A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila

Cytokinesis ensures the successful completion of the cell cycle and distribution of chromosomes, organelles, and cytoplasm between daughter cells. It is accomplished by formation and constriction of an actomyosin contractile ring that drives the progression of a cleavage furrow. Microinjection experiments and in vitro transfection assays have suggested a requirement for small GTPases of the Rho family in cytokinesis. Yet, the identity of proteins regulating Rho signaling pathways during cytokinesis remains unknown. Here we show that in Drosophila, Pebble (Pbl), a putative exchange factor for Rho GTPases (RhoGEF), is required for the formation of the contractile ring and initiation of cytokinesis. The dynamics of Pbl expression and its distribution during mitosis, as well as structure-function analysis, indicate that it is a key regulatory component of the pathway. pbl interacts genetically with Rho1, but not with Rac1 or Cdc42, and Pbl and Rho1 proteins interact in vivo in yeast. Similar to mutations in pbl, loss of Rho1 or expression of a dominant-negative Rho1 blocks cytokinesis. Our results identify Pbl as a RhoGEF specifically required for cytokinesis and linked through Rho1 activity to the reorganization of the actin cytoskeleton at the cleavage furrow.     

RNA-binding protein is involved in aggregation of light neurofilament protein and is implicated in the pathogenesis of motor neuron degeneration

Abnormal protein aggregation is emerging as a common theme in the pathogenesis of neurodegenerative disease. Our previous studies have shown that overexpression of untranslated light neurofilament (NF-L) RNA causes motor neuron degeneration in transgenic mice, leads to accumulation of ubiquitinated aggregates in degenerating cultured motor neurons and triggers aggregation of NF-L protein and co-aggregation of mutant SOD1 protein in neuronal cells. Here, we report that p190RhoGEF, an RNA-binding protein that binds to a destabilizing element in NF-L mRNA, is involved in aggregation of NF-L protein and is implicated in the pathogenesis of motor neuron degeneration. We show that p190RhoGEF co-aggregates with unassembled NF-L protein and that co-aggregation is associated with down-regulation of parent NF-L mRNA in neuronal cells. Co-expression of NF-M increases NF assembly and reduces RNA-triggered aggregation as well as loss of solubility of NF-L protein. siRNA-induced down-regulation of p190RhoGEF not only reduces aggregation and promotes assembly of NF-L and NF-M, but also causes reversal of aggregation and recovery of NF assembly in transfected cells. Examination of transgenic models of motor neuron disease shows that prominent aggregates of p190RhoGEF and NF-L and down-regulation of NF-L expression occur in degenerating motor neurons of mice expressing untranslated NF-L RNA or a G93A mutant SOD1 transgene. Moreover, aggregates of p190RhoGEF and NF-L appear as early pathological changes in presymptomatic G93A mutant SOD1 transgenic mice. Together, the findings indicate that p190RhoGEF is involved in aggregation of NF-L protein and support a working hypothesis that aggregation of p190RhoGEF and NF-L is an upstream event triggering neurotoxicity in motor neuron disease.     

Role of the Rho GTPase-activating protein RICS in neurite outgrowth

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS(-/-) neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS(-/-) mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.     

Unilateral focal polymicrogyria in a patient with classical Aarskog-Scott syndrome due to a novel missense mutation in an evolutionary conserved RhoGEF domain of the faciogenital dysplasia gene FGD1

Faciogenital dysplasia or Aarskog-Scott syndrome (AAS) is an X-linked disorder characterized by craniofacial, skeletal, and urogenital malformations and short stature. Mutations in the only known causative gene FGD1 are found in about one-fifth of the cases with the clinical diagnosis of AAS. FGD1 is a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase Cdc42 via its RhoGEF domain. The Cdc42 pathway is involved in skeletal formation and multiple aspects of neuronal development. We describe a boy with typical AAS and, in addition, unilateral focal polymicrogyria (PMG), a feature hitherto unreported in AAS. Sequencing of the FGD1 gene in the index case and his mother revealed the presence of a novel mutation (1396A>G; M466V), located in the evolutionary conserved alpha-helix 4 of the RhoGEF domain. M466V was not found in healthy family members, in >300 healthy controls and AAS patients, and has not been reported in the literature or mutation databases to date, indicating that this novel missense mutation causes AAS, and possibly PMG. Brain cortex malformations such as PMG could be initiated by mutations in the evolutionary conserved RhoGEF domain of FGD1, by perturbing the signaling via Rho GTPases such as Cdc42 known to cause brain malformation.     

The Lsc RhoGEF mediates signaling from thromboxane A2 to actin polymerization and apoptosis in thymocytes

The Lsc RhoGEF (also known as p115-RhoGEF) is a GTP exchange factor (GEF), an activator of GTPases of the Rho family. Lsc has a RhoGEF domain specific for Rho GTPase and a regulator of G protein signaling (RGS) domain specific for Galpha(12/13) subunits. One G protein receptor that can couple to Galpha(12/13) subunits is the receptor for thromboxane A(2 )(TXA(2)), thromboxane-prostanoid (called TP), which is highly expressed in immature thymocytes. TXA(2) has been implicated in thymocyte apoptosis. We found that Lsc(-/-) mice on a BALB/c background show thymic hyperplasia due to increased numbers of thymocytes and that these numbers further increase with the age of the mice. To investigate a role for Lsc in TXA(2) signaling, we analyzed activation of primary thymocytes by TXA(2) in vitro. TXA(2)-induced apoptosis of double-positive thymocytes and Rho activation required Lsc, and TXA(2) stimulation of actin polymerization and cofilin phosphorylation required both Lsc and Rho kinase (ROCK). Additionally, in the absence of Lsc, phosphorylation of the survival kinase Akt in response to TXA(2) was greatly enhanced. Together, these data demonstrate that Lsc is essential for mediating TXA(2 )signaling involved in apoptosis and actin organization and suggest that TXA(2) regulates thymic cellularity via Lsc.     

Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degeneration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease. Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular weight neurofilament mRNA and affects its stability via 3′ untranslated region destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell signaling pathway molecule.     

Axon specification in hippocampal neurons

Neurons are the most highly polarized cells, comprised of two structurally and functionally distinct parts, axons and dendrites. This asymmetry enables a vectorial flow of signaling within neurons. One of the most fundamental questions still to be answered in neuroscience is how these two specialized processes initially develop. The first manifestation of polarization occurs when one of the immature neurites acquires axonal characteristics. We review recent advances that have highlighted the involvement of several cellular events in the initial formation of the axon, including membrane traffic and cytoskeletal rearrangement. We then discuss the molecular mechanisms underlying axon formation, focusing on the Rho family small GTPases and an axon-inducing neuronal protein, CRMP-2.     

The role of the Rho GTPases in neuronal development

Our brain serves as a center for cognitive function and neurons within the brain relay and store information about our surroundings and experiences. Modulation of this complex neuronal circuitry allows us to process that information and respond appropriately. Proper development of neurons is therefore vital to the mental health of an individual, and perturbations in their signaling or morphology are likely to result in cognitive impairment. The development of a neuron requires a series of steps that begins with migration from its birth place and initiation of process outgrowth, and ultimately leads to differentiation and the formation of connections that allow it to communicate with appropriate targets. Over the past several years, it has become clear that the Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance. Given the importance of these molecules in these processes, it is therefore not surprising that mutations in genes encoding a number of regulators and effectors of the Rho GTPases have been associated with human neurological diseases. This review will focus on the role of the Rho GTPases and their associated signaling molecules throughout neuronal development and discuss how perturbations in Rho GTPase signaling may lead to cognitive disorders.     

The RhoGEF Zizimin-related acts in the Drosophila cellular immune response via the Rho GTPases Rac2 and Cdc42

Zizimin-related (Zir), a Rho guanine nucleotide exchange factor (RhoGEF) homologous to the mammalian Dock-C/Zizimin-related family, was identified in a screen to find new genes involved in the Drosophila melanogaster cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. RhoGEFs activate Rho-family GTPases, which are known to be central regulators of cell migration, spreading and polarity. When a parasitoid wasp is recognized as foreign, multiple layers of circulating immunosurveillance cells (haemocytes) should attach to the egg. In Zir mutants this process is disrupted and lamellocytes, a haemocyte subtype, fail to properly encapsulate the wasp egg. Furthermore, macrophage-like plasmatocytes exhibit a strong reduction in their ability to phagocytise Escherichia coli and Staphylococcus aureus bacteria. During encapsulation and phagocytosis Zir genetically interacts with two Rho-family GTPases, Rac2 and Cdc42. Finally, Zir is dispensable for the humoral immune response against bacteria. We propose that Zir is necessary to activate the Rho-family GTPases Rac2 and Cdc42 during the Drosophila cellular immune response.     

Small GTPase Rnd1 is involved in neuronal activity-dependent dendritic development in hippocampal neurons

Rho family small GTPases are key regulators for neuronal morphogenesis including dendritogenesis. We recently have shown that Rnd1, a member of the Rho family, is highly expressed in brain during the synaptogenic stage and is involved in dendritic spine formation. However, the mechanism by which Rnd1 regulates dendritic development including spine morphogenesis remains unknown. Here we report that Rnd1, a member of the Rho family, plays a critical role in neuronal activity-dependent dendritic development in hippocampal neurons. Overexpression of Rnd1 promoted dendritic growth and branching in cultured hippocampal neurons. On the other hand, suppression of endogenous Rnd1 expression by RNA interference significantly inhibited neuronal activity-dependent dendritic development and this inhibitory effect was canceled by inhibition of RhoA effector ROCK. In addition, knockdown of Rnd1 also abolished dendritic development promoted by treatment with brain-derived neurotrophic factor in hippocampal neurons. Our findings demonstrate that Rnd1 is involved in signaling pathways of neuronal activity-dependent dendritic development.     

The first CH domain of affixin activates Cdc42 and Rac1 through alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor

Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of alphaPIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of alphaPIX, alphaPIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation.     

Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.     

Cytoplasmic p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal cord injury in rats

p21(Cip1/WAF1), known as a cell-cycle inhibitory protein, facilitates neurite outgrowth from neurons when present in the cytoplasm. The molecular mechanism of this action is that p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity. As myelin-derived inhibitors of axonal outgrowth act on neurons by activating Rho, that is responsible for the lack of spontaneous regeneration of the injured central nervous system (CNS), Rho-kinase may be a good molecular target against injuries in the CNS. In this study, we delivered TAT-fusion protein of cytoplasmic p21(Cip1/WAF1) locally after dorsal hemisection of the thoracic spinal cord in rats. The treatment significantly stimulated axonal regeneration and recovery of hindlimb function, and inhibited the cavity formation in the spinal cord after the injury. Cytoplasmic p21(Cip1/WAF1) may provide a potential therapeutic agent that produces functional regeneration following CNS injuries.     

p190RhoGAP can act to inhibit PDGF-induced gliomas in mice: a putative tumor suppressor encoded on human chromosome 19q13.3

p190RhoGAP and Rho are key regulators of oligodendrocyte differentiation. The gene encoding p190RhoGAP is located at 19q13.3 of the human chromosome, a locus that is deleted in 50%-80% of oligodendrogliomas. Here we provide evidence that p190RhoGAP may suppress gliomagenesis by inducing a differentiated glial phenotype. Using a cell culture model of autocrine loop PDGF stimulation, we show that reduced Rho activity via p190RhoGAP overexpression or Rho kinase inhibition induced cellular process extension, a block in proliferation, and reduced expression of the neural precursor marker nestin. In vivo infection of mice with retrovirus expressing PDGF and the p190 GAP domain caused a decreased incidence of oligodendrogliomas compared with that observed with PDGF alone. Independent experiments revealed that the retroviral vector insertion site in 3 of 50 PDGF-induced gliomas was within the p190RhoGAP gene. This evidence strongly suggests that p190 regulates critical components of PDGF oncogenesis and can act as a tumor suppressor in PDGF-induced gliomas by down-regulating Rho activity.     

Regulation of dopamine-induced Na+ current response by small G-protein RhoB or C and phospholipase D in Aplysia neurons

A family of GTP-binding proteins, Rho, plays critical roles in cell migration, morphological change, cytokinesis, and smooth muscle contraction. Furthermore, evidence has recently been accumulating for an involvement in regulation of receptor-operated ionic channels. We previously reported that stimulation of D1-like receptor by dopamine (DA) induces a slow Na+ current response in the identified neurons of Aplysia under voltage-clamp. To further study a regulatory mechanism of the DA-induced response, we examined possible involvement of small G-proteins and subsequent enzymes. The Na+ current response to DA was gradually and irreversibly depressed after the intracellular injection of either Clostridium difficile toxin B, an inhibitor for all Rho family G-proteins, or Clostridium botulinum C3 exoenzyme, a specific blocker for RhoA-C. Intacellular injection of active RhoA had no significant effect on the response. However, injection of GAP domain of p50RhoGAP significantly depressed the DA-induced response, while application of GEF domain of RhoGEF Dbs increased the response. In addition, either intracellular injection of alpha-synuclein or extracellular application of 1-butanol, inhibitors for phospholipase D (PLD), significantly depressed the DA-induced response. These results suggest that the DA-induced Na+ current response may be facilitated by the activation of Rho family G-protein RhoB or C but not RhoA, and subsequent PLD.     

The X-linked mental retardation protein oligophrenin-1 is required for dendritic spine morphogenesis

Of 11 genes involved in nonspecific X-linked mental retardation (MRX), three encode regulators or effectors of the Rho GTPases, suggesting an important role for Rho signaling in cognitive function. It remains unknown, however, how mutations in Rho-linked genes lead to MRX. Here we report that oligophrenin-1, a Rho-GTPase activating protein that is absent in a family affected with MRX, is required for dendritic spine morphogenesis. Using RNA interference and antisense RNA approaches, we show that knock-down of oligophrenin-1 levels in CA1 neurons in rat hippocampal slices significantly decreases spine length. This phenotype can be recapitulated using an activated form of RhoA and rescued by inhibiting Rho-kinase, indicating that reduced oligophrenin-1 levels affect spine length by increasing RhoA and Rho-kinase activities. We further demonstrate an interaction between oligophrenin-1 and the postsynaptic adaptor protein Homer. Our findings provide the first insight into how mutations in a Rho-linked MRX gene may compromise neuronal function.     

Rac1b increases with progressive tau pathology within cholinergic nucleus basalis neurons in Alzheimer's disease

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.     

Pericyte Rho GTPase mediates both pericyte contractile phenotype and capillary endothelial growth state

Pericytes regulate microvascular development and maturation through the control of endothelial cell motility, proliferation, and differentiation. The Rho GTPases have recently been described as key regulators of pericyte shape and contractile phenotype by signaling through the actin cytoskeleton in an isoactin-specific manner. In this report, we reveal that Rho GTPase-dependent signal transduction not only influences pericyte shape and contractile potential but also modulates capillary endothelial proliferative status and pericyte-endothelial interactions in vitro. We provide evidence that overexpression of mutant Rho GTPases, but not other Ras-related small GTPases, significantly alters pericyte shape, contractility, and endothelial growth state in microvascular cell co-cultures. In particular, we describe the use of a silicon substrate deformation assay to demonstrate that pericyte contractility is Rho GTP- and Rho kinase-dependent; further, we describe a novel in vitro system for examining pericyte-mediated endothelial growth arrest and show that control pericytes are capable of growth-arresting capillary endothelial cells in a cell contact-dependent manner, whereas pericytes overexpressing dominant-active and -negative Rho GTPase are comparably incompetent. These data strongly suggest that signaling through the pericyte Rho GTPase pathway may provide critical cues to the processes of microvascular stabilization, maturation, and contractility during development and disease.     

Neuregulin‐1/glial growth factor stimulates Schwann cell migration by inducing α5 β1 integrin–ErbB2–focal adhesion kinase complex formation

After peripheral nerve injury, Schwann cells gain a migratory phenotype and remodel their extracellular matrix to provide a supportive environment for axonal regeneration. The soluble neuregulin‐1 isoform, that is, glial growth factor (GGF), is expressed in regenerating axons of injured peripheral nerves and regulates Schwann cell motility by activating the ErbB family of tyrosine kinase receptors, but how GGF/ErbB signaling contributes to Schwann cell motility remains unclear. Here, we show that GGF stimulates Schwann cell migration by inducing the formation of a protein complex containing the fibronectin receptor α5β1 integrin, ErbB2, and focal adhesion kinase (FAK). ErbB2 co‐localizes and co‐immunoprecipitates with the focal complex members including α5β1 integrin and FAK after GGF treatment. These effects of GGF appear to involve FAK activation, which occurs downstream of ErbB2 stimulation. RNAi‐mediated down‐regulation of α5 integrin expression in primary cultured Schwann cells resulted in significantly decreased interaction between FAK and ErbB2, as well as decreased GGF‐induced migration. An increase in the α5β1 integrin–ErbB2–FAK complex formation was observed in injured nerve Schwann cells, but not uninjured control. Taken together, these data suggest that GGF plays an important modulatory role in Schwann cell migration after nerve crush by inducing α5β1 integrin–ErbB2–FAK complex formation.     

High frequency of development of B cell lymphoproliferation and diffuse large B cell lymphoma in Dbl knock-in mice

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, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.