The Inhibition of RasGRF2, But Not RasGRF1, Alters Cocaine Reward in Mice.

Ras/Raf/MEK/ERK (Ras-ERK) signaling has been implicated in the effects of drugs of abuse. Inhibitors of MEK1/2, the kinases upstream of ERK1/2, have been critical in defining the role of the Ras-ERK cascade in drug-dependent alterations in behavioral plasticity, but the Ras family of small GTPases has not been extensively examined in drug-related behaviors. We examined the role of Ras Guanine Nucleotide Releasing Factor 1 (RasGRF1) and 2 (RasGRF2), upstream regulators of the Ras-ERK signaling cascade, on cocaine self-administration (SA) in male mice. We first established a role for Ras-ERK signaling in cocaine SA, demonstrating that pERK1/2 is upregulated following SA in C57BL/6N mice in striatum. We then compared RasGRF1 and RasGRF2 KO mouse lines, demonstrating that cocaine SA in RasGRF2 KO mice was increased relative to WT controls, whereas RasGRF1 KO and WT mice did not differ. This effect in RasGRF2 mice is likely mediated by the Ras-ERK signaling pathway, as pERK1/2 upregulation following cocaine SA was absent in RasGRF2 KO mice. Interestingly, the lentiviral knockdown of RasGRF2 in the NAc had the opposite effect to that in RasGRF2 KO mice, reducing cocaine SA. We subsequently demonstrated that the MEK inhibitor PD325901 administered peripherally prior to cocaine SA increased cocaine intake, replicating the increase seen in RasGRF2 KO mice, whereas PD325901 administered into the NAc decreased cocaine intake, similar to the effect seen following lentiviral knockdown of RasGRF2. These data indicate a role for RasGRF2 in cocaine SA in mice that is ERK-dependent, and suggest a differential effect of global versus site-specific RasGRF2 inhibition.SIGNIFICANCE STATEMENT Exposure to drugs of abuse activates a variety of intracellular pathways, and following repeated exposure, persistent changes in these pathways contribute to drug dependence. Downstream components of the Ras-ERK signaling cascade are involved in the acute and chronic effects of drugs of abuse, but their upstream mediators have not been extensively characterized. Here we show, using a combination of molecular, pharmacological, and lentiviral techniques, that the guanine nucleotide exchange factor RasGRF2 mediates cocaine self-administration via an ERK-dependent mechanism, whereas RasGRF1 has no effect on responding for cocaine. These data indicate dissociative effects of mediators of Ras activity on cocaine reward and expand the understanding of the contribution of Ras-ERK signaling to drug-taking behavior.

Age-dependent role for Ras-GRF1 in the late stages of adult neurogenesis in the dentate gyrus.

The dentate gyrus of the hippocampus plays a pivotal role in pattern separation, a process required for the behavioral task of contextual discrimination. One unique feature of the dentate gyrus that contributes to pattern separation is adult neurogenesis, where newly born neurons play a distinct role in neuronal circuitry. Moreover,the function of neurogenesis in this brain region differs in adolescent and adult mice. The signaling mechanisms that differentially regulate the distinct steps of adult neurogenesis in adolescence and adulthood remain poorly understood. We used mice lacking RASGRF1(GRF1), a calcium-dependent exchange factor that regulates synaptic plasticity and participates in contextual discrimination performed by mice, to test whether GRF1 plays a role in adult neurogenesis.We show Grf1 knockout mice begin to display a defect in neurogenesis at the onset of adulthood (~2 months of age), when wild-type mice first acquire the ability to distinguish between closely related contexts. At this age, young hippocampal neurons in Grf1 knockout mice display severely reduced dendritic arborization. By 3 months of age, new neuron survival is also impaired. BrdU labeling of new neurons in 2-month-old Grf1 knockout mice shows they begin to display reduced survival between 2 and 3 weeks after birth, just as new neurons begin to develop complex dendritic morphology and transition into using glutamatergic excitatory input. Interestingly, GRF1 expression appears in new neurons at the developmental stage when GRF1 loss begins to effect neuronal function. In addition, we induced a similar loss of new hippocampal neurons by knocking down expression of GRF1 solely in new neurons by injecting retrovirus that express shRNA against GRF1 into the dentate gyrus. Together, these findings show that GRF1 expressed in new neurons promotes late stages of adult neurogenesis. Overall our findings show GRF1 to be an age-dependent regulator of adult hippocampal neurogenesis, which contributes to ability of mice to distinguish closely related contexts.

The RasGrf family of mammalian guanine nucleotide exchange factors.

RasGrf1 and RasGrf2 are highly homologous mammalian guanine nucleotide exchange factors which are able to activate specific Ras or Rho GTPases. The RasGrf genes are preferentially expressed in the central nervous system, although specific expression of either locus may also occur elsewhere. RasGrf1 is a paternally-expressed, imprinted gene that is expressed only after birth. In contrast, RasGrf2 is not imprinted and shows a wider expression pattern. A variety of isoforms for both genes are also detectable in different cellular contexts. The RasGrf proteins exhibit modular structures composed by multiple domains including CDC25H and DHPH motifs responsible for promoting GDP/GTP exchange, respectively, on Ras or Rho GTPase targets. The various domains are essential to define their intrinsic exchanger activity and to modulate the specificity of their functional activity so as to connect different upstream signals to various downstream targets and cellular responses. Despite their homology, RasGrf1 and RasGrf2 display differing target specificities and non overlapping functional roles in a variety of signaling contexts related to cell growth and differentiation as well as neuronal excitability and response or synaptic plasticity. Whereas both RasGrfs are activatable by glutamate receptors, G-protein-coupled receptors or changes in intracellular calcium concentration, only RasGrf1 is reported to be activated by LPA, cAMP, or agonist-activated Trk and cannabinoid receptors. Analysis of various knockout mice strains has uncovered a specific functional contribution of RasGrf1 in processes of memory and learning, photoreception, control of post-natal growth and body size and pancreatic ß-cell function and glucose homeostasis. For RasGrf2, specific roles in lymphocyte proliferation, T-cell signaling responses and lymphomagenesis have been described.

Differential roles of NMDA Receptor Subtypes NR2A and NR2B in dendritic branch development and requirement of RasGRF1.

N-methyl-D-aspartate receptors (NMDARs) are known to regulate axonal refinement and dendritic branching. However, because NMDARs are abundantly present as tri-heteromers (e.g., NR1/NR2A/NR2B) during development, the precise role of the individual subunits NR2A and NR2B in these processes has not been elucidated. Ventral spinal cord neurons (VSCNs) provide a unique opportunity to address this problem, because the expression of both NR2A and NR2B (but not NR1) is downregulated in culture. Exogenous NR2A or NR2B were introduced into these naturally NR2-null neurons at 4 DIV, and electrophysiological recordings at 11 DIV confirmed that synaptic NR1NR2A receptors and NR1NR2B receptors were formed, respectively. Analysis of the dendritic architecture showed that introduction of NR2B, but not NR2A, dramatically increased the number of secondary and tertiary dendritic branches of VSCNs. Whole cell patch-clamp recordings further indicated that the newly formed branches in NR2B-expressing neurons were able to establish functional synapses because the frequency of miniature AMPA-receptor synaptic currents was increased. Using previously described mutants, we also found that disruption of the interaction between NR2B and RasGRF1 dramatically impaired dendritic branch formation in VSCNs. The differential role of the NR2A and NR2B subunits and the requirement for RasGRF1 in regulating branch formation was corroborated in hippocampal cultures. We conclude that the association between NR1NR2B-receptors and RasGRF1 is needed for dendritic branch formation in VSCNs and hippocampal neurons in vitro. The dominated NR2A expression and the limited interactions of this subunit with the signaling protein RasGRF1 may contribute to the restricted dendritic arbor development in the adult CNS.

The environment versus genetics in controlling the contribution of MAP kinases to synaptic plasticity.

BACKGROUND: A challenge in biomedical research is to design experimental paradigms that reflect a natural setting. Even when freshly isolated tissues are used, they are almost always derived from animals housed in cages that poorly reflect the animal's native environment. This issue is highlighted by studies on brain function, where mice housed in a more natural "enriched environment" display enhanced learning and memory and delayed onset of symptoms of neurodegenerative diseases compared to mice housed conventionally. How the environment mediates its effects on brain function is poorly understood. RESULTS: We show that after exposure of adolescent mice to an "enriched environment," the induction of long-term potentiation (LTP), a form of synaptic plasticity that is thought to contribute to learning and memory, involves a novel signal transduction pathway that is nonfunctional in comparable mice housed conventionally. This environmentally gated signaling pathway, which rescues defective LTP induction in adolescent Ras-GRF knockout mice, consists of NMDA glutamate receptor activation of p38, a MAP kinase that does not contribute to LTP in mice housed conventionally. Interestingly, the same exposure to environmental enrichment does not have this effect in adult mice. CONCLUSIONS: This study reveals a new level of cell signaling control whereby environmental factors gate the efficacy of a specific signaling cascade to control how LTP is induced in adolescent animals. The suppression of this gating mechanism in mature animals represents a new form of age-dependent decline in brain plasticity.

Rgnef promotes ovarian tumor progression and confers protection from oxidative stress.

Ovarian cancer is the fifth-leading cause of cancer death among women. The dissemination of ovarian tumors and growth as spheroids accompanies late-stage disease. In cell culture, ovarian tumor cell spheroids can exhibit elevated resistance to environmental stressors, such as reactive oxygen species. Homeostatic balance of the antioxidant response is a protective mechanism that prevents anoikis, a form of programmed cell death. Signaling pathways activated by integrin receptors suppress anoikis. Rgnef (ARHGEF28/p190RhoGEF) is a guanine nucleotide exchange factor that is activated downstream of integrins. We find that Rgnef protein levels are elevated in late-stage serous ovarian cancer, high Rgnef mRNA levels are associated with decreased progression-free and overall survival, and genomic ARHGEF28 loss is associated with increased patient survival. Using transgenic and transplantable Rgnef knockout mouse models, we find that Rgnef is essential for supporting three-dimensional ovarian spheroid formation in vitro and tumor growth in mice. Using RNA-sequencing and bioinformatic analyses, we identify a conserved Rgnef-supported anti-oxidant gene signature including Gpx4, Nqo1, and Gsta4; common targets of the NF-kB transcription factor. Antioxidant treatment enhanced growth of Rgnef-knockout spheroids and Rgnef re-expression facilitated NF-κB-dependent tumorsphere survival. These studies reveal a new role for Rgnef in ovarian cancer to facilitate NF-κB-mediated gene expression protecting cells from oxidative stress.

Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application.

In plants, the G2/M control of cell cycle remains an elusive issue as doubts persist about activatory dephosphorylation--in other eukaryotes provided by CDC25 phosphatase and serving as a final all-or-nothing mitosis regulator. We report on the effects of tobacco (Nicotiana tabacum L., cv. Samsun) transformation with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on cell characteristics. Transformed cell suspension cultures showed higher dry mass accumulation during the exponential phase and clustered more circular cell phenotypes compared to chains of elongated WT cells. Similar cell parameters, as in the transformants, can be induced in WT by cytokinins. Spcdc25 cells, after cytokinin treatment, showed giant cell clusters and growth inhibition. In addition, Spcdc25 expression led to altered carbohydrate status: increased starch and soluble sugars with higher sucrose:hexoses ratio, inducible in WT by cytokinin treatment. Taken together, the Spcdc25 transformation had a cytokinin-like effect on studied characteristics. However, endogenous cytokinin determination revealed markedly lower cytokinin levels in Spcdc25 transformants. This indicates that the cells sense Spcdc25 expression as an increased cytokinin availability, manifested by changed cell morphology, and in consequence decrease endogenous cytokinin levels. Clearly, the results on cell growth and morphology are consistent with the model of G2/M control including cytokinin-regulated activatory dephosphorylation. Nevertheless, no clear link is obvious between Spcdc25 transformation and carbohydrate status and thus the observed cytokinin-like effect on carbohydrate levels poses a problem. Hence, we propose that Spcdc25-induced higher CDK(s) activity at G2/M generates a signal-modifying carbohydrate metabolism to meet high energy and C demands of forthcoming cell division.

Spatial learning and long-term memory impairments in RasGrf1 KO, Pttg1 KO, and double KO mice.

BACKGROUND: RasGrf1 is a guanine-nucleotide releasing factor that enhances Ras activity. Human PTTG1 is an oncoprotein found in pituitary tumors and later identified as securin, a protein isolated from yeast with a reported role in chromosome separation. It has been suggested that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and their physiological response. At memory formation level, there are contradictory data regarding the role of RasGrf1, while Pttg1 has not been previously studied. Both proteins are expressed in the mammalian hippocampus, which is one of the key brain areas for spatial learning and memory. OBJECTIVE: The aim of this work was to study a potential link between RasGrf1 and Pttg1 in memory formation. METHOD: Spatial learning and memory test in the Pttg1 KO, RasGrf1 KO, and Pttg1-RasGrf1 double KO and their correspondent WT mice using a Barnes maze. RESULTS: In comparison with the WT control mice, Pttg1 KO mice learned how to solve the task in a less efficient way, suggesting problems in memory consolidation. RasGrf1 KO mice performance was similar to controls, and they learned to use the best searching strategy. Double KO mice reached a better spatial learning level than WT. CONCLUSION: A role for Pttg1 in memory consolidation/formation is suggested, while our RasGrf1 KO mice do not show hippocampus associated memory defects.

Distinct roles for Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 in the induction of long-term potentiation and long-term depression.

NMDA-type glutamate receptors (NMDARs) contribute to many forms of long-term potentiation (LTP) and long-term depression (LTD). NMDARs are heteromers containing calcium-permeating neuronal receptor 1 (NR1) subunits and a variety of NR2 subunits. Evidence suggests that, in the CA1 region of the hippocampus, NR2A-containing NMDARs promote LTP whereas NR2B-containing receptors promote LTD. However, the calcium sensors that distinguish between these signals to promote the appropriate form of synaptic plasticity are not known. Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 are highly similar calcium-stimulated exchange factors that activate Ras and Rac GTPases. Here, using a set of Ras-GRF knock-out mice, we show that Ras-GRF2 contributes predominantly to the induction of NMDAR-dependent LTP, whereas Ras-GRF1 contributes predominantly to the induction of NMDAR-dependent LTD in the CA1 region of the hippocampus of postpubescent mice (postnatal days 25-36). In contrast, neither Ras-GRF protein influences synaptic plasticity in prepubescent mice (postnatal days 14-18). Ras-GRF2 mediates signaling from (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl-phosphonic acid-sensitive (NVP-AAM077-sensitive) (NR2A-containing) NMDARs to the Ras effector extracellular signal-related protein kinase 1/2 (Erk1/2) mitogen-activated protein (MAP) kinase, a promoter of NMDAR-induced LTP at this site. In contrast, Ras-GRF1 mediates signaling from ifenprodil-sensitive (NR2B-containing) NMDARs to the Rac effector p38 MAP kinase, a promoter of LTD. These findings show that, despite their similar functional domain organization, Ras-GRF1 and Ras-GRF2 mediate opposing forms of synaptic plasticity by coupling different classes of NMDARs to distinct MAP kinase pathways. Moreover, the postnatal appearance of Ras-GRF-dependent LTP and LTD coincides with the emergence of hippocampal-dependent behavior, implying that Ras-GRF proteins contribute to forms of synaptic plasticity that are required specifically for mature hippocampal function.

A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks.

Targeted disruption of Ras-Grf2 shows its dispensability for mouse growth and development.

The mammalian Grf1 and Grf2 proteins are Ras guanine nucleotide exchange factors (GEFs) sharing a high degree of structural homology, as well as an elevated expression level in central nervous system tissues. Such similarities raise questions concerning the specificity and/or redundancy at the functional level between the two Grf proteins. grf1-null mutant mice have been recently described which showed phenotypic growth reduction and long-term memory loss. To gain insight into the in vivo function of Grf2, we disrupted its catalytic CDC25-H domain by means of gene targeting. Breeding among grf2(+/-) animals gave rise to viable grf2(-/-) adult animals with a normal Mendelian pattern, suggesting that Grf2 is not essential for embryonic and adult mouse development. In contrast to Grf1-null mice, analysis of grf2(-/-) litters showed similar size and weight as their heterozygous or wild-type grf2 counterparts. Furthermore, adult grf2(-/-) animals reached sexual maturity at the same age as their wild-type littermates and showed similar fertility levels. No specific pathology was observed in adult Grf2-null animals, and histopathological studies showed no observable differences between null mutant and wild-type Grf2 mice. These results indicate that grf2 is dispensable for mouse growth, development, and fertility. Furthermore, analysis of double grf1/grf2 null animals did not show any observable phenotypic difference with single grf1(-/-) animals, further indicating a lack of functional overlapping between the two otherwise highly homologous Grf1 and Grf2 proteins.

Rasgrf1 mRNA expression in myopic eyes of guinea pigs.

BACKGROUND: Genome-wide association studies of patients have linked the Rasgrf1 gene with myopia. The aim of this study was to investigate the messenger RNA (mRNA) expression of Rasgrf1 in the eyes of guinea pigs with induced myopia. METHODS: The myopia was induced by form deprivation in 24 guinea pigs, while additional 12 animals served as a control. Biometric measurements were used to monitor myopic progression. The animals were sacrificed at two, three and four weeks after beginning of the monocular form deprivation, followed by dissection of the retina, and the sclera, as well as mRNA isolation from both layers. A quantitative reverse transcriptase-polymerase chain reaction was performed to detect the expression of Rasgrf1. RESULTS: The spherical equivalent in eyes subjected to form deprivation differed from the fellow eyes, with measurements of -3.80 ± 0.08 D, -3.96 ± 0.94 D and -4.00 ± 0.94 D at the two-, three- and four-week times, respectively, significantly more myopia than the inter-ocular difference in the control group (p < 0.05). The form-deprived eyes also had a longer axial length compared with the fellow eye: 1.37 ± 0.76 mm, 1.32 ± 0.65 mm and 0.92 ± 0.80 mm at two, three and four weeks, respectively, significantly different from the control group (p < 0.05). In contrast, there was no difference in the corneal curvature, anterior chamber depth or lens thickness between the two eyes at any time (p > 0.05). The increase of Rasgrf1 expression was observed in the sclera, with a fold change of 6.596, 4.379 after three weeks and 6.788, 5.711 after four weeks of treatment, compared with the fellow eyes and the control group, respectively (p < 0.05). CONCLUSION: Rasgrf1 up-regulation was found in the sclera of myopic eyes; however, further investigation is needed to determine whether Rasgrf1 plays a causative role or is a consequence of myopia-induced scleral remodelling.

CDC25(Mm)/Ras-GRF1 regulates both Ras and Rac signaling pathways.

The Ras-GRF1 exchange factor molecule contains in addition to the catalytic domain two pleckstrin homology (PH1 and PH2), one IQ and one Dbl homology (DH) domains. In this study we investigated the role of such additional domains. We found that a Ras-GRF1 mutant lacking PH1 and IQ domains is sufficient to activate c-fos promoter in response to lysophosphatidic acid (LPA). The same mutant did not increase external stimuli-regulated kinase (ERK) activity, suggesting an additional mechanism for the induction of gene transcription. Isolated DH-PH2 module activates c-Jun NH(2)-terminal kinase and the c-fos promoter in response to LPA, providing the basis for an ERK-independent mechanism. These results provide evidence that Ras-GRF1 acts as a bifunctional molecule on both ERK-dependent and independent pathways.

Hippocampus-dependent learning and memory is impaired in mice lacking the Ras-guanine-nucleotide releasing factor 1 (Ras-GRF1).

Previous results have suggested that the Ras signaling pathway is involved in learning and memory. Ras is activated by nucleotide exchange factors, such as the calmodulin-activated guanine-nucleotide releasing factor 1 (Ras-GRF1). To test whether Ras-GRF1 is required for learning and memory, we inactivated the Ras-GRF1 gene in mice. These mutants performed normally in a rota-rod motor coordination task, and in two amygdala-dependent tasks (inhibitory avoidance and contextual conditioning). In contrast the mutants were impaired in three hippocampus-dependent learning tasks: contextual discrimination, the social transmission of food preferences, and the hidden-platform version of the Morris water maze. These studies indicate that Ras-GRF1 plays a role in hippocampal-dependent learning and memory.

At acidic pH, the diminished hypoxic expression of the SRP1/TIR1 yeast gene depends on the GPA2-cAMP and HOG pathways.

The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein, which is shown to be necessary for yeast growth at acidic pH in the presence of sodium dodecyl sulfate. However, the hypoxic expression of SRP1 is shown to be downregulated at acidic pH. The stress-responsive HOG pathway appeared necessary to maintain hypoxic SRP1 expression, but only at acidic pH. However, unlike known HOG pathway-dependent genes, SRP1 was under positive cAMP control and was positively modulated by protein kinase A at neutral and acidic pH. In addition, the HOG-independent hypoxic HEM13 gene was also positively regulated by cAMP levels. Therefore, the positive cAMP control of the hypoxic SRP1 and HEM13 genes was uncoupled from the HOG pathway. Surprisingly, this positive cAMP control was found to be mediated by GPA2 but not by RAS2, so the Gpa2p requirement appears critical at acidic pH. Although RAS2 is not involved in the regulation of SRP1 expression, the guanine nucleotide exchange factor Cdc25, which is known to control the GTP/GDP ratio on the Ras proteins, was nevertheless required for hypoxic SRP1 expression. Furthermore, the Ras proteins did not compensate for Gpa2p requirement in a delta gpa2 mutated strain. These results suggest that the Cdc25 factor might also control Gpa2p.

Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator.

During the last decade, the cell cycle and its control by cyclin-dependent kinases (CDKs) has been extensively studied in eukaryotes. The regulation of CDK activity includes, among others, its activation by Cdc25 phosphatase at G2/M. However, within the plant kingdom studies of this regulation have lagged behind and a plant cdc25 homologue has not been identified yet. Here, we report on the effects of transformation of tobacco (Nicotiana tabacum L., cv. Samsun) with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on de novo plant organ formation, a process dependent on rate and orientation of cell division. On shoot-inducing medium (low 1-naphthylacetic acid (NAA), high 6-benzylaminopurine (BAP)) the number of shoots formed on internode segments cultured from transgenic plants was substantially higher than in the non-transformed controls. Anatomical observations indicated that the shoot formation process was accelerated but with no changes in the quality and sequence of shoot development. Surprisingly, and in contrast to the controls, when on root-inducing medium (high NAA, low BAP) cultured segments from transgenic plants failed to initiate hardly any roots. Instead, they continued to form shoots at low frequencies. Moreover, in marked contrast to the controls, stem segments from transgenic plants were able to form shoots even without the addition of exogenous growth regulators to the medium. The results indicate that Spcdc25 expression in culture tobacco stem segments mimicked the developmental effects caused by an exogenous hormone balance shifted towards cytokinins. The observed cytokinin-like effects of Spcdc25 transformation are consistent with the concept of an interaction between cell cycle regulators and phytohormones during plant development.

Phosphatases driving mitosis: pushing the gas and lifting the brakes.

Entry into and progression through mitosis depends critically on the establishment and maintenance of protein phosphorylation. For this reason, studies on mitotic progression have focused heavily on the activation of MPF (M phase promoting factor), a cyclin-dependent kinase responsible for phosphorylating proteins that execute the dynamic events of mitosis. Recent work, however, has significantly expanded our understanding of mechanisms that allow accumulation of phosphoproteins at M phase, suggesting that mitotic entry relies not only on MPF activation but also on the inhibition of antimitotic phosphatases. It is now clear that there exists a separate, albeit equally important, signaling pathway for the inactivation of protein phosphatases at the G2/M transition. This pathway, which is governed by the kinase Greatwall is essential for both entry into and maintenance of M phase. This chapter will outline the molecular events regulating entry into mitosis, specifically highlighting the role that protein phosphorylation plays in triggering both MPF activation and the inhibition of phosphatase activity that would otherwise prevent accumulation of mitotic phosphoproteins. These intricate regulatory pathways are essential for maintaining normal cell division and preventing inappropriate cell proliferation, a central hallmark of cancer cells.

Transient neonatal diabetes mellitus gene Zac1 impairs insulin secretion in mice through Rasgrf1.

The biallelic expression of the imprinted gene ZAC1/PLAGL1 underlies ≈ 60% of all cases of transient neonatal diabetes mellitus (TNDM) that present with low perinatal insulin secretion. Molecular targets of ZAC1 misexpression in pancreatic ß cells are unknown. Here, we identified the guanine nucleotide exchange factor Rasgrf1 as a direct Zac1/Plagl1 target gene in murine ß cells. Doubling Zac1 expression reduced Rasgrf1 expression, the stimulus-induced activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, and, ultimately, insulin secretion. Normalizing Rasgrf1 expression reversed this phenotype. Moreover, the transplantation of Zac1-overexpressing ß cells failed to reinstate euglycemia in experimental diabetic mice. In contrast, Zac1 expression did not interfere with the signaling of the glucagon-like peptide 1 receptor (GLP-1R), and the GLP-1 analog liraglutide improved hyperglycemia in transplanted experimental diabetic mice. This study unravels a mechanism contributing to insufficient perinatal insulin secretion in TNDM and raises new prospects for therapy.

RasGrf1 deficiency delays aging in mice.

RasGRF1 is a Ras-guanine nucleotide exchange factor implicated in a variety of physiological processes including learning and memory and glucose homeostasis. To determine the role of RASGRF1 in aging, lifespan and metabolic parameters were analyzed in aged RasGrf1(-/-) mice. We observed that mice deficient for RasGrf1(-/-) display an increase in average and most importantly, in maximal lifespan (20% higher than controls). This was not due to the role of Ras in cancer because tumor-free survival was also enhanced in these animals. Aged RasGrf1(-/-) displayed better motor coordination than control mice. Protection against oxidative stress was similarly preserved in old RasGrf1(-/-). IGF-I levels were lower in RasGrf1(-/-) than in controls. Furthermore, SIRT1 expression was increased in RasGrf1(-/-) animals. Consistent with this, the blood metabolomic profiles of RasGrf1-deficient mice resembled those observed in calorie-restricted animals. In addition, cardiac glucose consumption as determined PET was not altered by aging in the mutant model, indicating that RasGrf1-deficient mice display delayed aging. Our observations link Ras signaling to lifespan and suggest that RasGrf1 is an evolutionary conserved gene which could be targeted for the development of therapies to delay age-related processes.

A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25.

Myopia and hyperopia are at opposite ends of the continuum of refraction, the measure of the eye's ability to focus light, which is an important cause of visual impairment (when aberrant) and is a highly heritable trait. We conducted a genome-wide association study for refractive error in 4,270 individuals from the TwinsUK cohort. We identified SNPs on 15q25 associated with refractive error (rs8027411, P = 7.91 × 10⁻8). We replicated this association in six adult cohorts of European ancestry with a combined 13,414 individuals (combined P = 2.07 × 10⁻9). This locus overlaps the transcription initiation site of RASGRF1, which is highly expressed in neurons and retina and has previously been implicated in retinal function and memory consolidation. Rasgrf1(-/-) mice show a heavier average crystalline lens (P = 0.001). The identification of a susceptibility locus for refractive error on 15q25 will be important in characterizing the molecular mechanism responsible for the most common cause of visual impairment.