Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse.

OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.

Altered fronto-striatal functions in the Gdi1-null mouse model of X-linked Intellectual Disability.

RAB-GDP dissociation inhibitor 1 (GDI1) loss-of-function mutations are responsible for a form of non-specific X-linked Intellectual Disability (XLID) where the only clinical feature is cognitive impairment. GDI1 patients are impaired in specific aspects of executive functions and conditioned response, which are controlled by fronto-striatal circuitries. Previous molecular and behavioral characterization of the Gdi1-null mouse revealed alterations in the total number/distribution of hippocampal and cortical synaptic vesicles as well as hippocampal short-term synaptic plasticity, and memory deficits. In this study, we employed cognitive protocols with high translational validity to human condition that target the functionality of cortico-striatal circuitry such as attention and stimulus selection ability with progressive degree of complexity. We previously showed that Gdi1-null mice are impaired in some hippocampus-dependent forms of associative learning assessed by aversive procedures. Here, using appetitive-conditioning procedures we further investigated associative learning deficits sustained by the fronto-striatal system. We report that Gdi1-null mice are impaired in attention and associative learning processes, which are a key part of the cognitive impairment observed in XLID patients.

RhoGDI2 confers gastric cancer cells resistance against cisplatin-induced apoptosis by upregulation of Bcl-2 expression.

Rho GDP dissociation inhibitor (RhoGDI)2 has been identified as a regulator of Rho family GTPase. Recently, we suggested that RhoGDI2 could promote tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that RhoGDI2 contributes to another important feature of aggressive cancers, i.e., resistance to chemotherapeutic agents such as cisplatin. Forced expression of RhoGDI2 attenuated cisplatin-induced apoptosis, whereas RhoGDI2 depletion showed opposite effects in vitro. Moreover, the increased anti-apoptotic effect of RhoGDI2 on cisplatin was further validated in RhoGDI2-overexpressing SNU-484 xenograft model in nude mice. Furthermore, we identified Bcl-2 as a major determinant of RhoGDI2-mediated cisplatin resistance in gastric cancer cells. Depletion of Bcl-2 expression significantly increased cisplatin-induced apoptosis in RhoGDI2-overexpressing gastric cancer cells, whereas overexpression of Bcl-2 blocked cisplatin-induced apoptosis in RhoGDI2-depleted gastric cancer cells. Overall, these findings establish RhoGDI2 as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk in gastric cancer.

RhoGDI-1 modulation of the activity of monomeric RhoGTPase RhoA regulates endothelial barrier function in mouse lungs.

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1(-/-) mice. We observed that basal endothelial permeability in lungs of RhoGDI-1(-/-) mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1(-/-) lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1-mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

In vivo dynamics of Rac-membrane interactions.

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.

Characterization of Rho-GDIgamma and Rho-GDIalpha mRNA in the developing and mature brain with an analysis of mice with targeted deletions of Rho-GDIgamma.

Rho-GDIs are a family of Rho GDP-dissociation inhibitors that are critical in modulating the activity of the small GTPases, Cdc42 and RhoA. Two Rho-GDI isoforms are expressed in the brain, Rho-GDIgamma and Rho-GDIalpha. Here, we describe the expression of both of these isoforms in the developing and mature brain. The mRNA expression patterns of Rho-GDIgamma and Rho-GDIalpha were almost identical in the brain with expression in the developing and mature cerebral cortex, striatum, and hippocampus. In addition, we generated mice with targeted deletions of Rho-GDIgamma that are viable and fertile and have no obvious phenotypic abnormalities. Mutant brains looked histologically normal and demonstrated normal patterns of dendritogenesis and neuronal layering as determined by Golgi staining. Mutant mice had normal sleep/wake patterns and sleep EEGs and showed normal hippocampal-dependent learning as assayed by the Morris water maze task. Based on the co-expression of Rho-GDIalpha and Rho-GDIgamma in identical populations of cells in the brain, the lack of phenotype caused by targeted deletion of Rho-GDIgamma may not be surprising given that Rho-GDIalpha may compensate for the loss of Rho-GDIgamma. Whether deletion of both Rho-GDIalpha and Rho-GDIgamma, thereby eliminating all GDI activity in the brain, would produce an observable phenotype remains to be determined.

Rho GDP dissociation inhibitor protects cancer cells against drug-induced apoptosis.

Rho GDP dissociation inhibitor (RhoGDI) plays an essential role in control of a variety of cellular functions through interactions with Rho family GTPases, including Rac1, Cdc42, and RhoA. RhoGDI is frequently overexpressed in human tumors and chemo-resistant cancer cell lines, raising the possibility that RhoGDI might play a role in the development of drug resistance in cancer cells. We found that overexpression of RhoGDI increased resistance of cancer cells (MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells) to the induction of apoptosis by two chemotherapeutic agents: etoposide and doxorubicin. Conversely, silencing of RhoGDI expression by DNA vector-mediated RNA interference (small interfering RNA) sensitized MDA-MB-231 cells to drug-induced apoptosis. Resistance to apoptosis was restored by reintroduction of RhoGDI protein expression. The mechanism for the anti-apoptotic activity of RhoGDI may derive from its ability to inhibit caspase-mediated cleavage of Rac1 GTPase, which is required for maximal apoptosis to occur in response to cytotoxic drugs. Taken together, the data show that RhoGDI is an anti-apoptotic molecule that mediates cellular resistance to these chemotherapy agents.

Mice deficient for the synaptic vesicle protein Rab3a show impaired spatial reversal learning and increased explorative activity but none of the behavioral changes shown by mice deficient for the Rab3a regulator Gdi1.

Rab proteins are small GTPases involved in intracellular trafficking. Among the 60 different Rab proteins described in mammals, Rab3a is the most abundant in brain, where it is involved in synaptic vesicle fusion and neurotransmitter release. Rab3a constitutive knockout mice (Rab3a(-/-)) are characterized by deficient short- and long-term synaptic plasticity in the mossy fiber pathway and altered circadian motor activity, while no effects on spatial learning have been reported so far for these mice. The goals of this study were to analyse possible behavioral consequences of the lack of synaptic plasticity in the mossy fiber pathway using a broad battery of sensitive behavioral measures that has been used previously to analyse the behavior of Gdi1 mice lacking a protein thought to regulate Rab3a. Rab3a(-/-) mice showed normal acquisition but moderately impaired platform reversal learning in the water maze including reference memory and episodic-like memory tasks. A mild deficit in spatial working memory was also observed when tested in the radial maze. Analysis of explorative behavior revealed increased locomotor activity and enhanced exploratory activity in open field, O-maze, dark/light box and novel object tests. Spontaneous activity in normal home cage settings was unaffected but Rab3a(-/-) mice showed increased motor activity when the home cage was equipped with a wheel. No differences were found for delayed and trace fear conditioning or for conditioned taste aversion learning. Congruent with earlier data, these results suggest that Rab3a-dependent synaptic plasticity might play a specific role in the reactivity to novel stimuli and behavioral stability rather than being involved in memory processing. On the other hand, the phenotypic changes in the Rab3a(-/-) mice bore no relation to the behavioral changes as observed in the Gdi1 mice. Such divergence in phenotypes implies that the putative synaptic interaction between Gdi1 and Rab3a should be reconsidered and re-analysed.

Defects in cytokinesis, actin reorganization and the contractile vacuole in cells deficient in RhoGDI.

Rho GDP-dissociation inhibitors (RhoGDIs) modulate the cycling of Rho GTPases between active GTP-bound and inactive GDP-bound states. We identified two RhoGDI homologues in DICTYOSTELIUM: GDI1 shares 51-58% similarity to RhoGDIs from diverse species. GDI2 is more divergent (40-44% similarity) and lacks the N-terminal regulatory arm characteristic for RhoGDI proteins. Both are cytosolic proteins and do not relocalize upon reorganization of the actin cytoskeleton. Using a two-hybrid approach, we identified Rac1a/1b/1c, RacB, RacC and RacE as interacting partners for GDI1. Cells lacking GDI1 are multinucleate, grow slowly and display a moderate pinocytosis defect, but rates of phagocytosis are unaffected. Mutant cells present prominent actin-rich protrusions, and large vacuoles that are continuous with the contractile vacuole system. The actin polymerization response upon stimulation with cAMP was reduced, but the motile behavior toward the chemoattractant was unaffected. Our results indicate that GDI1 plays a central role in the regulation of signal transduction cascades mediated by Rho GTPases.

Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDIalpha.

The Rho small G protein family members regulate various actin cytoskeleton-dependent cell functions. The Rho GDI (GDP dissociation inhibitor) family, consisting of Rho GDIalpha, -beta, and -gamma, is a regulator that keeps the Rho family members in the cytosol as the GDP-bound inactive form and translocates the GDP-bound form from the membranes to the cytosol after the GTP-bound form accomplishes their functions. Rho GDIalpha is ubiquitously expressed in mouse tissues and shows GDI activity on all the Rho family members in vitro. We have generated mice lacking Rho GDIalpha by homologous recombination to clarify its in vivo function. Rho GDIalpha -/- mice showed several abnormal phenotypes. Firstly, Rho GDIalpha -/- mice were initially viable but developed massive proteinuria mimicking nephrotic syndrome, leading to death due to renal failure within a year. Histologically, degeneration of tubular epithelial cells and dilatation of distal and collecting tubules were readily detected in the kidneys. Secondly, Rho GDIalpha -/- male mice were infertile and showed impaired spermatogenesis with vacuolar degeneration of seminiferous tubules in their testes. Thirdly, Rho GDIalpha -/- embryos derived from Rho GDIalpha -/- female mice were defective in the postimplantation development. In addition, these morphological and functional abnormalities showed age-dependent progression. These results suggest that the signaling pathways of the Rho family members regulated by Rho GDIalpha play important roles in maintaining the structure and physiological function of at least kidneys and reproductive systems in adult mice.