Upregulation of RasGRF1 ameliorates spatial cognitive dysfunction in mice after chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH)-mediated cognitive impairment is a serious problem worldwide. However, given its complexity, the underlying mechanisms by which CCH induces cognitive dysfunction remain unclear, resulting in a lack of effective treatments. In this study, we aimed to determine whether changes in the expression of RasGRF1, an important protein associated with cognition and synaptic plasticity, underlie the associated impairments in cognition after CCH. We found that RasGRF1 levels markedly decreased following CCH. Through prediction and validation studies, we observed that miRNA-323-3p was upregulated after CCH and could bind to the 3'-untranslated region of Rasgrf1 mRNA and regulate its expression in vitro. Moreover, the inhibition of miRNA-323-3p upregulated Rasgrf1 expression in the hippocampus after CCH, which was reversed by Rasgrf1 siRNA. This suggests that miRNA-323-3p is an important regulator of Rasgrf1. The Morris water maze and Y maze tests showed that miRNA-323-3p inhibition and Rasgrf1 upregulation improved spatial learning and memory, and electrophysiological measurements revealed deficits in long-term potentiation after CCH that were reversed by Rasgrf1 upregulation. Dendritic spine density and mature mushroom spine density were also improved after miRNA-323-3p inhibition and Rasgrf1 upregulation. Furthermore, Rasgrf1 upregulation by miRNA-323-3p inhibition improved dendritic spine density and mature mushroom spine density and ameliorated the deterioration of synapses and postsynaptic density. Overall, RasGRF1 regulation attenuated cognitive impairment, helped maintain structural and functional synaptic plasticity, and prevented synapse deterioration after CCH. These results suggest that Rasgrf1 downregulation by miRNA-323-3p plays an important role in cognitive impairment after CCH. Thus, RasGRF1 and miRNA-323-3p may represent potential therapeutic targets for cognitive impairment after CCH.

Loss of MLL Induces Epigenetic Dysregulation of Rasgrf1 to Attenuate Kras-Driven Lung Tumorigenesis.

Menin is necessary for the formation of the menin/mixed lineage leukemia (MLL) complex and is recruited directly to chromatin. Menin is an important tumor suppressor in several cancer types, including lung cancer. Here, we investigated the role of MLL in menin-regulated lung tumorigenesis. Ablation of MLL suppressed KrasG12D-induced lung tumorigenesis in a genetically engineered mouse model. MLL deficiency decreased histone H3 lysine 4 trimethylation (H3K4me3) and subsequently suppressed expression of the Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1) gene. Rasgrf1 was essential for the GTP-bound active state of Kras and the activation of Kras downstream pathways as well as their cancer-promoting activities. MI-3, a small-molecule inhibitor targeting MLL, specifically inhibited the growth of Kras-mutated lung cancer cells in vitro and in vivo with minimal effect on wild-type Kras lung cancer growth. Together, these results demonstrate a novel tumor promoter function of MLL in mutant Kras-induced lung tumorigenesis and further indicate that specific blockade of the MLL-Rasgrf1 pathway may be a potential therapeutic strategy for the treatment of tumors containing Kras mutations. SIGNIFICANCE: Activation of mutant Kras is dependent on MLL-mediated epigenetic regulation of Rasgrf1, conferring sensitivity to small-molecule inhibition of MLL in Kras-driven lung cancer.

Median Nerve Stimulation Attenuates Traumatic Brain Injury-Induced Comatose State by Regulating the Orexin-A/RasGRF1 Signaling Pathway.

BACKGROUND: Despite the arousal effect of median nerve stimulation (MNS) being well documented in the clinical treatment of coma patients with traumatic brain injury (TBI), the mechanisms underlying the observed effect are still not completely understood. This study aimed to evaluate the protective effects and potential mechanism of MNS in comatose rats with TBI. METHODS: A total of 60 rats were randomly divided into 5 groups: the control group, sham-stimulated group, MNS group, orexins receptor type 1 (OX1R) antagonist group, and antagonist control group. The free-fall drop method was used to establish a TBI model. After administrating MNS or OX1R antagonist, consciousness was evaluated. Protein levels in the prefrontal cortex were measured using an enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence. RESULTS: In the MNS group, tissue damage and consciousness state was markedly improved compared with that in the sham-stimulated group. Administration of the OX1R antagonist attenuated the beneficial effects of MNS in TBI-induced comatose rats. Additionally, MNS also significantly enhanced the expression of orexin-A/OX1R and the activation of Ras guanine nucleotide-releasing factor 1 (RasGRF1). CONCLUSIONS: These data show that MNS exerts its wake-promoting effect by activating the OX1R-RasGRF1 pathway in TBI-induced comatose rats.

In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density.

Cells control the properties of the cytoplasm to ensure proper functioning of biochemical processes. Recent studies showed that cytoplasmic density varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here, we study how a cell cycle oscillator comprising cyclin-dependent kinase (Cdk1) responds to changes in cytoplasmic density by systematically diluting or concentrating cycling Xenopus egg extracts in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density: as low as 0.2× to more than 1.22× relative cytoplasmic density (RCD). A further dilution or concentration from these values arrested the system in a low or high steady state of Cdk1 activity, respectively. Interestingly, diluting an arrested cytoplasm of 1.22× RCD recovers oscillations at lower than 1× RCD. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of tuning, forming a hysteresis loop. We propose a mathematical model which recapitulates these observations and predicts that the Cdk1/Wee1/Cdc25 positive feedback loops do not contribute to the observed robustness, supported by experiments. Our system can be applied to study how cytoplasmic density affects other cellular processes.

The Tyrosine Phosphatase hPTPRß Controls the Early Signals and Dopaminergic Cells Viability via the P2X7 Receptor.

ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X7 receptor (P2X7R) and stimulates neuronal cell death. Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X7R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRß and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson's Disease.

Proteomic signature of the Dravet syndrome in the genetic Scn1a-A1783V mouse model.

BACKGROUND: Dravet syndrome is a rare, severe pediatric epileptic encephalopathy associated with intellectual and motor disabilities. Proteomic profiling in a mouse model of Dravet syndrome can provide information about the molecular consequences of the genetic deficiency and about pathophysiological mechanisms developing during the disease course. METHODS: A knock-in mouse model of Dravet syndrome with Scn1a haploinsufficiency was used for whole proteome, seizure, and behavioral analysis. Hippocampal tissue was dissected from two- (prior to epilepsy manifestation) and four- (following epilepsy manifestation) week-old male mice and analyzed using LC-MS/MS with label-free quantification. Proteomic data sets were subjected to bioinformatic analysis including pathway enrichment analysis. The differential expression of selected proteins was confirmed by immunohistochemical staining. RESULTS: The findings confirmed an increased susceptibility to hyperthermia-associated seizures, the development of spontaneous seizures, and behavioral alterations in the novel Scn1a-A1873V mouse model of Dravet syndrome. As expected, proteomic analysis demonstrated more pronounced alterations following epilepsy manifestation. In particular, proteins involved in neurotransmitter dynamics, receptor and ion channel function, synaptic plasticity, astrogliosis, neoangiogenesis, and nitric oxide signaling showed a pronounced regulation in Dravet mice. Pathway enrichment analysis identified several significantly regulated pathways at the later time point, with pathways linked to synaptic transmission and glutamatergic signaling dominating the list. CONCLUSION: In conclusion, the whole proteome analysis in a mouse model of Dravet syndrome demonstrated complex molecular alterations in the hippocampus. Some of these alterations may have an impact on excitability or may serve a compensatory function, which, however, needs to be further confirmed by future investigations. The proteomic data indicate that, due to the molecular consequences of the genetic deficiency, the pathophysiological mechanisms may become more complex during the course of the disease. As a result, the management of Dravet syndrome may need to consider further molecular and cellular alterations. Ensuing functional follow-up studies, this data set may provide valuable guidance for the future development of novel therapeutic approaches.

RasGRF1 participates in the protective effect of tanshinone IIA on depressive like behaviors of a chronic unpredictable mild stress induced mouse model.

Tanshinone IIA (Tan IIA) is reported to have neuroprotective effects to suppress cell apoptosis of cortical neurons induced by Aß25-35 through inhibiting oxidative stress. Nevertheless, few studies have investigated the effects of Tan IIA on depressive disorder. Here, we aimed to measure the effects of Tan IIA on chronic unpredictable mild stress (CUMS) induced mouse model and its underlying mechanism. For 28 days, mice were subjected to CUMS while Tan IIA was administered once daily at doses of 0, 1, 2.5, 5, or 10 mg/kg. CUMS exposure increased depressive-like behaviors, as indicated by increased immobility time in the forced swim and tail suspension tests, decreased sucrose preference in the sucrose preference test, and reduced exploratory behavior in the open field test. All of these behaviors were reversed dose-dependently by Tan IIA treatment. Oxidative stress was determined by measuring malondialdehyde, glutathione peroxidase, and superoxide dismutase activity and total antioxidant capacity. Levels of pro-inflammatory factors IL-1ß and IL-18, cAMP response element binding protein and brain derived neurotrophic factor were detected by ELISA and western blot assay, respectively. The results showed that CUMS increased oxidative stress and pro-inflammatory factors and decreased levels of cAMP response element binding protein and brain-derived neurotrophic factor. Tan IIA treatment again reversed these effects. Importantly, RasGRF1 expression increased in CUMS-exposed mice but decreased after Tan IIA administration. Using RasGRF1-/- mice to determine the role of RasGRF1 in mice exposed to CUMS, we found that knockdown of RasGRF1 reversed the effects of CUMS on mice, just like Tan IIA. These results indicate that Tan IIA may reverse depressive-like behaviors in CUMS-exposed mice by regulating RasGRF1.

Identification of a RAS-activating TMEM87A-RASGRF1 Fusion in an Exceptional Responder to Sunitinib with Non-Small Cell Lung Cancer.

PURPOSE: We pursued genomic analysis of an exceptional responder with non-small cell lung cancer (NSCLC) through a multi-platform effort to discover novel oncogenic targets. EXPERIMENTAL DESIGN: In this open-label, single-arm phase II study (NCT01829217), an enriched cohort of patients with advanced NSCLC was treated with the multi-kinase inhibitor sunitinib. The primary endpoint was objective response rate. Tissue was collected for multi-platform genomic analysis of responders, and a candidate oncogene was validated using in vitro models edited by CRISPR-Cas9. RESULTS: Of 13 patients enrolled, 1 patient (8%), a never smoker, had a partial response lasting 33 months. Genomic analysis of the responder identified no oncogenic variant using multi-platform DNA analysis including hotspot allelotyping, massively parallel hybrid-capture next-generation sequencing, and whole-exome sequencing. However, bulk RNA-sequencing (RNA-seq) revealed a novel fusion, TMEM87A-RASGRF1, with high overexpression of the fusion partners. RASGRF1 encodes a guanine exchange factor which activates RAS from GDP-RAS to GTP-RAS. Oncogenicity was demonstrated in NIH/3T3 models with intrinsic TMEM87A-RASGRF1 fusion. In addition, activation of MAPK was shown in PC9 models edited to express this fusion, although sensitivity to MAPK inhibition was seen without apparent sensitivity to sunitinib. CONCLUSIONS: Sunitinib exhibited limited activity in this enriched cohort of patients with advanced NSCLC. Nonetheless, we find that RNA-seq of exceptional responders represents a potentially underutilized opportunity to identify novel oncogenic targets including oncogenic activation of RASGRF1.

RASGRF1 in CRF cells controls the early adolescent female response to repeated stress.

RASGRF1 (GRF1) is a calcium-stimulated guanine-nucleotide exchange factor that activates RAS and RAC GTPases. In hippocampus neurons, it mediates the action of NMDA and calcium-permeable AMPA glutamate receptors on specific forms of synaptic plasticity, learning, and memory in both male and female mice. Recently, we showed GRF1 also regulates the HPA axis response to restraint stress, but only in female mice before puberty. In particular, we found that after 7 days of restraint stress (7DRS) (30 min/day) both elevated serum CORT levels and induction of an anxiolytic phenotype normally observed in early adolescent (EA) female mice are blocked in GRF1-knockout mice. In contrast, no effects were observed in EA male or adult females. Here, we show this phenotype is due, at least in part, to GRF1 loss in CRF cells of the paraventricular nucleus of the hypothalamus, as GRF1 knockout specifically in these cells suppressed 7DRS-induced elevation of serum CORT levels specifically in EA females, but only down to levels found in comparably stressed EA males. Nevertheless, it still completely blocked the 7DRS-induced anxiolytic phenotype observed in EA females. Interestingly, loss of GRF1 in CRF cells had no effect after only three restraint stress exposures, implying a role for GRF1 in 7DRS stress-induced plasticity of CRF cells that appears to be specific to EA female mice. Overall, these findings indicate that GRF1 in CRF cells makes a key contribution to the distinct response EA females display to repeated stress.

Benzothiazole amphiphiles promote RasGRF1-associated dendritic spine formation in human stem cell-derived neurons.

Synaptic dysfunction has been implicated as an early cause of cognitive decline in neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD). Methods to slow down or reverse the loss of functional synapses, therefore, represent a promising avenue to explore for treating NDDs. We have previously reported the development of a class of benzothiazole amphiphiles (BAMs) that exhibited the capability to improve memory and learning both in wild-type mice and in an AD rodent model, putatively through promoting RasGRF1-associated formation of dendritic spines in hippocampal neurons. While these results represent a good first step in exploring a new approach to treating NDDs, the capability of these compounds to increase spine density has not been previously examined in a human neuronal model. Here, we found that neurons derived from differentiated human induced pluripotent stem cells exhibited both an increase in RasGRF1 expression and a phenotypic increase in the density of postsynaptic density protein 95-positive puncta (which we use to provide an estimate of dendritic spine density) in BAM-treated vs. control neurons. These results demonstrate that the previously observed spinogenic effects of BAMs in rodent neurons can be recapitulated in a human neuronal model, which further supports the potential utility of BAM agents for treating human diseases associated with spine deficits such as AD or other NDDs.

Characterisation of HRas local signal transduction networks using engineered site-specific exchange factors.

Ras GTPases convey signals from different types of membranes. At these locations, different Ras isoforms, interactors and regulators generate different biochemical signals and biological outputs. The study of Ras localisation-specific signal transduction networks has been hampered by our inability to specifically activate each of these Ras pools. Here, we describe a new set of site-specific tethered exchange factors, engineered by fusing the RasGRF1 CDC25 domain to sub-localisation-defining cues, whereby Ras pools at specific locations can be precisely activated. We show that the CDC25 domain has a high specificity for activating HRas but not NRas and KRas. This unexpected finding means that our constructs mainly activate endogenous HRas. Hence, their use enabled us to identify distinct pathways regulated by HRas in endomembranes and plasma membrane microdomains. Importantly, these new constructs unveil different patterns of HRas activity specified by their subcellular localisation. Overall, the targeted GEFs described herein constitute ideal tools for dissecting spatially-defined HRas biochemical and biological functions.

Arp2/3-Branched Actin Maintains an Active Pool of GTP-RhoA and Controls RhoA Abundance.

Small GTPases regulate cytoskeletal dynamics, cell motility, and division under precise spatiotemporal control. Different small GTPases exhibit cross talks to exert feedback response or to act in concert during signal transduction. However, whether and how specific cytoskeletal components' feedback to upstream signaling factors remains largely elusive. Here, we report an intriguing finding that disruption of the Arp2/3-branched actin specifically reduces RhoA activity but upregulates its total protein abundance. We further dissect the mechanisms underlying these circumstances and identify the altered cortactin/p190RhoGAP interaction and weakened CCM2/Smurf1 binding to be involved in GTP-RhoA reduction and total RhoA increase, respectively. Moreover, we find that cytokinesis defects induced by Arp2/3 inhibition can be rescued by activating RhoA. Our study reveals an intricate feedback from the actin cytoskeleton to the small GTPase. Our work highlights the role of Arp2/3-branched actin in signal transduction aside from its function in serving as critical cytoskeletal components to maintain cell morphology and motility.

Over-expression of p190RhoGEF enhances B-cell activation and germinal center formation in T-cell-dependent humoral immune responses.

Previously, we reported induced expression of the p190 Rho guanine nucleotide exchange factor (p190RhoGEF, ARHGEF28) following CD40 stimulation of B cells isolated from mouse spleen. We also reported that p190RhoGEF and a downstream effector molecule RhoA are required for B-cell differentiation, especially for the induction of the plasma cell (PC) differentiation. This study investigates the role of p190RhoGEF in B-cell biology in vivo, using p190RhoGEF transgenic (TG) mice that overexpress a wild-type full gene in B cells. Immunization of these mice with T-cell-dependent antigen showed that populations of germinal center B cells and PCs were significantly increased in TG mice. Furthermore, similar results were shown in recombination activating 1 (Rag1) knockout mice that were reconstituted with B cells isolated from TG mice in combination with T cells isolated from littermate control mice. Analyses of isotype class switching and transcription factors involved in a germinal center reaction and PC differentiation also supported the findings from the cellular responses. These results suggest that p190RhoGEF may play a role in the stage of PC differentiation during T-cell-dependent humoral immune responses.

The Interdependent Activation of Son-of-Sevenless and Ras.

The guanine-nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) plays a critical role in metazoan signaling by converting Ras•GDP (guanosine diphosphate) to Ras•GTP (guanosine triphosphate) in response to tyrosine kinase activation. Structural studies have shown that SOS differs from other Ras-specific GEFs in that SOS is itself activated by Ras•GTP binding to an allosteric site, distal to the site of nucleotide exchange. The activation of SOS involves membrane recruitment and conformational changes, triggered by lipid binding, that open the allosteric binding site for Ras•GTP. This is in contrast to other Ras-specific GEFs, which are activated by second messengers that more directly affect the active site. Allosteric Ras•GTP binding stabilizes SOS at the membrane, where it can turn over other Ras molecules processively, leading to an ultrasensitive response that is distinct from that of other Ras-specific GEFs.

TrkB Regulates N-Methyl-D-Aspartate Receptor Signaling by Uncoupling and Recruiting the Brain-Specific Guanine Nucleotide Exchange Factor, RasGrf1.

Brain-derived neurotrophic factor (BDNF) facilitates multiple aspects of neuronal differentiation and cellular physiology by activating the high-affinity receptor tyrosine kinase, TrkB. While it is known that both BDNF and TrkB modulate cellular processes involved in learning and memory, exactly how TrkB cross-talks and modulates signaling downstream of excitatory ionotropic receptors, such as the NMDA receptor (NMDAR), are not well understood. A model that we have investigated involves the signaling molecule RasGrf1, a guanine nucleotide exchange factor for both Ras and Rac. We previously identified RasGrf1 as a novel Trk binding partner that facilitates neurite outgrowth in response to both nerve growth factor (NGF) (Robinson et al. in J Biol Chem 280:225-235, 2005) and BDNF (Talebian et al. in J Mol Neurosci 49:38-51, 2013); however, RasGrf1 can also bind the NR2B subunit of the NMDAR (Krapivinsky et al. in Neuron 40:775-784, 2003) and stimulate long-term depression (LTD) (Li et al. in J Neurosci 26:1721-1729, 2006). We have addressed a model that TrkB facilitates learning and memory via two processes. First, TrkB uncouples RasGrf1 from NR2B and facilitates a decrease in NMDA signaling associated with LTD (p38-MAPK). Second, the recruitment of RasGrf1 to TrkB enhances neurite outgrowth and pERK activation and signaling associated with learning and memory. We demonstrate that NMDA recruits RasGrf1 to NR2B; however, co-stimulation with BDNF uncouples this association and recruits RasGrf1 to TrkB. In addition, activation of TrkB stimulates the tyrosine phosphorylation of RasGrf1 which increases neurite outgrowth (Talebian et al. in J Mol Neurosci 49:38-51, 2013), and the tyrosine phosphorylation of NR2B (Tyr1472) (Nakazawa et al. in J Biol Chem 276:693-699, 2001) which facilitates NMDAR cell surface retention (Zhang et al. in J Neurosci 28:415-24, 2008). Collectively, these data demonstrate that TrkB alters NMDA signaling by a dual mechanism that uncouples LTD and, in turn, stimulates neuronal growth and the signaling pathways associated with learning and memory.

Deletion of RasGRF1 Attenuated Interstitial Fibrosis in Streptozotocin-Induced Diabetic Cardiomyopathy in Mice through Affecting Inflammation and Oxidative Stress.

BACKGROUND: Diabetic cardiomyopathy (DCM) is characterized by cardiac fibrosis and stiffness, which often develops into heart failure. This study investigated the role of Ras protein-specific guanine nucleotide releasing factor 1 (RasGRF1) in the development of DCM. METHODS: Forty-eight mice were divided into four groups (n = 12 per group): Group 1: Wild-type (WT) mice, Group 2: RasGRF1 deficiency (RasGRF1-/-) mice. Group 3: Streptozotocin (STZ)-induced diabetic WT mice, Group 4: STZ-induced diabetic RasGRF1-/- mice. Myocardial functions were assessed by cardiac echography. Heart tissues from all of the mice were investigated for cardiac fibrosis, inflammation, and oxidative stress markers. RESULTS: Worse impaired diastolic function with elevation serum interleukin (IL)-6 was found in the diabetic group compared with the non-diabetic groups. Serum IL-6 levels were found to be elevated in the diabetic compared with the non-diabetic groups. However, the diabetic RasGRF1-/- mice exhibited lower serum IL-6 levels and better diastolic function than the diabetic WT mice. The diabetic RasGRF1-/- mice were associated with reduced cardiac inflammation, which was shown by lower invading inflammation cells, lower expression of matrix metalloproteinase 9, and less chemokines compared to the diabetic WT mice. Furthermore, less oxidative stress as well as extracellular matrix deposition leading to a reduction in cardiac fibrosis was also found in the diabetic RasGRF1-/- mice compared with the diabetic WT mice. CONCLUSION: The deletion of RasGRF1 attenuated myocardial fibrosis and improved cardiac function in diabetic mice through inhibiting inflammation and oxidative stress.

RASgrf1, a Potential Methylatic Mediator of Anti-epileptogenesis?

Epileptogenesis, induced by status epilepticus (SE), is a chronic process, and intervention in this progress may prevent chronic epilepsy. It has been proposed that DNA methylation might be related with epileptogenesis. RASgrf1 has a differentially methylated region at the promoter which can silence gene expression. We have previously observed the down-regulation of RASgrf1 in epilepsy patients and proved that hypermethylation of RASgrf1 reaches maximal level at the latent period in mice after kainate-induced SE (KA mice), with corresponding alteration of RASgrf1 expression. In the present study, N-phthalyl-L-tryptophan (RG108), a DNA methyltransferase inhibitor, was applied in KA mice at latent phase and the behavior, electroencephalogram and pathological changes were observed in chronic phase. Methylation and expression of RASgrf1 were determined by polymerase chain reaction (PCR), western blotting, and bisulfite sequencing PCR. The results showed that the incidence of spontaneous recurrent seizures (SRS) was significantly lower in the RG108 group than the normal saline (NS) group. Subgroup analysis showed significant hypermethylation and lower expression of RASgrf1 in the RG108-SRS subgroup and the NS-SRS subgroup but not in the RG108-NSRS (no SRS) subgroup and the NS-NSRS subgroup compared with the control group. No significant difference was found between the RG108-SRS and NS-SRS subgroups. Meanwhile, hippocampal neuronal loss was observed in RG108-SRS and NS-SRS subgroups. We thus demonstrated that RG108 could modify the progression of epileptogenesis after KA induced SE and prevent chronic epilepsy. Meanwhile, hypermethylation of RASgrf1 after KA induced SE could be reversed with corresponding changes of RASgrf1 expression. Additionally, we speculated that RASgrf1 might be a potential epigenetic mediator in epileptogenesis and chronic epilepsy.

Swe1 and Mih1 regulate mitotic spindle dynamics in budding yeast via Bik1.

The mitotic spindle is a very dynamic structure that is built de novo and destroyed at each round of cell division. In order to perform its fundamental function during chromosome segregation, mitotic spindle dynamics must be tightly coordinated with other cell cycle events. These changes are driven by several protein kinases, phosphatases and microtubule-associated proteins. In budding yeast, the kinase Swe1 and the phosphatase Mih1 act in concert in controlling the phosphorylation state of Cdc28, the catalytic subunit of Cdk1, the major regulator of the cell cycle. In this study we show that Swe1 and Mih1 are also involved in the control of mitotic spindle dynamics. Our data indicate that Swe1 and the Polo-like kinase Cdc5 control the balance between phosphorylated and unphosphorylated forms of Mih1, which is, in turn, important for mitotic spindle elongation. Moreover, we show that the microtubule-associated protein Bik1 is a phosphoprotein, and that Swe1 and Mih1 are both involved in controlling phosphorylation of Bik1. These results uncover new players and provide insights into the complex regulation of mitotic spindle dynamics.

Fructose-1,6-bisphosphate couples glycolytic flux to activation of Ras.

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.

Ras-GRF2 regulates nestin-positive stem cell density and onset of differentiation during adult neurogenesis in the mouse dentate gyrus.

Various parameters of neurogenesis were analyzed in parallel in the two neurogenic areas (the Dentate Gyrus[DG] and the Subventricular Zone[SVZ]/Rostral Migratory Stream[RMS]/Main Olfactory Bulb[MOB] neurogenic system) of adult WT and KO mouse strains for the Ras-GRF1/2 genes (Ras-GRF1-KO, Ras-GRF2-KO, Ras-GRF1/2-DKO). Significantly reduced numbers of doublecortin[DCX]-positive cells were specifically observed in the DG, but not the SVZ/RMS/MOB neurogenic region, of Ras-GRF2-KO and Ras-GRF1/2-DKO mice indicating that this novel Ras-GRF2-dependent phenotype is spatially restricted to a specific neurogenic area. Consistent with a role of CREB as mediator of Ras-GRF2 function in neurogenesis, the density of p-CREB-positive cells was also specifically reduced in all neurogenic regions of Ras-GRF2-KO and DKO mice. Similar levels of early neurogenic proliferation markers (Ki67, BrdU) were observed in all different Ras-GRF genotypes analyzed but significantly elevated levels of nestin-immunolabel, particularly of undifferentiated, highly ramified, A-type nestin-positive neurons were specifically detected in the DG but not the SVZ/RMS/MOB of Ras-GRF2-KO and DKO mice. Together with assays of other neurogenic markers (GFAP, Sox2, Tuj1, NeuN), these observations suggest that the deficit of DCX/p-CREB-positive cells in the DG of Ras-GRF2-depleted mice does not involve impaired neuronal proliferation but rather delayed transition from the stem cell stage to the differentiation stages of the neurogenic process. This model is also supported by functional analyses of DG-derived neurosphere cultures and transcriptional characterization of the neurogenic areas of mice of all relevant Ras-GRF genotypes suggesting that the neurogenic role of Ras-GRF2 is exerted in a cell-autonomous manner through a specific transcriptional program.