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.

Correlation between High Myopia Susceptibility and Polymorphisms of RASGRF1 Gene among College Students in Zhejiang.

Objective: The aim of the study is to analyze the correlation between high myopia susceptibility and Ras protein-specific guanine nucleotide-releasing factor-1(RASGRF1) gene polymorphism among college students in Zhejiang. Methods: A stratified whole-group sampling method was used to select 218 cases of college students in Zhejiang who met the inclusion and exclusion criteria from January, 2019, to December, 2021, and they were divided into 77 cases (154 eyes) in the high myopia group and 141 cases (282 eyes) in the medium-low myopia group according to the degree of myopia, and 109 cases of college volunteers without myopia from the same period of medical examination in the region were included in the control group. The single nucleotide polymorphisms (SNPs) located in functional regions were selected by searching the literature and genetic databases, and the base sequences of rs939658, rs4778879, and rs8033417 loci were obtained by genotyping candidate SNPs using multiplex ligase detection reaction technique. The cardinality test was used to compare the differences in genotype frequency distribution of each locus of the RASGRF1 gene between the high myopia group and the low to moderate myopia group and the control group. Results: The genotype frequencies and allele frequencies of the RASGRF1 gene rs939658 locus in the high myopia group compared with the moderate-low myopia group and the control group were not statistically significant (P > 0.05). The genotype frequencies and allele frequencies of the rs4778879 locus of the RASGRF1 gene were compared among the three groups, and the differences were not statistically significant (P > 0.05). The genotype frequency and allele frequency of the rs8033417 locus of the RASGRF1 gene differed significantly among the three groups (P < 0.05). Conclusion: The polymorphism of the rs8033417 locus of the RASGRF1 gene was significantly correlated with the susceptibility of high myopia among college students in Zhejiang.

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.

DNA Methylation Analysis of Imprinted Genes in the Cortex and Hippocampus of Cross-Fostered Mice Selectively Bred for Increased Voluntary Wheel-Running.

We have previously shown that high runner (HR) mice (from a line genetically selected for increased wheel-running behavior) have distinct, genetically based, neurobiological phenotypes as compared with non-selected control (C) mice. However, developmental programming effects during early life, including maternal care and parent-of-origin-dependent expression of imprinted genes, can also contribute to variation in physical activity. Here, we used cross-fostering to address two questions. First, do HR mice have altered DNA methylation profiles of imprinted genes in the brain compared to C mice? Second, does maternal upbringing further modify the DNA methylation status of these imprinted genes? To address these questions, we cross-fostered all offspring at birth to create four experimental groups: C pups to other C dams, HR pups to other HR dams, C pups to HR dams, and HR pups to C dams. Bisulfite sequencing of 16 imprinted genes in the cortex and hippocampus revealed that the HR line had altered DNA methylation patterns of the paternally imprinted genes, Rasgrf1 and Zdbf2, as compared with the C line. Both fostering between the HR and C lines and sex modified the DNA methylation profiles for the paternally expressed genes Mest, Peg3, Igf2, Snrpn, and Impact. Ig-DMR, a gene with multiple paternal and maternal imprinted clusters, was also affected by maternal upbringing and sex. Our results suggest that differential methylation patterns of imprinted genes in the brain could contribute to evolutionary increases in wheel-running behavior and are also dependent on maternal upbringing and sex.

The Cdc25/Ras/cAMP-dependent protein kinase A signaling pathway regulates proline utilization in wine yeast Saccharomyces cerevisiae under a wine fermentation model.

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes.

All About That Ras: Novel Fusion Drives Ras Pathway Activation in Lung Cancer.

Lung cancers in never- and light-smokers often harbor targetable oncogenic mutations in Ras pathway genes. Here, a novel OCLN-RASGRF1 fusion is identified in an otherwise Ras wild-type lung tumor. Studying this and other RASGRF1 fusions, the authors show that these fusions lead to malignant phenotypes that can be reversed by MEK inhibition. See related article by Hunihan et al., p. 3091.

Expression of a RhoA-Specific Guanine Nucleotide Exchange Factor, p190RhoGEF, in Mouse Macrophages Negatively Affects M1 Polarization and Inflammatory Responses.

A RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, was first cloned and identified in neuronal cells. In immune cells, we first reported the role of p190RhoGEF in B cells: expression of p190RhoGEF increased after CD40 stimulation and was required for CD40-mediated B cell activation and differentiation. We also showed that over-expression of p190RhoGEF negatively affected dendritic cell function in response to bacterial lipopolysaccharide (LPS). In this study, we examined the role of p190RhoGEF in macrophages using p190RhoGEF over-expressing transgenic (TG) mice. We found macrophages from TG mice to be more round than those from control mice, with enriched polymerized actin at the edge attached to the glass. TG macrophages also responded less to LPS: production of reactive oxygen species, phagocytosis, chemokine-dependent migration, and pro-inflammatory cytokine secretion were all reduced compared with the responses of macrophages from littermate (LTM) control mice. Furthermore, the classical M1 subset population was observed less in the peritoneal macrophages of TG mice than the LTM control mice during LPS-elicited peritoneal inflammation. When the activity of RhoA was inhibited in TG macrophages, their morphology and LPS responses became similar to those of the LTM macrophages. These results suggest that over-expression of p190RhoGEF in macrophages could reduce M1 polarization and inflammatory responses by regulating the actin cytoskeleton.

RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition.

PURPOSE: The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies, including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history. EXPERIMENTAL DESIGN: We performed genomic characterization of 103 LUADs from patients with ≤10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA sequencing in search of established and previously uncharacterized candidate drivers. RESULTS: We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo. CONCLUSIONS: Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations. See related commentary by Moorthi and Berger, p. 2983.

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.

RASGRF1-rearranged Cutaneous Melanocytic Neoplasms With Spitzoid Cytomorphology: A Clinicopathologic and Genetic Study of 3 Cases.

Spitz neoplasms, according to 2018 WHO Blue Book, are morphologically defined by spindled and/or epithelioid melanocytes and genetically by either HRAS mutations or kinase gene fusions. The terminology "spitzoid" refers to lesions with similar morphology but with alternate or undefined genetic anomalies. Herein, we present 3 melanocytic neoplasms with a spitzoid cytomorphology, variable nuclear atypia, and harboring undescribed fusions involving RASGRF1. Two cases presented as unpigmented papules on the heel of a 26-year-old female (case 1) and the forearm of a 13-year-old boy (case 2). They were classified as low-grade melanocytomas (WHO 2018). The third case appeared as a pigmented ulcer on the sole of a 72-year-old female (case 3) that displayed diagnostic features of an invasive melanoma (Breslow thickness 6 mm, Clark level V). A wide skin reexcision identified an epidermotropic metastasis, and sentinel lymph node biopsy displayed multiple subcapsular metastatic deposits. RNA sequencing revealed CD63::RASGRF1, EHBP1::RASGRF1, and ABCC2::RASGRF1 fusions in cases 1 to 3, respectively. They were confirmed by a RASGRF1 break-apart fluorescence in situ hybridization technique. Translocations of RASGRF1, a gene coding a guanine nucleotide exchange factor but not a kinase, have rarely been reported in tumors. While all these cases showed spitzoid cytomorphology, it is too early to tell if they are true Spitz neoplasms as currently defined.

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.

Intratumoral heterogeneity and genetic characteristics of prostate cancer.

Prostate cancer is a heterogeneous disease and optimum gene targeting treatment is often impermissible. We aim to determine the intratumoral genomic heterogeneity of prostate cancer and explore candidate genes for targeted therapy. Exome sequencing was performed on 37 samples from 16 patients with prostate cancer. Somatic variant analysis, copy number variant (CNV) analysis, clonal evolution analysis and variant spectrum analysis were used to study the intratumoral genomic heterogeneity and genetic characteristics of metastatic prostate cancer. Our study confirmed the high intratumoral genetic heterogeneity of prostate cancer in many aspects, including number of shared variants, tumor mutation burden (TMB), variant genes, CNV burden, weighted genome instability index (wGII), CNV profiles, clonal evolutionary process, variant spectrum and mutational signatures. Moreover, we identified several common genetic characteristics of prostate cancer. Alterations of DNA damage repair genes, RTK/RAS pathway associated gene RASGRF1 and autophagy gene EPG5 may be involved in tumorigenesis in prostate cancer. CNV burden and DNA damage repair (DDR) genes may be associated with metastasis of prostate cancer.

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.