RhoE deficiency produces postnatal lethality, profound motor deficits and neurodevelopmental delay in mice

Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt) are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD) 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders.     

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (> 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

Rnd1 regulates axon extension by enhancing the microtubule destabilizing activity of SCG10

The GTPase Rnd1 affects actin dynamics antagonistically to Rho and has been implicated in the regulation of neurite outgrowth, dendrite development, and axon guidance. Here we show that Rnd1 interacts with the microtubule regulator SCG10. This interaction requires a central domain of SCG10 comprising about 40 amino acids located within the N-terminal-half of a putative alpha-helical domain and is independent of phosphorylation at the four identified phosphorylation sites that regulate SCG10 activity. Rnd1 enhances the microtubule destabilizing activity of SCG10 and both proteins colocalize in neurons. Knockdown of Rnd1 or SCG10 by RNAi suppressed axon extension, indicating a critical role for both proteins during neuronal differentiation. Overexpression of Rnd1 in neurons induces the formation of multiple axons. The effect of Rnd1 on axon extension depends on SCG10. These results indicate that SCG10 acts as an effector downstream of Rnd1 to regulate axon extensions by modulating microtubule organization.     

The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18

The polarization of a neuron generally results in the formation of one axon and multiple dendrites, allowing for the establishment of neuronal circuitry. The molecular mechanisms involved in priming one neurite to become the axon, particularly those regulating the microtubule network, remain elusive. Here we report the identification of DOCK7, a member of the DOCK180-related protein superfamily, as a Rac GTPase activator that is asymmetrically distributed in unpolarized hippocampal neurons and selectively expressed in the axon. Knockdown of DOCK7 expression prevents axon formation, whereas overexpression induces formation of multiple axons. We further demonstrate that DOCK7 and Rac activation lead to phosphorylation and inactivation of the microtubule destabilizing protein stathmin/Op18 in the nascent axon and that this event is important for axon development. Our findings unveil a pathway linking the Rac activator DOCK7 to a microtubule regulatory protein and highlight the contribution of microtubule network regulation to axon development.     

Rapostlin is a novel effector of Rnd2 GTPase inducing neurite branching

Rho family GTPases are central regulators of neuronal morphology. Recently, Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been identified as new members of Rho family GTPases. Of these, Rnd2 is specifically expressed in neurons in brain; however, the signaling pathways of Rnd2 are not known. Here we have performed a yeast two-hybrid screen using Rnd2 as a bait and identified a novel Rnd2-effector protein, expressed predominantly in brain. We named it Rapostlin (apostle of Rnd2). Rapostlin has two functional domains, Fer-CIP4 homology (FCH) domain at the amino terminus and SH3 (Src homology 3) domain at the carboxyl terminus. In in vitro binding assays, Rapostlin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner, and the Rnd2-binding domain of Rapostlin is localized between FCH and SH3 domains. Rapostlin directly binds to microtubules, and the amino-terminal region containing the FCH domain of Rapostlin is essential for this interaction. In PC12 cells, Rapostlin induces neurite branching in response to Rnd2, and at least the amino-terminal region of Rapostlin is necessary for this activity. Therefore, Rapostlin is the first effector of Rnd2, regulating neurite branch formation.     

A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.     

COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression

During corticogenesis, late-born callosal projection neurons (CPNs) acquire their laminar position through glia-guided radial migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of CPNs are poorly defined. Here, we show that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching the cortical plate and have morphological defects during migration. Interestingly, we observed that the rate of neuronal migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This gradient is strongly impaired in COUP-TFI(-/-) brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. We show that COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring correct Rnd2 levels in COUP-TFI(-/-) brains cell-autonomously rescues neuron radial migration and morphological transitions. We also observed impairments in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by lowering Rnd2 expression levels. Thus, our data demonstrate that COUP-TFI modulates late-born neuron migration and favours proper differentiation of CPNs by finely regulating Rnd2 expression levels.     

Rho proteins: their function in neurons

Neurons possess a polarized morphology. In general, each neuron has several dendrites but only one axon. Such morphology is the basis for directionalized rapid signaling, information flowing from the short dendrites to the long axon. The mechanisms involved in the establishment of the neuronal polarity remain largely unknown. However, recently, members of Rho family proteins have been implicated in the regulation of neuronal morphology especially development of neuronal polarity, axon outgrowth and guidance, dendritic tree elaboration and synapse formation. Moreover, the Rho GTPases have been reported to be directly or indirectly involved in some neurological conditions such as X-linked mental retardation as well as Alzheimer's and Parkinson's diseases. These findings demonstrate the importance of Rho GTPases in the development, maintenance and function of the nervous system.     

p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.     

Differential sensitivity of skeletal and fusimotor neurons to Bcl-2-mediated apoptosis during neuromuscular development

Proper development of the nervous system requires that a carefully controlled balance be maintained between both proliferation and neuronal survival. The process of programmed cell death is believed to play a key role in regulating levels of neuronal survival, in large part through the action of antiapoptotic proteins, such as Bcl-2. Consistent with this, Bcl-2 has been shown to be a key regulator of apoptotic signaling in post-mitotic neurons. However, we still know remarkably little regarding the role that Bcl-2 plays in regulating the survival of specific motor neuron populations. In the present study, we have examined somatic motor neurons of the lumbar spinal cord, and branchiomotor neurons of the facial nucleus in bcl-2-null mice to determine the differential dependence among motor neuron populations with respect to Bcl-2-mediated survival. Examination of neuronal and axon number, axonal area, and the distribution of axonal loss in bcl-2-null mice demonstrates that, in contrast to the great majority of alpha motor neurons, gamma motor neurons exhibit a unique dependence upon bcl-2 for survival. These results demonstrate, for the first time, the connection between Bcl-2 expression, motor neuron survival, and the establishment of different motor populations.     

Role of LKB1-SAD/MARK pathway in neuronal polarization

The formation of axon/dendrite polarity is critical for the neuron to perform its signaling function in the brain. Recent advance in our understanding of cellular and molecular mechanisms underlying the development and maintenance of neuronal polarity has been greatly facilitated by the use of the culture system of dissociated hippocampal neurons. Among many polarization-related proteins, we here focus on the mammalian LKB1, the counterpart of the C. elegans Par-4, which is an upstream regulator among six Par (partitioning-defective) genes that act as master regulators of cell polarity in different cell types across evolutionary distant species. Recent studies have identified LKB1 and its downstream targets SAD/MARK kinases (mammalian homologs of Par-1) as key regulators of neuronal polarization and axon development in cultured neurons and in developing cortical neurons in vivo. We will review the properties of and interactions among proteins in this LKB1-SAD/MARK pathway, drawing upon information obtained from both neuronal and non-neuronal systems. Due to central role of the protein kinase A-dependent phosphorylation of LKB1 in the activation of this pathway, we will review recent findings on how cAMP and cGMP signaling may serve as antagonistic second messengers for axon/dendrite development, and how these cyclic nucleotides may mediate the action of extracellular polarizing factors by modulating the activity of the LKB1-SAD/MARK pathway.     

RhoE participates in the stimulation of the inflammatory response induced by ethanol in astrocytes

Astroglial cells are involved in the neuropathogenesis of several inflammatory diseases of the brain, where the activation of inflammatory mediators and cytokines plays an important role. We have previously demonstrated that ethanol up-regulates inflammatory mediators in both brain and astroglial cells. Since Rho GTPases are involved in inflammatory responses of astrocytes where loss of stress fibers takes place and RhoE/Rnd3 disorganizes the actin cytoskeleton, the aim of the present study was to investigate the implication of this protein in the stimulation of inflammatory signaling induced by ethanol. Our findings show that RhoE expression induces a decrease in both RhoA and Rac. In addition, RhoE not only induces actin cytoskeleton disorganization but it also stimulates both the IRAK/ERK/NF-kappaB pathway and the COX-2 expression associated with the inflammatory response in these cells. Our results also show that ethanol exposure induces RhoE signaling in astrocytes. Preincubation of astrocytes with GF109203X, an inhibitor of PKCs, reduces the RhoE levels and abolishes the ethanol-induced activation of IRAK, NF-kappaB and the COX-2 expression. Furthermore, RhoE overexpression restores ethanol responses in astrocytes treated with the PKCs inhibitor. Altogether, our findings suggest that this small GTPase is involved in the stimulation of the inflammatory signaling induced by ethanol in astrocytes. These findings provide new insights into the molecular mechanism involved in the inflammatory responses in astrocytes.     

Specific roles of Akt iso forms in apoptosis and axon growth regulation in neurons

Akt is a member of the AGC kinase family and consists of three isoforms. As one of the major regulators of the class I PI3 kinase pathway, it has a key role in the control of cell metabolism, growth, and survival. Although it has been extensively studied in the nervous system, we have only a faint knowledge of the specific role of each isoform in differentiated neurons. Here, we have used both cortical and hippocampal neuronal cultures to analyse their function. We characterized the expression and function of Akt isoforms, and some of their substrates along different stages of neuronal development using a specific shRNA approach to elucidate the involvement of each isoform in neuron viability, axon development, and cell signalling. Our results suggest that three Akt isoforms show substantial compensation in many processes. However, the disruption of Akt2 and Akt3 significantly reduced neuron viability and axon length. These changes correlated with a tendency to increase in active caspase 3 and a decrease in the phosphorylation of some elements of the mTORC1 pathway. Indeed, the decrease of Akt2 and more evident the inhibition of Akt3 reduced the expression and phosphorylation of S6. All these data indicate that Akt2 and Akt3 specifically regulate some aspects of apoptosis and cell growth in cultured neurons and may contribute to the understanding of mechanisms of neuron death and pathologies that show deregulated growth.