Defective dendrite elongation but normal fertility in mice lacking the Rho-like GTPase activator Dbl

Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.     

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.     

RhoG GTPase Controls a Pathway That Independently Activates Rac1 and Cdc42Hs

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.     

Immunological function of kidney-infiltrating lymphocytes from aged NZB mice

A striking aspect of autoimmune kidney disease in the NZB mouse strain is the perivascular infiltration of lymphoid cells. Upon release by enzymatic digestion of kidney tissue from animals 6 months of age or older, these cells have been found to exhibit a high level of immunologic activity not seen in younger mice or in nonautoimmune strains. Kidney-derived cells were found to respond to T and B cell mitogens at levels ranging up to those observed for peripheral blood, and in some cases splenic lymphocytes, from the same animals. An enhanced proliferative response to autologous and allogeneic stimulation was observed compared to these other lymphoid sources. Both spontaneous and LPS-stimulated immunoglobulin synthesis were noted with all three populations, which could be totally or partially blocked by cycloheximide. Selective localization of autoantibody-producing cell populations was observed, with anti-erythrocyte antibody restricted to splenocytes and PBL, and the anti-dsDNA implicated in immune complex formation found only in kidney-derived cell culture supernatants.     

Rho GTPase function in tumorigenesis

Malignant tumor cells display uncontrolled proliferation, loss of epithelial cell polarity, altered interactions with neighboring cells and the surrounding extracellular matrix, and enhanced migratory properties. Proteins of the Rho GTPase family regulate all these processes in cell culture and, for that reason, Rho GTPases, their regulators, and their effectors have been suggested to control tumor formation and progression in humans. However, while the tumor-relevant functions of Rho GTPases are very well documented in vitro, we are only now beginning to assess their contribution to cancer in human patients and in animal models. This review will give a very brief overview of Rho GTPase function in general and then focus on in vivo evidence for a role of Rho GTPases in malignant tumors, both in human patients and in genetically modified mice.     

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.     

Reproductive function in female mice lacking the gene for endothelial nitric oxide synthase.

Nitric oxide (NO) acts as a neuronal messenger in both the central and peripheral nervous systems and has been implicated in reproductive physiology and behavior. Pharmacological inhibition of nitric oxide synthase (NOS) with the nonspecific NOS inhibitor, l-N(G)-nitro-Arg-methyl ester (l-NAME), induced deficits in both the number of ovarian rupture sites and the number of oocytes recovered in the oviducts of mice. Female neuronal NOS knockout (nNOS-/-) mice have normal numbers of rupture sites, but reduced numbers of oocytes recovered following systemic injections of gonadotropins, suggesting that NO produced by nNOS accounts, in part, for deficits in ovulatory efficiency observed after l-NAME administration. Additionally, endothelial NOS knockout (eNOS-/-) mice have reduced numbers of ovulated oocytes after superovulation. Because endothelial NOS has been identified in ovarian follicles, and because of the noted reduced breeding efficiency of eNOS-/- mice, the present study sought to determine the role of NO from eNOS in mediating the number of rupture sites present after ovulation. Estrous cycle length and variability were consistently reduced in eNOS-/- females. The number of rupture sites was normal in eNOS-/- mice under natural conditions and after administration of exogenous GnRH. After exogenous gonadotropin administration, eNOS-/- females displayed a significant reduction in the number of ovarian rupture sites. Female eNOS-/- mice also produced fewer pups/litter compared to WT mice. These data suggest that NO from endothelial sources might play a role in mediating rodent ovulation and may be involved in regulation of the timing of the estrous cycle.     

The Human Rho-GEF trio and its target GTPase RhoG are involved in the NGF pathway, leading to neurite outgrowth

Rho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange. Trio displays two Rho-GEF domains, GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA, and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated. Here, we show that human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, we demonstrate that Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, we show that Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling.     

Immunological function of food-restricted germfree and specific pathogen-free mice.

The effects of food restriction on immune function was investigated in germfree (GF) and specific pathogen-free (SPF) mice. They were maintained from five weeks of age under either full-fed or food-restricted conditions to 4.5 grams per day (equivalent to approximately 80% of full-fed intake) of a commercial diet. Longest survival rate was attained in food-restricted SPF mice followed by food-restricted GF, full-fed GF, and full-fed SPF animals. Food-restricted GF mice showed shorter survival rate than their SPF counterparts. This result suggests that food restriction may be just as effective as GF status for extending life span. Immune function declined significantly with age in full-fed groups of GF and SPF mice. In both food-restricted GF and SPF mice, mitogenic response to concanavalin A or lipopolysaccharide and antibody response to sheep red blood cells were lower early in life and became higher later in life as compared with full-fed mice. Hence, the maintenance of effective immunological function until old age may be the reason for food-restricted groups to live slightly longer than full-fed groups.     

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.     

Hair loss and defective T- and B-cell function in mice lacking ORAI1

ORAI1 is a pore subunit of the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1^−/− mice. Orai1^−/− mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1^−/− mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1^−/− mice, but B cells showed a substantial decrease in Ca²⁺ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1^−/− T cells showed substantial reductions in store-operated Ca²⁺ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.     

Rab GTPase function in Golgi trafficking

The Rab, ARF, and Arl members of the Ras superfamily of small GTPases work together to control specific intracellular trafficking pathways. Here we focus on their roles in protein transport to and within the Golgi apparatus.     

Rac GTPase regulation of 3D invasion in neuroblastomas lacking MYCN amplification

Neuroblastomas are highly invasive tumors that occur in pediatric patients and treatment of invasive disease remains a challenge. The study of cells invading in 3-dimensional (3D) hydrogels has revealed morphologically distinct modes of invasion by which cancer cells adapt to the local tissue environment in order to invade local tissue. Specifically, the small G protein Rac GTPase has been implicated as regulating the elongated/mesenchymal mode of cell invasion. In the present study we demonstrate an inverse association between Rac expression and amplification of MYCN, a well-established prognostic indicator in neuroblastoma. Moreover, the association further tracks with previously described morphological variants of neuroblastoma. Importantly, while MYCN amplification is associated with universally poor prognosis, the clinical course of patients whose tumors lack MYCN amplification are more difficult to predict. Therefore, we analyzed the role that Rac plays in regulating the invasive behavior of neuroblastoma cells lacking MYCN amplification. Using siRNA targeting Rac in single cell suspensions in 3D collagen gels and Rac inhibition of multicellular spheroids (MCS) embedded in collagen gels, we find that the high Rac-expressing lines differ in their morphological response to Rac depletion and inhibition. Live cell imaging of embedded MCS reveals distinct individual and collective modes of invasion between the cell lines. Critically, Rac inhibition blocked both individual and collective invasion in 2 of the 3 high Rac expressing cell lines. Our study suggests that Rac activity may be an important determinant of metastatic capability in subsets of neuroblastoma cells lacking MYCN amplification.     

Renal function of gene-targeted mice lacking both SGK1 and SGK3

Serum- and glucocorticoid-inducible kinase (SGK) 1 and SGK3 share the ability to upregulate several ion channels, including the epithelial Na(+) channel. Whereas SGK1 is under genomic control of mineralocorticoids and glucocorticoids, SGK3 is constitutively expressed. The SKG1-knockout (sgk1(-/-)) mouse is seemingly normal when it is fed a standard diet, but its ability to retain NaCl is impaired when it is fed a salt-deficient diet. In the SGK3-knockout (sgk3(-/-)) mouse fed standard and salt-deficient diets, hair growth is strikingly delayed but NaCl excretion is normal. Thus the possibility was considered that SGK1 and SGK3 could mutually replace each other, thus preventing severe NaCl loss in sgk1(-/-) and sgk3(-/-) mice. We crossed SGK1- and SGK3-knockout mice and compared renal electrolyte excretion of the double mutants (sgk1(-/-)/sgk3(-/-)) with that of their wild-type littermates (sgk1(+/+)/sgk3(+/+)). Similar to sgk3(-/-) mice, the sgk1(-/-)/sgk3(-/-) mice display delayed hair growth. Blood pressure was slightly, but significantly (P < 0.03), lower in sgk1(-/-)/sgk3(-/-) (102 +/- 4 mmHg) than in sgk1(+/+)/sgk3(+/+) (114 +/- 3 mmHg) mice, a difference that was maintained in mice fed low- and high-salt diets. Plasma aldosterone concentrations were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) than in sgk1(+/+)sgk3(+/+) mice fed control (511 +/- 143 vs. 143 +/- 32 pg/ml) and low-salt (1,325 +/- 199 vs. 362 +/- 145 pg/ml) diets. During salt depletion, absolute and fractional excretions of Na(+) were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) (1.2 +/- 0.2 micromol/24 h g body wt, 0.12 +/- 0.03%) than in sgk1(+/+)/sgk3(+/+) (0.4 +/- 0.1 micromol/24 h g body wt, 0.04 +/- 0.01%) mice. The sgk1(-/-)/sgk3(-/-) mice share the delayed hair growth with sgk3(-/-) mice and the modestly impaired renal salt retention with sgk1(-/-) mice. Additional lack of the isoform kinase does not substantially compound the phenotype for either property.     

PIKE GTPase signaling and function

PIKE (PI 3-Kinase Enhancer) is a recently identified brain specific nuclear GTPase, which binds PI 3-kinase and stimulates its lipid kinase activity. Nerve growth factor treatment leads to PIKE activation by triggering the nuclear translocation of phospholipase C-gamma1 (PLC-gamma1), which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. To date, three forms of PIKE have been characterized: PIKE-S, PIKE-L and PIKE-A. PIKE-S is initially identified shorter isoform. PIKE-L, a longer isoform of PIKE gene, differs from PIKE-S by C-terminal extension containing Arf-GAP (ADP ribosylation factor-GTPase Activating Protein) and two ankyrin repeats domains. In contrast to the exclusive nuclear localization of PIKE-S, PIKE-L occurs in both the nucleus and the cytoplasm. PIKE-L physiologically associates with Homer 1, an mGluR I binding adaptor protein. The Homer/PIKE-L complex couples PI 3-kinase to mGluR I and regulates a major action of group I mGluRs, prevention of neuronal apoptosis. More recently, a third PIKE isoform, PIKE-A was identified in human glioblastoma multiforme brain cancers. Unlike the brain specific PIKE-L and -S isoforms, PIKE-A distributes in various tissues. PIKE-A contains the same domains present in PIKE-L but lacks N-terminal proline-rich domain (PRD), which binds PI 3-kinase and PLC-gamma1. Instead, PIKE-A specifically binds to active Akt and upregulates its activity in a GTP-dependent manner, mediating human cancer cell invasion and preventing apoptosis. Thus, PIKE extends its roles from the nucleus to the cytoplasm, mediating cellular processes from cell invasion to programmed cell death.     

Immunological development and cardiovascular function are normal in annexin VI null mutant mice

Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function.     

Cerebral hypoplasia and craniofacial defects in mice lacking heparan sulfate Ndst1 gene function

Mutant mice bearing a targeted disruption of the heparan sulfate (HS) modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) exhibit severe developmental defects of the forebrain and forebrain-derived structures, including cerebral hypoplasia, lack of olfactory bulbs, eye defects and axon guidance errors. Neural crest-derived facial structures are also severely affected. We show that properly synthesized heparan sulfate is required for the normal development of the brain and face, and that Ndst1 is a modifier of heparan sulfate-dependent growth factor/morphogen signalling in those tissues. Among the multiple heparan sulfate-binding factors potentially affected in Ndst1 mutant embryos, the facial phenotypes are consistent with impaired sonic hedgehog (Shh) and fibroblast growth factor (Fgf) interaction with mutant heparan sulfate. Most importantly, the data suggest the possibility that defects in heparan sulfate synthesis could give rise to or contribute to a number of developmental brain and facial defects in humans.