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.

Novel RasGRF1-derived Tat-fused peptides inhibiting Ras-dependent proliferation and migration in mouse and human cancer cells.

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.

Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin.

Tiam1 is a ubiquitous guanine nucleotide exchange factor (GEF) that activates the Rac GTPase. We have shown previously that the N terminus of Tiam1 contributes to the signaling specificity of its downstream target Rac via association with IB2, a scaffold that promotes Rac activation of a p38 kinase cascade. Here we show that the N terminus of Tiam1 can influence Rac signaling specificity in a different way by interaction with spinophilin, a scaffold that binds to p70 S6 kinase, another protein regulated by Rac. In particular, spinophilin binding promotes the plasma membrane localization of Tiam1 and enhances the ability of Tiam1 to activate p70 S6 kinase. In contrast, spinophilin binding suppresses the ability of Tiam to activate Pak1, a different Rac effector. Finally, a mutant spinophilin that cannot bind to Tiam1 suppresses serum-induced p70 S6 kinase activation in cells, suggesting that a Tiam1/spinophilin complex contributes to p70 S6 kinase regulation by extracellular signals. These findings add to a growing body of evidence supporting the concept that some Rac-GEFs not only activate Rac GTPases but also participate in the selection of Rac effector by binding to particular scaffolds that complex with components of specific Rac effector pathways.