0d.

Dysfunction of calcium-regulated exocytosis at a single-cell level causes catecholamine hypersecretion in patients with pheochromocytoma.

Neuroendocrine tumors constitute a heterogeneous group of tumors arising from hormone-secreting cells and are generally associated with a dysfunction of secretion. Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible for an excess of catecholamine secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, while the hypersecretory activity of Pheo is well known to pathologists and clinicians, it has never been carefully explored at the cellular and molecular levels. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheos, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. In most patients, catecholamine secretion recordings from single Pheo cells revealed a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.

Somatostatin analogue pasireotide (SOM230) inhibits catecholamine secretion in human pheochromocytoma cells.

Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patients with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.

Nestorone® , a 19nor-progesterone derivative boosts remyelination in an animal model of demyelination.

INTRODUCTION: We previously showed that Nestorone® (NES), a synthetic progestin structurally related to progesterone, stimulated remyelination of the corpus callosum in a Cuprizone (CUP) mouse model of demyelination in intact females by promoting replenishment with mature oligodendrocytes (OL) (Glia. 2015;63:104-117). Here, we further investigated the underlying mechanisms of this promyelinating effect. METHODS: We explored whether NES, applied subcutaneously through Alzet mini-osmotic pumps, regulates specific transcription factors involved in oligodendrocyte progenitor cell (OPC) proliferation and their differentiation into mature OL, using RT-qPCR and Western Blot analysis. RESULTS: Our present data show that in comparison to controls, a one-week treatment with NES, through Alzet mini-osmotic pumps, enhanced the production of three relevant transcription factor mRNAs encoding Olig2, Myt1, and Sox17. After 3 weeks, NES treatment reversed the effect of CUP on the levels of corresponding Olig2, Myt1, and Sox17 proteins. Moreover, in mice receiving NES + Estradiol (E2) co-treatment, levels of Olig2, Myt1, and Sox17 proteins did not change as compared to NES alone. CONCLUSION: NES alone or with E2 increased the levels of transcription factors, essential for myelin synthesis.

Clozapine counteracts a ketamine-induced depression of hippocampal-prefrontal neuroplasticity and alters signaling pathway phosphorylation.

Single sub-anesthetic doses of ketamine can exacerbate the symptoms of patients diagnosed with schizophrenia, yet similar ketamine treatments rapidly reduce depressive symptoms in major depression. Acute doses of the atypical antipsychotic drug clozapine have also been shown to counteract ketamine-induced psychotic effects. In the interest of understanding whether these drug effects could be modeled with alterations in neuroplasticity, we examined the impact of acutely-administered ketamine and clozapine on in vivo long-term potentiation (LTP) in the rat's hippocampus-to-prefrontal cortex (H-PFC) pathway. We found that a low dose of ketamine depressed H-PFC LTP, whereas animals that were co-administrated the two drugs displayed LTP that was similar to a saline-treated control. To address which signaling molecules might mediate such effects, we also examined phosphorylation and total protein levels of GSK3ß, GluA1, TrkB, ERK, and mTOR in prefrontal and hippocampal sub-regions. Among the statistically significant effects that were detected (a) both ketamine and clozapine increased the phosphorylation of Ser9-GSK3ß throughout the prefrontal cortex and of Ser2481-mTOR in the dorsal hippocampus (DH), (b) clozapine increased the phosphorylation of Ser831-GluA1 throughout the prefrontal cortex and of Ser845-GluA1 in the ventral hippocampus, (c) ketamine treatment increased the phosphorylation of Thr202/Tyr204-ERK in the medial PFC (mPFC), and (d) clozapine treatment was associated with decreases in the phosphorylation of Tyr705-TrkB in the DH and of Try816-TrkB in the mPFC. Further analyses involving phosphorylation effect sizes also suggested Ser831-GluA1 in the PFC displayed the highest degree of clozapine-responsivity relative to ketamine. These results provide evidence for how ketamine and clozapine treatments affect neuroplasticity and signaling pathways in the stress-sensitive H-PFC network. They also demonstrate the potential relevance of H-PFC pathway neuroplasticity for modeling ketamine-clozapine interactions in regards to psychosis.

Nestorone® as a Novel Progestin for Nonoral Contraception: Structure-Activity Relationships and Brain Metabolism Studies.

Nestorone® (NES) is a potent nonandrogenic progestin being developed for contraception. NES is a synthetic progestin that may possess neuroprotective and myelin regenerative potential as added health benefits. In receptor transactivation experiments, NES displayed greater potency than progesterone to transactivate the human progesterone receptor (PR). This was confirmed by docking experiments where NES adopts the same docking position within the PR ligand-binding domain (LBD) as progesterone and forms additional stabilizing contacts between 17α-acetoxy and 16-methylene groups and PR LBD, supporting its higher potency than progesterone. The analog 13-ethyl NES also establishes similar contacts as NES with Met909, leading to comparable potency as NES. In contrast, NES is not stabilized within the human androgen receptor LBD, leading to negligible androgen receptor transactivation. Because progesterone acts in the brain by both PR binding and indirectly via binding of the metabolite allopregnanolone to γ-aminobutyric acid type A receptor (GABAAR), we investigated if NES is metabolized to 3α, 5α-tetrahydronestorone (3α, 5α-THNES) in the brain and if this metabolite could interact with GABAAR. In female mice, low concentrations of reduced NES metabolites were identified by gas chromatography/mass spectrometry in both plasma and brain. Electrophysiological studies showed that 3α, 5α-THNES exhibited only limited activity to enhance GABAAR-evoked responses with WSS-1 cells and did not modulate synaptic GABAARs of mouse cortical neurons. Thus, the inability of reduced metabolite of NES (3α, 5α-THNES) to activate GABAAR suggests that the neuroprotective and myelin regenerative effects of NES are mediated via PR binding and not via its interaction with the GABAAR.

Chronic cannabinoid exposure during adolescence leads to long-term structural and functional changes in the prefrontal cortex.

In many species, adolescence is a critical phase in which the endocannabinoid system can regulate the maturation of important neuronal networks that underlie cognitive function. Therefore, adolescents may be more susceptible to the neural consequences of chronic cannabis abuse. We reported previously that chronically exposing adolescent rats to the synthetic cannabinoid agonist CP55,940 leads to impaired performances in adulthood i.e. long-lasting deficits in both visual and spatial short-term working memories. Here, we examined the synaptic structure and function in the prefrontal cortex (PFC) of adult rats that were chronically treated with CP55,940 during adolescence. We found that chronic cannabinoid exposure during adolescence induces long-lasting changes, including (1) significantly altered dendritic arborization of pyramidal neurons in layer II/III in the medial PFC (2) impaired hippocampal input-induced synaptic plasticity in the PFC and (3) significant changes in the expression of PSD95 (but not synaptophysin or VGLUT3) in the medial PFC. These changes in synaptic structure and function in the PFC provide key insight into the structural, functional and molecular underpinnings of long-term cognitive deficits induced by adolescent cannabinoid exposure. They suggest that cannabinoids may impede the structural maturation of neuronal circuits in the PFC, thus leading to impaired cognitive function in adulthood.