Cocaine-Dependent Acquisition of Locomotor Sensitization and Conditioned Place Preference Requires D1 Dopaminergic Signaling through a Cyclic AMP, NCS-Rapgef2, ERK, and Egr-1/Zif268 Pathway.

Elucidation of the mechanism of dopamine signaling to ERK that underlies plasticity in dopamine D1 receptor-expressing neurons leading to acquired cocaine preference is incomplete. NCS-Rapgef2 is a novel cAMP effector, expressed in neuronal and endocrine cells in adult mammals, that is required for D1 dopamine receptor-dependent ERK phosphorylation in mouse brain. In this report, we studied the effects of abrogating NCS-Rapgef2 expression on cAMP-dependent ERK→Egr-1/Zif268 signaling in cultured neuroendocrine cells; in D1 medium spiny neurons of NAc slices; and in either male or female mouse brain in a region-specific manner. NCS-Rapgef2 gene deletion in the NAc in adult mice, using adeno-associated virus-mediated expression of cre recombinase, eliminated cocaine-induced ERK phosphorylation and Egr-1/Zif268 upregulation in D1-medium spiny neurons and cocaine-induced behaviors, including locomotor sensitization and conditioned place preference. Abrogation of NCS-Rapgef2 gene expression in mPFC and BLA, by crossing mice bearing a floxed Rapgef2 allele with a cre mouse line driven by calcium/calmodulin-dependent kinase IIα promoter also eliminated cocaine-induced phospho-ERK activation and Egr-1/Zif268 induction, but without effect on the cocaine-induced behaviors. Our results indicate that NCS-Rapgef2 signaling to ERK in dopamine D1 receptor-expressing neurons in the NAc, but not in corticolimbic areas, contributes to cocaine-induced locomotor sensitization and conditioned place preference. Ablation of cocaine-dependent ERK activation by elimination of NCS-Rapgef2 occurred with no effect on phosphorylation of CREB in D1 dopaminoceptive neurons of NAc. This study reveals a new cAMP-dependent signaling pathway for cocaine-induced behavioral adaptations, mediated through NCS-Rapgef2/phospho-ERK activation, independently of PKA/CREB signaling.SIGNIFICANCE STATEMENT ERK phosphorylation in dopamine D1 receptor-expressing neurons exerts a pivotal role in psychostimulant-induced neuronal gene regulation and behavioral adaptation, including locomotor sensitization and drug preference in rodents. In this study, we examined the role of dopamine signaling through the D1 receptor via a novel pathway initiated through the cAMP-activated guanine nucleotide exchange factor NCS-Rapgef2 in mice. NCS-Rapgef2 in the NAc is required for activation of ERK and Egr-1/Zif268 in D1 dopaminoceptive neurons after acute cocaine administration, and subsequent enhanced locomotor response and drug seeking behavior after repeated cocaine administration. This novel component in dopamine signaling provides a potential new target for intervention in psychostimulant-shaped behaviors, and new understanding of how D1-medium spiny neurons encode the experience of psychomotor stimulant exposure.

PACAP signaling in stress: insights from the chromaffin cell.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified in hypothalamus, based on its ability to elevate cyclic AMP in the anterior pituitary. PACAP has been identified as the adrenomedullary neurotransmitter in stress through a combination of ex vivo, in vivo, and in cellula experiments over the past two decades. PACAP causes catecholamine secretion, and activation of catecholamine biosynthetic enzymes, during episodes of stress in mammals. Features of PACAP signaling allowing stress transduction at the splanchnicoadrenomedullary synapse have yielded insights into the contrasting roles of acetylcholine's and PACAP's actions as first messengers at the chromaffin cell, via differential release at low and high rates of splanchnic nerve firing, and differential signaling pathway engagement leading to catecholamine secretion and chromaffin cell gene transcription. Secretion stimulated by PACAP, via calcium influx independent of action potential generation, is under active investigation in several laboratories both at the chromaffin cell and within autonomic ganglia of both the parasympathetic and sympathetic nervous systems. PACAP is a neurotransmitter important in stress transduction in the central nervous system as well, and is found at stress-transduction nuclei in brain including the paraventricular nucleus of hypothalamus, the amygdala and extended amygdalar nuclei, and the prefrontal cortex. The current status of PACAP as a master regulator of stress signaling in the nervous system derives fundamentally from the establishment of its role as the splanchnicoadrenomedullary transmitter in stress. Experimental elucidation of PACAP action at this synapse remains at the forefront of understanding PACAP's role in stress signaling throughout the nervous system.

Rapgef2, a guanine nucleotide exchange factor for Rap1 small GTPases, plays a crucial role in adherence junction (AJ) formation in radial glial cells through ERK-mediated upregulation of the AJ-constituent protein expression.

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and ß-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs.

Separate cyclic AMP sensors for neuritogenesis, growth arrest, and survival of neuroendocrine cells.

Dividing neuroendocrine cells differentiate into a neuronal-like phenotype in response to ligands activating G protein-coupled receptors, leading to the elevation of the second messenger cAMP. Growth factors that act at receptor tyrosine kinases, such as nerve growth factor, also cause differentiation. We report here that two aspects of cAMP-induced differentiation, neurite extension and growth arrest, are dissociable at the level of the sensors conveying the cAMP signal in PC12 and NS-1 cells. Following cAMP elevation, neuritogenic cyclic AMP sensor/Rapgef2 is activated for signaling to ERK to mediate neuritogenesis, whereas Epac2 is activated for signaling to the MAP kinase p38 to mediate growth arrest. Neither action of cAMP requires transactivation of TrkA, the receptor for NGF. In fact, the differentiating effects of NGF do not require activation of any of the cAMP sensors protein kinase A, Epac, or neuritogenic cyclic AMP sensor/Rapgef2 but, rather, depend on ERK and p38 activation via completely independent signaling pathways. Hence, cAMP- and NGF-dependent signaling for differentiation are also completely insulated from each other. Cyclic AMP and NGF also protect NS-1 cells from serum withdrawal-induced cell death, again by two wholly separate signaling mechanisms, PKA-dependent for cAMP and PKA-independent for NGF.

ERK-dependent induction of the immediate-early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs-GPCR signaling for neuritogenesis.

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6 h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50, upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis.

Cyclic AMP-dependent activation of ERK via GLP-1 receptor signalling requires the neuroendocrine cell-specific guanine nucleotide exchanger NCS-RapGEF2.

Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signalling initiated by the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), via the Gs -protein coupled receptor (Gs PCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in Neuroscreen-1 (NS-1) neuroendocrine cells. We examined the expression of other Family B Gs PCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist vasoactive intestinal polypeptide, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably as a result of insufficient receptor protein expression. Vasoactive intestinal polypeptide and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK-dependent (blocked by U0126), indicating its use of the cAMP→RapGEF2→ERK neuritogenic signalling pathway previously identified for PACAP/PAC1 signalling in NS-1 cells. NCS-RapGEF2 is expressed in the rodent insulinoma cell lines MIN6 and INS-1, as well as in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after RapGEF2-shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after 1 hour of exposure to exendin-4 revealed an immediate early-gene response that was composed of both ERK-dependent and ERK-independent signalling targets. We propose that cAMP signalling initiated by glucagon-like peptide 1 (GLP-1) in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS-RapGEF2, Epac and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation.

Epigenomic regulation by labile iron.

Iron is an essential micronutrient metal for cellular functions but can generate highly reactive oxygen species resulting in oxidative damage. For these reasons its uptake and metabolism is highly regulated. A small but dynamic fraction of ferrous iron inside the cell, termed intracellular labile iron, is redox-reactive and ready to participate multiples reactions of intracellular enzymes. Due to its nature its determination and precise quantification has been a roadblock. However, recent progress in the development of intracellular labile iron probes are allowing the reevaluation of our current understanding and unmasking new functions. The role of intracellular labile iron in regulating the epigenome was recently discovered. This chapter examine how intracellular labile iron can modulate histone and DNA demethylation and how its pool can mediate a signaling pathway from cAMP serving as a sensor of the metabolic needs of the cells.

A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

What's New in Endocrinology: The Chromaffin Cell.

Recent advances in understanding the intracellular and intercellular features of adrenal chromatin cells as stress transducers are reviewed here, along with their implications for endocrine function in other tissues and organs participating in endocrine regulation in the mammalian organism.

Differential Pharmacophore Definition of the cAMP Binding Sites of Neuritogenic cAMP Sensor-Rapgef2, Protein Kinase A, and Exchange Protein Activated by cAMP in Neuroendocrine Cells Using an Adenine-Based Scaffold.

We recently reported that the adenylate cyclase (AC) inhibitor SQ22,536 (9-tetrahydrofuranyl-adenine) also has inhibitory activity against the neuroendocrine-specific neuritogenic cAMP sensor-Rapgef2 (NCS-Rapgef2), a guanine nucleotide exchanger and activator for the small effector GTPase Rap1. Cell-based assays that distinguish signaling through the three intracellular cAMP sensors NCS-Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA), as well as AC, were used. These, collectively, assess the activities of adenine (6-amino-purine) derivatives modified at several positions to enhance selectivity for NCS-Rapgef2 by decreasing affinity for adenylate cyclase (AC), without increasing affinity for PKA or Epac. Testing of each adenine derivative in whole-cell assays incorporates features of cell permeability, target selectivity, and intrinsic potency into a single EC50 or IC50, making robust extrapolation to compound activity in vivo more likely. N6-MBC-cAMP is a selective PKA activator (EC50 = 265 µM) with low efficacy at NCS-Rapgef2. 8-CPT-2'-O-Me-cAMP and ESI-09 are confirmed as Epac-selective, for stimulation and inhibition, respectively, versus both PKA and NCS-Rapgef2. The compound N6-Phe-cAMP is a full agonist of NCS-Rapgef2 (EC50 = 256 µM). It has little or no activity against Epac or PKA. The compound N6-phenyl-9-tetrahydrofuranyladenine is a novel and potent NCS-Rapgef2 inhibitor without activity at PKA, Epac, or ACs, as assayed in the neuroendocrine NS-1 cell line. This line has been engineered to allow high-content screening for activation and inhibition of AC, PKA, Epac, and NCS-Rapgef2 and the cellular activities initiated by these signaling pathway protein components.

cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool.

It is widely accepted that cAMP regulates gene transcription principally by activating the protein kinase A (PKA)-targeted transcription factors. Here, we show that cAMP enhances the generation of 5-hydroxymethylcytosine (5hmC) in multiple cell types. 5hmC is converted from 5-methylcytosine (5mC) by Tet methylcytosine dioxygenases, for which Fe(II) is an essential cofactor. The promotion of 5hmC was mediated by a prompt increase of the intracellular labile Fe(II) pool (LIP). cAMP enhanced the acidification of endosomes for Fe(II) release to the LIP likely through RapGEF2. The effect of cAMP on Fe(II) and 5hmC was confirmed by adenylate cyclase activators, phosphodiesterase inhibitors, and most notably by stimulation of G protein-coupled receptors (GPCR). The transcriptomic changes caused by cAMP occurred in concert with 5hmC elevation in differentially transcribed genes. Collectively, these data show a previously unrecognized regulation of gene transcription by GPCR-cAMP signaling through augmentation of the intracellular labile Fe(II) pool and DNA hydroxymethylation.

Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex.

Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2(flox/flox) brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1(G12V). These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells.

A role for miR-19 in the migration of adult-born neurons and schizophrenia.

The latest miRNA database (Release 21) annotated 2588 and 1915 miRNAs in the human and mouse genomes, respectively.1 However, the biological roles of miRNAs in vivo remain largely unknown. In particular, the physiological and pathological roles of individual microRNAs in the brain have not been investigated extensively although expression profiles of microRNAs have been reported in many given conditions. In a recent study,2 we identified miR-19, which is enriched in adult hippocampal neural progenitor cells (NPCs), as a key regulator for adult hippocampal neurogenesis. miR-19 is an intrinsic factor regulating the migration of newborn neurons by modulating expression level of RAPGEF2. After observing the abnormal expression patterns of miR-19 and RAPGEF2 in NPCs derived from induced pluripotent stem cells of schizophrenic patients, which display aberrant cell migration, we proposed miR-19 as a molecule associated with schizophrenia. The results illustrate that a single microRNA has the potential to impact the functions of the brain. Identifying miRNA-mediated posttranscriptional gene regulation in the brain will expand our understanding of brain development and functions and the etiologies of several brain disorders.

Functional Implications of miR-19 in the Migration of Newborn Neurons in the Adult Brain.

Altered microRNA profiles have been implicated in human brain disorders. However, the functional contribution of individual microRNAs to neuronal development and function is largely unknown. Here, we report biological functions for miR-19 in adult neurogenesis. We determined that miR-19 is enriched in neural progenitor cells (NPCs) and downregulated during neuronal development in the adult hippocampus. By manipulating miR-19 in NPCs for gain- and loss-of-function studies, we discovered that miR-19 regulates cell migration by directly targeting Rapgef2. Concordantly, dysregulation of miR-19 in NPCs alters the positioning of newborn neurons in the adult brain. Furthermore, we found abnormal expression of miR-19 in human NPCs generated from schizophrenic patient-derived induced pluripotent stem cells (iPSCs) that have been described as displaying aberrant migration. Our study demonstrates the significance of posttranscriptional gene regulation by miR-19 in preventing the irregular migration of adult-born neurons that may contribute to the etiology of schizophrenia.