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Rho GTPases and activity-dependent dendrite development
Rho GTPases and activity-dependent dendrite development Linda Van Aelst and Hollis T Cline Dendritic morphology has an important influence on neuronal information processing. Multiple environmental cues, including neuronal activity, the neurotrophin family of growth factors, and extracellular guidance molecules have been shown to influence dendritic size, shape, and development. The Rho GTPases have emerged as key integrators of these environmental cues to regulate the underlying dendritic cytoskeleton. Addresses 1 Bungtown Rd, Cold Spring Harbor Lab, Cold Spring Harbor, New York, New York 11724, USA e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:297–304 This review comes from a themed issue on Signalling mechanisms Edited by Richard L Huganir and S Lawrence Zipursky Available online 20th May 2004 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.05.012 Abbreviations BDNF brain derived neurotrophic factor EphB ephrin B receptor GAP GTPase activating proteins GEF guanylate exchange factor GFP green fluorescent protein GST glutathione S transferase GTP guanine trinucleotide phosphate MB mushroom body NMDA N-methyl-D-aspartate NT neurotrophin PAK P21 activated kinase WASP Wiskott Aldrich syndrome protein Wnt Wingless
Introduction Neurons are highly polarized cells composed of specialized extensions. They have a main axon that typically relays information to other neurons, and dendrites that typically receive inputs from other neurons. The dendrites of neurons are often highly branched and the extent of the arborization of the dendritic tree correlates with the number and distribution of inputs that the neuron can receive and process. Consequently, the mechanisms that govern the development of dendritic arbors have a fundamental impact on the establishment and function of neuronal circuits in the brain. Recent evidence indicates that several different types of activity in the brain affect dendritic arbor development and plasticity and this litwww.sciencedirect.com
erature has been recently reviewed [1,2]. The purpose of this review is to present recent evidence linking the Rho guanine trinucleotide phosphate (GTP)ases with activityinduced dendritic arbor development. Dendritic arbor structure is complex. In typical neurons, primary dendrites extend from the cell body and branch repeatedly to establish a distinctive tree. Some neurons have tiny protrusions or spines, which extend from the branches of the dendrites. For spiny neurons, spines are the sites of the majority of excitatory synaptic inputs, whereas inhibitory synapses typically form on the main shaft of dendritic branches. For non-spiny neurons, excitatory and inhibitory synapses are distributed along dendritic branches. Dendrites also extend filopodia, which might play a role in spine formation. The cytoskeleton of dendritic branches is composed principally of microtubules with a cortex of actin close to the plasma membrane. The cytoskeleton of spines and filopodia is thought to be composed entirely of actin. Defining how neurons acquire their size and shape during development and how learning and experience can influence morphological changes that alter the functional connectivity between presynaptic and postsynaptic cells is a major challenge for modern neuroscientists. Of particular relevance to this review are data from time-lapse imaging studies that have documented the structural changes that occur in dendrites as the arbor becomes more elaborate (Figure 1). Zebrafish and Xenopus laevis tadpoles are relatively transparent at early stages of development, so expression of fluorescent proteins in single neurons allows high-resolution time-lapse images of developing dendritic arbors within the brains of intact animals (reviewed by Cline [1]). These studies have shown that dendritic arbors follow a stereotyped sequence of events during their elaboration: many fine filopodial branches are added to the arbor, but the majority are rapidly retracted. The branches that stabilize extend and become a substrate for further branch addition and stabilization. Therefore, the arbor gradually becomes more complex by iterative branch addition, stabilization, and extension (Figure 1). It is currently believed that the cytoskeleton of newly added branches is entirely composed of actin and that microtubule invasion of the branch accompanies its stabilization, however, the cytoskeletal changes that underlie dendritic arbor development are not well defined. Many environmental cues influence dendritic size, shape, and development. Amongst them, neuronal activity seems to be particularly important for dendritic growth. The neurotrophin family of growth factors and previously Current Opinion in Neurobiology 2004, 14:297–304
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Figure 1
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Activity-dependent dendrite arbor growth. (a) In vivo images of GFP-expressing neurons in the absence and presence of visual stimulation show a stimulus-induced increase in arbor growth rate (top panel). Expression of dominant negative RhoAN19 increases arbor growth rate independent of visual stimulation and occludes stimulus-induced arbor growth. Expression of dominant negative RacN17 or Cdc42N17 blocks stimulus-induced growth, but does not affect the basal growth rate in the absence of visual stimulation. (b) Scheme of Rho GTPase-dependent stages of dendritic arbor growth. Three distinct behaviors are required for dendritic arbor growth. Branches must be added to the arbor and Rac stimulates branch additions. Because the majority of branches are rapidly retracted, an active mechanism must stabilize branches. This stabilization is also Rac-mediated. Finally, stabilized branches extend and these branches then support further branch additions in an iterative process [55]. Branch extension is mediated by a decrease in RhoA activity. High levels of RhoA deter branch stabilization. (c) Schematic signaling pathway activated by visual stimulation. Stimulation increases glutamate receptor activity that increases Rac and Cdc42 activity and decreases RhoA activity. Crosstalk among the GTPases can further affect their activities. Changes in GTPase activities alter actin and microtubule based cytoskeletal dynamics through downstream effectors.
identified axon guidance molecules have also come forward as key regulators of dendritic development and guidance. During recent years, some light has been shed on the mechanisms by which these cues signal to affect dendritic complexity. The Rho GTPases have emerged as key integrators of these environmental cues to regulate the underlying dendritic cytoskeleton. Below, we review the properties of the Rho GTPases and their signaling pathways and then discuss the potential mechanisms by which environmental cues might act through the Rho GTPases to affect dendritic arbor development.
Rho GTPases Genes of the Rho family of small GTPases encode low molecular weight guanine nucleotide binding proteins. These proteins function as molecular switches by cycling between an ‘active’ GTP-bound state and an ‘inactive’ guanine dinucleotide phosphate (GDP)-bound state. Only in their GTP-bound state are the proteins able to Current Opinion in Neurobiology 2004, 14:297–304
interact with downstream molecules that mediate their effects. The ratio of the two forms is regulated by the opposing effects of GTPase activating proteins (GAPs), which increase the intrinsic rate of hydrolysis of bound GTP, and guanine nucleotide exchange factors (GEFs), which enhance the exchange of bound GDP for GTP. The best studied Rho family members, Cdc42 (cell division cycle 42), Rac1 (Ras-related C3 botullinum toxin substrate 1), and RhoA (Ras homologous member A), have been reported to function as key regulators of the actin cytoskeleton and more recently of the microtubule cytoskeleton (reviewed by Van Aelst and D’SouzaSchorey [3]). Given that actin filaments and microtubules provide the structural basis for dendrites, the involvement of the Rho GTPases in mediating dendrite growth and remodeling has been intensively investigated. To date, numerous studies have demonstrated that altering the activity levels of RhoA, Rac1, and Cdc42 GTPases affects various aspects of dendritic development. www.sciencedirect.com
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Studies using vertebrate and invertebrate model systems have revealed a role for RhoA in regulating dendritic arbor growth. Expression of a constitutively active mutant form of RhoA in hippocampal neurons [4], Xenopus and chick retinal ganglion cells [5,6], Xenopus tectal neurons [7], and Drosophila melanogaster mushroom body (MB) neurons [8] generally resulted in a decrease in dendritic growth. Conversely, removal of Drosophila Rho in individual MB neurons led to dendrite overextension [8], whereas ectopic expression of a dominant negative mutant form of RhoA (RhoAN19) in Xenopus tectal cells resulted in an increase in total dendritic length [7]. Blocking RhoA function in several other systems, however, caused only a mild or no detectable phenotype. For example, in hippocampal neurons, expression of the RhoAN19 dominant negative mutant did not induce any observable phenotype [4]. In this case, a model was proposed in which the Rho signaling pathway is normally repressed under physiological conditions to allow dendritic growth, and is only locally activated when dendritic growth needs be limited.
The Rho GTPases are also involved in dendritic spine formation. Several lines of evidence support a role for Rac1 in both the formation and the maintenance of spines. Cerebellar Purkinje cells of transgenic mice engineered to express the constitutively active Rac mutant, Rac1V12, showed a large number of small supranumerary spines [12]. Consistent with these findings, expression of Rac1V12 in hippocampal or cortical neurons led to overproduction of abnormal spines and membrane ruffling, whereas expression of the dominant negative Rac1N17 mutant caused a reduction in spine density [4,13]. In contrast to Rac1, RhoA appears to block spine formation and maintenance as well as elongation. Activation of RhoA in hippocampal or cortical neurons in organotypic slices resulted in a decrease in spine number and spine length [4,13]. No obvious phenotype was observed when altering Cdc42 activity in these neurons, or in single cell clones of Drosophila MB neurons. However, analyses of mutant Cdc42 clones, which lack Cdc42 in VS neurons, demonstrated a requirement for Cdc42 in maintaining dendritic spine density [10].
Rac and to a lesser extent Cdc42 have emerged as key regulators of dendritic branching and remodeling. In Xenopus, the dendritic arbor complexity of retinal ganglion cells was reduced by expression of dominant negative mutant forms of Rac1 and Cdc42, but not RhoA. Conversely, an activated mutant form of Rac (but not of Cdc42) promoted an increase in dendritic complexity [5]. Time-lapse experiments in Xenopus tectal cells revealed that Rac1 promotes branch addition and stabilization, whereas increased RhoA activity inhibits branch extension [7]. Similar imaging experiments in chick retinal ganglion cells showed an increase in the addition of tertiary branches upon expression of constitutively activated mutant Rac [6]. In Drosophila, MB neurons mutant for all three Rac genes (Rac1, Rac2 and Mtl [Mig-2-like gene]) exhibited a significant reduction in dendritic length and number of branches [9]. Whereas no observable phenotypes were found in single cell clones of MB neurons lacking Cdc42, analyses of mutant Cdc42 clones in vertical system (VS) neurons, which are more complex and stereotyped than MB neurons, demonstrated a requirement for Cdc42 in regulating dendritic branching, guidance, and consistency of axon caliber [10]. In summary, the above studies are generally consistent with a key role for RhoA in controlling dendritic length, and key roles for Rac and Cdc42 in regulating branch dynamics. Importantly, studies in Xenopus tectal neurons demonstrated that crosstalk among Rho GTPases takes place within growing dendritic arbors [11]. This tight cross regulation of GTPase activities, triggered in response to extracellular cues, appears to define the overall complexity of the dendritic tree. In Xenopus tectal neurons, RhoA activity was increased by Rac activation and Cdc42 inhibition, whereas Rac was inhibited by activation of RhoA (see below).
For the large part, the effects of the Rho GTPases on dendritic development are brought about by their actions on the actin and/or microtubule cytoskeleton. For a more comprehensive review on Rho effectors, the reader is referred to other studies [3,14–18].
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Neuronal activity A major challenge has been to define the extracellular cues that trigger the activation of the Rho GTPases to bring about the appropriate cytoskeletal remodeling essential for the development of the complex dendritic tree (Figure 1). Although several studies have demonstrated that synaptic activity affects dendritic arbor development and spine morphogenesis [19–22], the links among afferent input, glutamate receptor activity, and Rho GTPases have only recently been explored. Sin et al. [23] used in vivo time lapse imaging to test whether or not a relatively short period (4 h) of enhanced visual stimulation would affect dendritic arbor development of Xenopus tectal neurons. Images were collected of green fluorescent protein (GFP)-expressing neurons at either 1 h or 4 h intervals over 8 h. During the first 4 h of the imaging protocol, the animals were returned to a dark chamber between imaging sessions. During the second 4 h period, the animals received enhanced visual stimulation. Dendritic arbor growth rate was increased 2.5 fold compared to the rate seen in the absence of visual stimulation. The enhanced growth rate was blocked by N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor blockers, which indicates that glutamatergic synaptic transmission is required for the enhanced arbor growth rate. An analysis of branch dynamics showed that the enhanced visual stimulation increased the rate of Current Opinion in Neurobiology 2004, 14:297–304
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branch additions and stabilizations as well as extension of pre-existing branches [23]. To examine the requirement for Rho GTPases in dendritic arbor development in vivo, Li et al. [7] co-expressed GFP and constitutively active and dominant negative mutant forms of Rac, RhoA, and Cdc42 in optic tectal neurons in the brains of intact animals. We collected time-lapse images of the developing arbor at 2 h intervals over 8 h or at daily intervals over several days. Animals were anesthetized during the brief periods of image collection, but otherwise were awake and receiving normal levels of visual stimulation. These two imaging protocols allowed us to determine whether or not the Rho GTPase activity had an impact on the overall growth of the dendritic arbor, and to determine the effect of the different GTPases on the dynamic branch behaviors that are known to be important for arbor growth. Rac activity and, to a lesser extent, Cdc42 activity promote rapid branch dynamics, specifically branch additions and retractions, whereas RhoA activity is required for extension of branches. As branch addition, stabilization, and extension are distinct cell biological processes that are required for dendritic arbor development, it was interesting to find that they are separately regulated by the different Rho GTPases. Despite the pleasing simplicity of these results, that different GTPases control different structural changes in the developing neuron, numerous reports suggested that there might be crosstalk between the upstream and the downstream effectors of the Rho GTPases. Given that neurons integrate and respond to several extracellular signals during dendritic arbor development, it seemed likely that crosstalk among the GTPases would have an impact on dendrite branch dynamics and growth. To address this question, we developed an in situ version of the popular pulldown assay of GTPase activity, in which tissue sections from the tadpole optic tectum were incubated with the GTPase binding domain of the downstream effectors, p21 activated kinase (PAK), Wiskott Aldrich syndrome protein (WASP), and Rhotekin, fused to glutathione S transferase (GST). Subsequent incubation of the sections with GST antibody then revealed increases in endogenous Rac, Cdc42, and RhoA activity [11]. We expressed constitutively active or dominant negative forms of Rac, Cdc42, or RhoA and tested whether or not changes in the activities of these GTPases increased the activity of endogenous GTPases through crosstalk. These experiments showed that in the intact brain, with all the normally complex interactions that govern dendritic arbor growth, that RhoA decreases Rac activity, whereas Rac activity increases RhoA activity. This suggests a regulatory feedback loop among the GTPases and that the final outcome with respect to morphological changes in the dendrite reflects the balance of activities of the different GTPases. Indeed, our Current Opinion in Neurobiology 2004, 14:297–304
in vivo imaging data showed that exposure to lysophosphatidic acid (LPA), which activates RhoA, counteracts the increased branch dynamics normally seen when expressing active Rac. To determine whether or not glutamate receptor activity regulates the Rho GTPases, Li et al. [11] adapted the in situ GST-binding assay for use in intact brains and tested whether or not optic nerve stimulation altered endogenous GTPase activity in the optic tectum. Optic nerve stimulation increased endogenous Rac activity and decreased endogenous RhoA activity in the tectum. Furthermore, the changes in GTPase activities were downstream of glutamatergic synaptic inputs. This was the first clear demonstration that endogenous GTPase activity is regulated by synaptic input. Because this assay reports the combined activity in presynaptic retinal axon arbors and postsynaptic tectal cell dendrites, it will be important to obtain data on the effect of retinal input activity on endogenous GTPase activities with greater spatial and temporal resolution. A further link between visual input activity and Rho GTPase-mediated dendritic arbor development was provided by Sin et al. [23]. This study demonstrated directly that visual stimulation enhanced dendritic arbor development using a mechanism that required both glutamate receptor activity and Rho GTPase activity. As mentioned above, we first demonstrated that visual stimulation increases the rate of dendritic arbor development [23]. We then found that the visual stimulationinduced arbor development was completely blocked by expression of dominant negative forms of Rac and Cdc42 and by constitutively active RhoA. It appears that endogenous RhoA activity is high in tectal cell dendrites and these levels are decreased downstream of glutamate receptor activation. Glutamate receptor activity also increases Rac and Cdc42 activity, which increase branch dynamics. These data fit well with the initial scheme of how branch dynamics lead to arbor growth. Increased rates of branch additions are triggered by glutamate receptor activity through increases in Rac and Cdc42 activity. Endogenous RhoA activity is low, and this is permissive for increased Rac activity, as our crosstalk studies indicate that RhoA activity decreases Rac activity. The increased dendrite branch additions lead to increased formation of trial connections with highly dynamic axon branches [24,25]. If these trial connections are maintained by virtue of increased adhesion and strengthened synaptic transmission, then the branches will be stabilized. The stabilized branches then extend under conditions of low RhoA activity. Although the molecular link between glutamate receptor activity and regulation of GTPase activity is not yet known, this is clearly an important area of future research. For instance, signal transduction pathways www.sciencedirect.com
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governing growth cone turning behaviors in response to guidance cues might reveal calcium-sensitive regulators of Rho GTPase activity. These same calcium sensitive regulators might be the missing link between calcium influx downstream of glutamate receptor activation and GTPase activity.
Neurotrophins and extracellular signaling molecules During recent years, neurotrophins and several of the well-characterized axon guidance factors including semaphorins, ephrins, and slit have been shown to also affect dendritic outgrowth and spine morphogenesis. More recent evidence also suggests that these extracellular signaling molecules or their signaling pathways are affected by neuronal activity. The underlying mechanisms by which these extrinsic factors exert their effects on dendritic growth and guidance remain largely unknown (see reviews by Wong and Ghosh, Jan and Jan, and Miller and Kaplan [2,26,27]), although accumulating studies that have been performed mainly in the context of axon outgrowth or guidance have revealed links between these factors and the activation of Rho GTPases (see Figure 2). Given the demonstrated role of the Rho GTPases in dendritic arbor development, is quite likely that they also mediate the actions of a variety of extracellular signaling molecules on dendritic outgrowth and guidance. We will therefore discuss some of the signaling pathways that link extracellular signaling molecules to the Rho GTPases with an emphasis on those that have been implicated in dendritic development and are regulated by or affect neuronal activity.
Neurotrophins, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and NT4/5 constitute a family of dimeric secretory proteins that signal through receptor tyrosine kinases, known as trkA, trkB, trkC and the low affinity trkA receptor. Several studies have demonstrated that the neurotrophins influence the development of dendritic arbors and synaptic plasticity [19,28–30]. BDNF is released from neurons in a calcium-dependent manner so its actions are likely to be downstream of neuronal activity. In addition, trkB receptors are trafficked to the cell surface by an activitydependent mechanism [31–33], which indicates that both the extracellular neurotrophin levels and the cell surface receptor levels are increased by neuronal activity. It is likely that activity-dependent mechanisms also control the release of other neurotrophins and the trafficking of their receptors to the cell surface. In a pioneering series of studies, McAllister and Katz [28,29] demonstrated that the neurotrophins, BDNF, NT3, and NT4/5 have differential growth regulating effects on cortical neuronal dendrites. They further demonstrated that the growth regulating effects of applied neurotrophins required NMDA receptor activity. BDNF reportedly enhances glutamatergic synaptic strength by an NMDA receptor mediated mechanism [30]. As synaptic input activity can also promote the elaboration of the dendritic arbor [22,23], through a mechanism requiring Rho GTPases [23], the cooperative interaction between glutamatergic synaptic transmission and BDNF on dendritic arbor growth in cortical neurons could arise by a neurotrophin-mediated increase in
Figure 2
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Intracellular signaling pathways linking neurotrophins and axon guidance factors to the activation of Rho GTPases in the context of axon outgrowth and guidance (see [14,26,49,50,51] and text). Given the recent implication of BDNF, NGF, Sem3A, and slit in dendritic development and/or function and the well-established role of Rho GTPases in these processes, similar molecular links in the context of dendritic development might be uncovered. www.sciencedirect.com
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synaptic strength followed by glutamate receptor mediated effects on the GTPases. Alternatively, NMDA receptor stimulation might increase the numbers of trkB receptors at postsynaptic sites. This, in turn, could increase BDNF signaling and activity of GTPasemediated dendrite growth. The importance of these signaling pathways for human brain function is demonstrated by a recent finding in which a mutation in BDNF in the human population prevents BDNF secretion and leads to poor episodic memory and deficient dendritic arbor development of hippocampal neurons [34]. Numerous studies have followed those of McAllister and Katz. Because cortical neurons have been used to examine the function of the Rho GTPases in dendritic arbor development, it is possible to map the effects of the neurotrophins on those of Rho GTPase-mediated changes in dendritic arbor structure. For instance, in cortical neurons, BDNF is likely to increase arbor complexity by increasing the rate of branch additions and branch stabilizations, which is comparable to the morphological changes seen with expression of Rac [4]. Similarly, insulin-like growth factor appears to promote dendritic arbor development in cortical neurons through a mechanism that might require Cdc42 [35]. The EphB2 receptor kinase is a receptor for B-type ephrins, which are membrane-bound ligands found at excitatory synapses in developing and mature neurons [36]. Recent data suggest that the EphB receptor plays a key part in synaptic development, plasticity, and spine morphogenesis [37–39]. The EphB receptor associates with the NMDA receptor, which might lead to clustering of the receptors at synaptic sites and enhanced NMDA receptor activity [40]. Two recent studies have implicated the Rho GTPases as crucial mediators of the effect of EphB2 on spine morphology. In one study, Penzes et al. [41] demonstrated that trans-synaptic EphrinB–EphB receptor activation resulted in the phosphorylation and recruitment of Kalirin-7 to synapses, as well as the phosphorylation of the serine/threonine kinase, PAK, a downstream effector of Rac and Cdc42. Kalirin-7 is the most prevalent isoform of the dual Rac1/RhoA-GEF Kalirin in the adult rat brain, containing the first Rac1–GEF domain, but lacking the second RhoA GEF domain. Indeed, Kalirin-7 was found to activate Rac1 [42]. Interfering with the function of the EphB receptor, Kalirin, Rac, or Pak eliminated EphrinB-induced spine development of hippocampal neurons [41]. These findings support a model in which activation of the EphB2 receptor induces the recruitment of Kalirin-7 to the synapse, which results in a local activation of Rac1 and its downstream effector Pak. The local activation of these proteins consequently triggers remodeling of the actin cytoskeleton in spines. Notably, ectopic expression of Kalirin-7 in cortical and hippocampal cultured neurons induces the formation of spine-like structures, whereas reducing levels of Current Opinion in Neurobiology 2004, 14:297–304
Kalirin by means of an antisense approach causes a reduction in spine density followed by simplification of the dendritic tree [42,43]. Signaling through the EphB receptor on spine structure (combined with NMDA receptor function) ensures a coordination of structural and functional aspects of synaptic plasticity. In an independent study, Irie and Yamaguchi [44] provided evidence for the involvement of Cdc42 in mediating the effect of EphB2 on dendritic spine development. They isolated intersectin-1, a GEF for Cdc42, together with the Cdc42 effector, N-WASP, in a complex with EphB2. Both N-WASP (by directly interacting with intersectin-1) and EphB2 were found to enhance the GEF activity of intersectin-1, which resulted in increased Cdc42–GTP levels. Notably, the finding that an effector of Rho GTPases can influence the activity of a regulator of these GTPases is not that uncommon (see Soderling et al. [45]). They also observed that dominant negative mutant forms of intersectin-1, N-WASP, and Cdc42 interfered with spine formation in cultured hippocampal neurons [44]. Whether or not these dominant negative forms interfere with EphrinB-induced spine morphology remains to be seen. On the basis of these findings, a model was proposed in which the EphB receptor in a complex with intersectin-1 and N-WASP triggers the activation of Cdc42, which in turn promotes actin polymerization through N-WASP and the Arp2/3 (actin related protein) complex, leading to spherical expansion of spine heads. Such an expansion of spine head size might be coordinated with recruitment of glutamate receptors to the spine and addition of other postsynaptic machinery during synaptic development and plasticity. A recent study by Yu and Malenka [46] has tied together signaling pathways in the regulation of dendritic arbor growth through experiments with the soluble factor Wnt (Wingless), the N-cadherin/b-catenin complex, Rho GTPases, and neuronal activity. Overexpression of bcatenin increased dendritic arbor growth by a mechanism that required the interaction of its binding partner, the transmembrane adhesion protein N-cadherin, and neuronal activity. Wnts are soluble factors that regulate cell proliferation and cell fate [47]. Wnt7b has recently been shown to affect synaptogenesis in the cerebellum [48]. Wnt signaling through the receptor, frizzled, leads to the dissociation of b-catenin from N-cadherin and subsequent enhancement of dendritic arbor growth. The arbor growth mediated by this pathway is positively regulated by neuronal activity, which also leads to a release of Wnt from the neurons, indicating positive feedback between neuronal activity- and Wnt-mediated arbor growth and synapse formation, which in turn results in further Wnt release and enhanced neuronal activity. The groups of Malenka [4,6] and Salinas (pers comm) have begun to investigate the role of Rho GTPases downstream from this multifaceted signaling sequence. It will be www.sciencedirect.com
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interesting to determine the mechanisms that integrate signals from neuronal activity, N-cadherin-mediated association with other synaptic partners, and Wnt/frizzled activity with the Rho GTPases.
Conclusions Taken together, the above studies reveal molecular links among neuronal activity, Rho GTPases, and signaling through neurotrophins and other extracellular molecules in the control of dendritic arbor development. Future experiments will be essential to define the signal transduction cascades by which these molecules control dendritic growth. As suggested above, these experiments are likely to be guided by the more detailed knowledge available on these pathways in axon growth cone guidance (see Figure 2 and the reviews cited here [14,26,49,50,51]). A second area of important research will be to determine the genetic link among Rho GTPases, behavior [52], and mental retardation [53,54].
Acknowledgements This work was supported by an endowment from the Charles Robertson family (to HT Cline), the National Institutes of Health (to HT Cline, L Van Aelst), and the Dana Foundation (to HT Cline, L Van Aelst).
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11. Li Z, Aizenman CD, Cline HT: Regulation of Rho GTPases by crosstalk and neuronal activity in vivo. Neuron 2002, 33:741-750. 12. Luo L, Hensch TK, Ackerman L, Barbel S, Jan LY, Jan YN: Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature 1996, 379:837-840. 13. Tashiro A, Minden A, Yuste R: Regulation of dendritic spine morphology by the rho family of small GTPases: antagonistic roles of Rac and Rho. Cereb Cortex 2000, 10:927-938. 14. Luo L: Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 2002, 18:601-635. 15. Bishop AL, Hall A: Rho GTPases and their effector proteins. Biochem J 2000, 348:241-255. 16. Meyer G, Feldman EL: Signaling mechanisms that regulate actin-based motility processes in the nervous system. J Neurochem 2002, 83:490-503. 17. Bokoch GM: Biology of the p21-activated kinases. Annu Rev Biochem 2003, 72:743-781. 18. Riento K, Ridley AJ: Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 2003, 4:446-456. 19. Engert F, Bonhoeffer T: Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 1999, 399:66-70. 20. Maletic-Savatic M, Malinow R, Svoboda K: Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 1999, 283:1923-1927. 21. Dunaevsky A, Tashiro A, Majewska A, Mason C, Yuste R: Developmental regulation of spine motility in the mammalian central nervous system. Proc Natl Acad Sci USA 1999, 96:13438-13443. 22. Rajan I, Cline HT: Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. J Neurosci 1998, 18:7836-7846. 23. Sin WC, Haas K, Ruthazer ES, Cline HT: Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 2002, 419:475-480. 24. Witte S, Stier H, Cline HT: In vivo observations of timecourse and distribution of morphological dynamics in Xenopus retinotectal axon arbors. J Neurobiol 1996, 31:219-234. 25. Ruthazer ES, Akerman CJ, Cline HT: Control of axon branch dynamics by correlated activity in vivo. Science 2003, 301:66-70. 26. Jan YN, Jan LY: The control of dendrite development. Neuron 2003, 40:229-242. 27. Miller FD, Kaplan DR: Signaling mechanisms underlying dendrite formation. Curr Opin Neurobiol 2003, 13:391-398. 28. McAllister AK, Katz LC, Lo DC: Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth. Neuron 1997, 18:767-778. 29. McAllister AK, Lo DC, Katz LC: Neurotrophins regulate dendritic growth in developing visual cortex. Neuron 1995, 15:791-803. 30. Schuman EM: Neurotrophin regulation of synaptic transmission. Curr Opin Neurobiol 1999, 9:105-109. 31. Balkowiec A, Katz DM: Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons. J Neurosci 2002, 22:10399-10407. 32. Balkowiec A, Katz DM: Activity-dependent release of endogenous brain-derived neurotrophic factor from primary sensory neurons detected by ELISA in situ. J Neurosci 2000, 20:7417-7423. 33. Meyer-Franke A, Wilkinson GA, Kruttgen A, Hu M, Munro E, Hanson MG Jr, Reichardt LF, Barres BA: Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons. Neuron 1998, 21:681-693. Current Opinion in Neurobiology 2004, 14:297–304
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34. Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Bertolino A, Zaitsev E, Gold B, Goldman D, Dean M et al.: The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 2003, 112:257-269.
b-catenin mediated arbor elaboration might require RhoA signaling, as dominant negative RhoA blocks the b-catenin mediated growth.
35. Cheng CM, Mervis RF, Niu SL, Salem N Jr, Witters LA, Tseng V, Reinhardt R, Bondy CA: Insulin-like growth factor 1 is essential for normal dendritic growth. J Neurosci Res 2003, 73:1-9.
48. Hall AC, Lucas FR, Salinas PC: Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell 2000, 100:525-535.
36. Torres R, Firestein BL, Dong H, Staudinger J, Olsen EN, Huganir RL, Bredt DS, Gale NW, Yancopoulos GD: PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 1998, 21:1453-1463.
49. Guan KL, Rao Y: Signalling mechanisms mediating neuronal responses to guidance cues. Nat Rev Neurosci 2003, 4:941-956.
37. Ethell IM, Irie F, Kalo MS, Couchman JR, Pasquale EB, Yamaguchi Y: EphB/syndecan-2 signaling in dendritic spine morphogenesis. Neuron 2001, 31:1001-1013. 38. Grunwald IC, Korte M, Aldermann G, Plueck A, Kullander K, Adams RH, Frotscher M, Bonhoeffer T, Klein R: Hippocampal plasticity requires postsynaptic ephrinBs. Nat Neurosci 2004, 7:33-40. 39. Henderson JT, Georgiou J, Jia Z, Robertson J, Elowe S, Roder JC, Pawson T: The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function. Neuron 2001, 32:1041-1056. 40. Murai KK, Pasquale EB: Can Eph receptors stimulate the mind? Neuron 2002, 33:159-162. 41. Penzes P, Beeser A, Chernoff J, Schiller MR, Eipper BA, Mains RE, Huganir RL: Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin. Neuron 2003, 37:263-274. 42. Penzes P, Johnson RC, Sattler R, Zhang X, Huganir RL, Kambampati V, Mains RE, Eipper BA: The neuronal Rho-GEF Kalirin-7 interacts with PDZ domain-containing proteins and regulates dendritic morphogenesis. Neuron 2001, 29:229-242. 43. Ma XM, Huang J, Wang Y, Eipper BA, Mains RE: Kalirin, a multifunctional Rho guanine nucleotide exchange factor, is necessary for maintenance of hippocampal pyramidal neuron dendrites and dendritic spines. J Neurosci 2003, 23:10593-10603. 44. Irie F, Yamaguchi Y: EphB receptors regulate dendritic spine development via intersectin, Cdc42 and N-WASP. Nat Neurosci 2002, 5:1117-1118. 45. Soderling SH, Binns KL, Wayman GA, Davee SM, Ong SH, Pawson T, Scott JD: The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Nat Cell Biol 2002, 4:970-975. 46. Yu X, Malenka RC: Beta-catenin is critical for dendritic morphogenesis. Nat Neurosci 2003, 6:1169-1177. This paper presents an elegant series of experiments examining bcatenin signal in dendritic arbor growth in cultured hippocampal neurons.
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47. Smalley MJ, Dale TC: Wnt signalling in mammalian development and cancer. Cancer Metastasis Rev 1999, 18:215-230.
50. Yuan XB, Jin M, Xu X, Song YQ, Wu CP, Poo MM, Duan S: Signalling and crosstalk of Rho GTPases in mediating axon guidance. Nat Cell Biol 2003, 5:38-45. 51. Liu BP, Strittmatter SM: Semaphorin-mediated axonal guidance via Rho-related G proteins. Curr Opin Cell Biol 2001, 13:619-626. 52. Lamprecht R, Farb CR, LeDoux JE: Fear memory formation involves p190 RhoGAP and ROCK proteins through a GRB2-mediated complex. Neuron 2002, 36:727-738. This study demonstrated that inhibition of the p190RhoGAP/Rho kinase (ROCK) pathway in the amygdala of intact animals blocks fear conditioned changes in behavior. This integrative study is one of the first to test the effect of altering GTPase function on behavior. It shows structural changes in neurons mediated by the Rho GTPases are required for normal mechanisms of learning and memory in the brain. 53. Govek E-E, Newey SE, Ackerman CJ, Cross JR, Van Der Veken L, Van Aelst L: The X-linked mental retardation protein oligophrenin-1 is required for dendritic spine morphogenesis. Nat Neurosci 2004, 7:364-372. The authors determined the function of the RhoGAP oligophrenin in regulating spine morphogenesis. By using RNA interference (RNAi) and antisense RNA approaches, the authors demonstrated that knock-down of oligophrenin-1 levels in CA1 pyramidal neurons in hippocampal slices results in a significant decrease in dendritic spine length. This phenotype was recapitulated by expression of a dominant active form of RhoA and was rescued by inhibiting Rho-kinase. These studies show that oligophrenin-1 controls spine structure by signaling through the Rho GTPases. Although several proteins in GTPase signaling pathways are mutant in various types of mental retardations, this study is the first to demonstrate a direct link between spine morphogenesis and altered GTPase signaling through a disease associated gene. 54. Newey SE, Van Aelst L: The Rho GTPases, dendritic stucture and mental retardation. J Neurobiol 2004, in press. 55. Niell CM, Meyer MP, Smith SJ: In vivo imaging of synapse formation on a growing dendritic arbor. Nat Neurosci 2004, 7:254-260. The authors analyze time lapse images of optic tectal neurons in zebrafish that express postsynaptic density protein 95 (PSD95)-GFP to label postsynaptic structures and dsRed to label the entire structure of the dendritic arbor. The authors show that stable dendritic branches contain PSD95, which suggests that synapses stabilize branches. Furthermore, branch additions tend to occur near synaptic sites, which suggests that synaptic inputs promote branch additions.
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Current Opinion in Neurobiology 2004, 14:297–304 This review comes from a themed issue on Signalling mechanisms Edited by Richard L Huganir and S Lawrence Zipursky Available online 20th May 2004 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.05.012 Abbreviations BDNF brain derived neurotrophic factor EphB ephrin B receptor GAP GTPase activating proteins GEF guanylate exchange factor GFP green fluorescent protein GST glutathione S transferase GTP guanine trinucleotide phosphate MB mushroom body NMDA N-methyl-D-aspartate NT neurotrophin PAK P21 activated kinase WASP Wiskott Aldrich syndrome protein Wnt Wingless
Introduction Neurons are highly polarized cells composed of specialized extensions. They have a main axon that typically relays information to other neurons, and dendrites that typically receive inputs from other neurons. The dendrites of neurons are often highly branched and the extent of the arborization of the dendritic tree correlates with the number and distribution of inputs that the neuron can receive and process. Consequently, the mechanisms that govern the development of dendritic arbors have a fundamental impact on the establishment and function of neuronal circuits in the brain. Recent evidence indicates that several different types of activity in the brain affect dendritic arbor development and plasticity and this litwww.sciencedirect.com
erature has been recently reviewed [1,2]. The purpose of this review is to present recent evidence linking the Rho guanine trinucleotide phosphate (GTP)ases with activityinduced dendritic arbor development. Dendritic arbor structure is complex. In typical neurons, primary dendrites extend from the cell body and branch repeatedly to establish a distinctive tree. Some neurons have tiny protrusions or spines, which extend from the branches of the dendrites. For spiny neurons, spines are the sites of the majority of excitatory synaptic inputs, whereas inhibitory synapses typically form on the main shaft of dendritic branches. For non-spiny neurons, excitatory and inhibitory synapses are distributed along dendritic branches. Dendrites also extend filopodia, which might play a role in spine formation. The cytoskeleton of dendritic branches is composed principally of microtubules with a cortex of actin close to the plasma membrane. The cytoskeleton of spines and filopodia is thought to be composed entirely of actin. Defining how neurons acquire their size and shape during development and how learning and experience can influence morphological changes that alter the functional connectivity between presynaptic and postsynaptic cells is a major challenge for modern neuroscientists. Of particular relevance to this review are data from time-lapse imaging studies that have documented the structural changes that occur in dendrites as the arbor becomes more elaborate (Figure 1). Zebrafish and Xenopus laevis tadpoles are relatively transparent at early stages of development, so expression of fluorescent proteins in single neurons allows high-resolution time-lapse images of developing dendritic arbors within the brains of intact animals (reviewed by Cline [1]). These studies have shown that dendritic arbors follow a stereotyped sequence of events during their elaboration: many fine filopodial branches are added to the arbor, but the majority are rapidly retracted. The branches that stabilize extend and become a substrate for further branch addition and stabilization. Therefore, the arbor gradually becomes more complex by iterative branch addition, stabilization, and extension (Figure 1). It is currently believed that the cytoskeleton of newly added branches is entirely composed of actin and that microtubule invasion of the branch accompanies its stabilization, however, the cytoskeletal changes that underlie dendritic arbor development are not well defined. Many environmental cues influence dendritic size, shape, and development. Amongst them, neuronal activity seems to be particularly important for dendritic growth. The neurotrophin family of growth factors and previously Current Opinion in Neurobiology 2004, 14:297–304
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Figure 1
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Activity-dependent dendrite arbor growth. (a) In vivo images of GFP-expressing neurons in the absence and presence of visual stimulation show a stimulus-induced increase in arbor growth rate (top panel). Expression of dominant negative RhoAN19 increases arbor growth rate independent of visual stimulation and occludes stimulus-induced arbor growth. Expression of dominant negative RacN17 or Cdc42N17 blocks stimulus-induced growth, but does not affect the basal growth rate in the absence of visual stimulation. (b) Scheme of Rho GTPase-dependent stages of dendritic arbor growth. Three distinct behaviors are required for dendritic arbor growth. Branches must be added to the arbor and Rac stimulates branch additions. Because the majority of branches are rapidly retracted, an active mechanism must stabilize branches. This stabilization is also Rac-mediated. Finally, stabilized branches extend and these branches then support further branch additions in an iterative process [55]. Branch extension is mediated by a decrease in RhoA activity. High levels of RhoA deter branch stabilization. (c) Schematic signaling pathway activated by visual stimulation. Stimulation increases glutamate receptor activity that increases Rac and Cdc42 activity and decreases RhoA activity. Crosstalk among the GTPases can further affect their activities. Changes in GTPase activities alter actin and microtubule based cytoskeletal dynamics through downstream effectors.
identified axon guidance molecules have also come forward as key regulators of dendritic development and guidance. During recent years, some light has been shed on the mechanisms by which these cues signal to affect dendritic complexity. The Rho GTPases have emerged as key integrators of these environmental cues to regulate the underlying dendritic cytoskeleton. Below, we review the properties of the Rho GTPases and their signaling pathways and then discuss the potential mechanisms by which environmental cues might act through the Rho GTPases to affect dendritic arbor development.
Rho GTPases Genes of the Rho family of small GTPases encode low molecular weight guanine nucleotide binding proteins. These proteins function as molecular switches by cycling between an ‘active’ GTP-bound state and an ‘inactive’ guanine dinucleotide phosphate (GDP)-bound state. Only in their GTP-bound state are the proteins able to Current Opinion in Neurobiology 2004, 14:297–304
interact with downstream molecules that mediate their effects. The ratio of the two forms is regulated by the opposing effects of GTPase activating proteins (GAPs), which increase the intrinsic rate of hydrolysis of bound GTP, and guanine nucleotide exchange factors (GEFs), which enhance the exchange of bound GDP for GTP. The best studied Rho family members, Cdc42 (cell division cycle 42), Rac1 (Ras-related C3 botullinum toxin substrate 1), and RhoA (Ras homologous member A), have been reported to function as key regulators of the actin cytoskeleton and more recently of the microtubule cytoskeleton (reviewed by Van Aelst and D’SouzaSchorey [3]). Given that actin filaments and microtubules provide the structural basis for dendrites, the involvement of the Rho GTPases in mediating dendrite growth and remodeling has been intensively investigated. To date, numerous studies have demonstrated that altering the activity levels of RhoA, Rac1, and Cdc42 GTPases affects various aspects of dendritic development. www.sciencedirect.com
Rho GTPases and activity-dependent dendrite development Van Aelst and Cline 299
Studies using vertebrate and invertebrate model systems have revealed a role for RhoA in regulating dendritic arbor growth. Expression of a constitutively active mutant form of RhoA in hippocampal neurons [4], Xenopus and chick retinal ganglion cells [5,6], Xenopus tectal neurons [7], and Drosophila melanogaster mushroom body (MB) neurons [8] generally resulted in a decrease in dendritic growth. Conversely, removal of Drosophila Rho in individual MB neurons led to dendrite overextension [8], whereas ectopic expression of a dominant negative mutant form of RhoA (RhoAN19) in Xenopus tectal cells resulted in an increase in total dendritic length [7]. Blocking RhoA function in several other systems, however, caused only a mild or no detectable phenotype. For example, in hippocampal neurons, expression of the RhoAN19 dominant negative mutant did not induce any observable phenotype [4]. In this case, a model was proposed in which the Rho signaling pathway is normally repressed under physiological conditions to allow dendritic growth, and is only locally activated when dendritic growth needs be limited.
The Rho GTPases are also involved in dendritic spine formation. Several lines of evidence support a role for Rac1 in both the formation and the maintenance of spines. Cerebellar Purkinje cells of transgenic mice engineered to express the constitutively active Rac mutant, Rac1V12, showed a large number of small supranumerary spines [12]. Consistent with these findings, expression of Rac1V12 in hippocampal or cortical neurons led to overproduction of abnormal spines and membrane ruffling, whereas expression of the dominant negative Rac1N17 mutant caused a reduction in spine density [4,13]. In contrast to Rac1, RhoA appears to block spine formation and maintenance as well as elongation. Activation of RhoA in hippocampal or cortical neurons in organotypic slices resulted in a decrease in spine number and spine length [4,13]. No obvious phenotype was observed when altering Cdc42 activity in these neurons, or in single cell clones of Drosophila MB neurons. However, analyses of mutant Cdc42 clones, which lack Cdc42 in VS neurons, demonstrated a requirement for Cdc42 in maintaining dendritic spine density [10].
Rac and to a lesser extent Cdc42 have emerged as key regulators of dendritic branching and remodeling. In Xenopus, the dendritic arbor complexity of retinal ganglion cells was reduced by expression of dominant negative mutant forms of Rac1 and Cdc42, but not RhoA. Conversely, an activated mutant form of Rac (but not of Cdc42) promoted an increase in dendritic complexity [5]. Time-lapse experiments in Xenopus tectal cells revealed that Rac1 promotes branch addition and stabilization, whereas increased RhoA activity inhibits branch extension [7]. Similar imaging experiments in chick retinal ganglion cells showed an increase in the addition of tertiary branches upon expression of constitutively activated mutant Rac [6]. In Drosophila, MB neurons mutant for all three Rac genes (Rac1, Rac2 and Mtl [Mig-2-like gene]) exhibited a significant reduction in dendritic length and number of branches [9]. Whereas no observable phenotypes were found in single cell clones of MB neurons lacking Cdc42, analyses of mutant Cdc42 clones in vertical system (VS) neurons, which are more complex and stereotyped than MB neurons, demonstrated a requirement for Cdc42 in regulating dendritic branching, guidance, and consistency of axon caliber [10]. In summary, the above studies are generally consistent with a key role for RhoA in controlling dendritic length, and key roles for Rac and Cdc42 in regulating branch dynamics. Importantly, studies in Xenopus tectal neurons demonstrated that crosstalk among Rho GTPases takes place within growing dendritic arbors [11]. This tight cross regulation of GTPase activities, triggered in response to extracellular cues, appears to define the overall complexity of the dendritic tree. In Xenopus tectal neurons, RhoA activity was increased by Rac activation and Cdc42 inhibition, whereas Rac was inhibited by activation of RhoA (see below).
For the large part, the effects of the Rho GTPases on dendritic development are brought about by their actions on the actin and/or microtubule cytoskeleton. For a more comprehensive review on Rho effectors, the reader is referred to other studies [3,14–18].
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Neuronal activity A major challenge has been to define the extracellular cues that trigger the activation of the Rho GTPases to bring about the appropriate cytoskeletal remodeling essential for the development of the complex dendritic tree (Figure 1). Although several studies have demonstrated that synaptic activity affects dendritic arbor development and spine morphogenesis [19–22], the links among afferent input, glutamate receptor activity, and Rho GTPases have only recently been explored. Sin et al. [23] used in vivo time lapse imaging to test whether or not a relatively short period (4 h) of enhanced visual stimulation would affect dendritic arbor development of Xenopus tectal neurons. Images were collected of green fluorescent protein (GFP)-expressing neurons at either 1 h or 4 h intervals over 8 h. During the first 4 h of the imaging protocol, the animals were returned to a dark chamber between imaging sessions. During the second 4 h period, the animals received enhanced visual stimulation. Dendritic arbor growth rate was increased 2.5 fold compared to the rate seen in the absence of visual stimulation. The enhanced growth rate was blocked by N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor blockers, which indicates that glutamatergic synaptic transmission is required for the enhanced arbor growth rate. An analysis of branch dynamics showed that the enhanced visual stimulation increased the rate of Current Opinion in Neurobiology 2004, 14:297–304
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branch additions and stabilizations as well as extension of pre-existing branches [23]. To examine the requirement for Rho GTPases in dendritic arbor development in vivo, Li et al. [7] co-expressed GFP and constitutively active and dominant negative mutant forms of Rac, RhoA, and Cdc42 in optic tectal neurons in the brains of intact animals. We collected time-lapse images of the developing arbor at 2 h intervals over 8 h or at daily intervals over several days. Animals were anesthetized during the brief periods of image collection, but otherwise were awake and receiving normal levels of visual stimulation. These two imaging protocols allowed us to determine whether or not the Rho GTPase activity had an impact on the overall growth of the dendritic arbor, and to determine the effect of the different GTPases on the dynamic branch behaviors that are known to be important for arbor growth. Rac activity and, to a lesser extent, Cdc42 activity promote rapid branch dynamics, specifically branch additions and retractions, whereas RhoA activity is required for extension of branches. As branch addition, stabilization, and extension are distinct cell biological processes that are required for dendritic arbor development, it was interesting to find that they are separately regulated by the different Rho GTPases. Despite the pleasing simplicity of these results, that different GTPases control different structural changes in the developing neuron, numerous reports suggested that there might be crosstalk between the upstream and the downstream effectors of the Rho GTPases. Given that neurons integrate and respond to several extracellular signals during dendritic arbor development, it seemed likely that crosstalk among the GTPases would have an impact on dendrite branch dynamics and growth. To address this question, we developed an in situ version of the popular pulldown assay of GTPase activity, in which tissue sections from the tadpole optic tectum were incubated with the GTPase binding domain of the downstream effectors, p21 activated kinase (PAK), Wiskott Aldrich syndrome protein (WASP), and Rhotekin, fused to glutathione S transferase (GST). Subsequent incubation of the sections with GST antibody then revealed increases in endogenous Rac, Cdc42, and RhoA activity [11]. We expressed constitutively active or dominant negative forms of Rac, Cdc42, or RhoA and tested whether or not changes in the activities of these GTPases increased the activity of endogenous GTPases through crosstalk. These experiments showed that in the intact brain, with all the normally complex interactions that govern dendritic arbor growth, that RhoA decreases Rac activity, whereas Rac activity increases RhoA activity. This suggests a regulatory feedback loop among the GTPases and that the final outcome with respect to morphological changes in the dendrite reflects the balance of activities of the different GTPases. Indeed, our Current Opinion in Neurobiology 2004, 14:297–304
in vivo imaging data showed that exposure to lysophosphatidic acid (LPA), which activates RhoA, counteracts the increased branch dynamics normally seen when expressing active Rac. To determine whether or not glutamate receptor activity regulates the Rho GTPases, Li et al. [11] adapted the in situ GST-binding assay for use in intact brains and tested whether or not optic nerve stimulation altered endogenous GTPase activity in the optic tectum. Optic nerve stimulation increased endogenous Rac activity and decreased endogenous RhoA activity in the tectum. Furthermore, the changes in GTPase activities were downstream of glutamatergic synaptic inputs. This was the first clear demonstration that endogenous GTPase activity is regulated by synaptic input. Because this assay reports the combined activity in presynaptic retinal axon arbors and postsynaptic tectal cell dendrites, it will be important to obtain data on the effect of retinal input activity on endogenous GTPase activities with greater spatial and temporal resolution. A further link between visual input activity and Rho GTPase-mediated dendritic arbor development was provided by Sin et al. [23]. This study demonstrated directly that visual stimulation enhanced dendritic arbor development using a mechanism that required both glutamate receptor activity and Rho GTPase activity. As mentioned above, we first demonstrated that visual stimulation increases the rate of dendritic arbor development [23]. We then found that the visual stimulationinduced arbor development was completely blocked by expression of dominant negative forms of Rac and Cdc42 and by constitutively active RhoA. It appears that endogenous RhoA activity is high in tectal cell dendrites and these levels are decreased downstream of glutamate receptor activation. Glutamate receptor activity also increases Rac and Cdc42 activity, which increase branch dynamics. These data fit well with the initial scheme of how branch dynamics lead to arbor growth. Increased rates of branch additions are triggered by glutamate receptor activity through increases in Rac and Cdc42 activity. Endogenous RhoA activity is low, and this is permissive for increased Rac activity, as our crosstalk studies indicate that RhoA activity decreases Rac activity. The increased dendrite branch additions lead to increased formation of trial connections with highly dynamic axon branches [24,25]. If these trial connections are maintained by virtue of increased adhesion and strengthened synaptic transmission, then the branches will be stabilized. The stabilized branches then extend under conditions of low RhoA activity. Although the molecular link between glutamate receptor activity and regulation of GTPase activity is not yet known, this is clearly an important area of future research. For instance, signal transduction pathways www.sciencedirect.com
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governing growth cone turning behaviors in response to guidance cues might reveal calcium-sensitive regulators of Rho GTPase activity. These same calcium sensitive regulators might be the missing link between calcium influx downstream of glutamate receptor activation and GTPase activity.
Neurotrophins and extracellular signaling molecules During recent years, neurotrophins and several of the well-characterized axon guidance factors including semaphorins, ephrins, and slit have been shown to also affect dendritic outgrowth and spine morphogenesis. More recent evidence also suggests that these extracellular signaling molecules or their signaling pathways are affected by neuronal activity. The underlying mechanisms by which these extrinsic factors exert their effects on dendritic growth and guidance remain largely unknown (see reviews by Wong and Ghosh, Jan and Jan, and Miller and Kaplan [2,26,27]), although accumulating studies that have been performed mainly in the context of axon outgrowth or guidance have revealed links between these factors and the activation of Rho GTPases (see Figure 2). Given the demonstrated role of the Rho GTPases in dendritic arbor development, is quite likely that they also mediate the actions of a variety of extracellular signaling molecules on dendritic outgrowth and guidance. We will therefore discuss some of the signaling pathways that link extracellular signaling molecules to the Rho GTPases with an emphasis on those that have been implicated in dendritic development and are regulated by or affect neuronal activity.
Neurotrophins, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and NT4/5 constitute a family of dimeric secretory proteins that signal through receptor tyrosine kinases, known as trkA, trkB, trkC and the low affinity trkA receptor. Several studies have demonstrated that the neurotrophins influence the development of dendritic arbors and synaptic plasticity [19,28–30]. BDNF is released from neurons in a calcium-dependent manner so its actions are likely to be downstream of neuronal activity. In addition, trkB receptors are trafficked to the cell surface by an activitydependent mechanism [31–33], which indicates that both the extracellular neurotrophin levels and the cell surface receptor levels are increased by neuronal activity. It is likely that activity-dependent mechanisms also control the release of other neurotrophins and the trafficking of their receptors to the cell surface. In a pioneering series of studies, McAllister and Katz [28,29] demonstrated that the neurotrophins, BDNF, NT3, and NT4/5 have differential growth regulating effects on cortical neuronal dendrites. They further demonstrated that the growth regulating effects of applied neurotrophins required NMDA receptor activity. BDNF reportedly enhances glutamatergic synaptic strength by an NMDA receptor mediated mechanism [30]. As synaptic input activity can also promote the elaboration of the dendritic arbor [22,23], through a mechanism requiring Rho GTPases [23], the cooperative interaction between glutamatergic synaptic transmission and BDNF on dendritic arbor growth in cortical neurons could arise by a neurotrophin-mediated increase in
Figure 2
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Intracellular signaling pathways linking neurotrophins and axon guidance factors to the activation of Rho GTPases in the context of axon outgrowth and guidance (see [14,26,49,50,51] and text). Given the recent implication of BDNF, NGF, Sem3A, and slit in dendritic development and/or function and the well-established role of Rho GTPases in these processes, similar molecular links in the context of dendritic development might be uncovered. www.sciencedirect.com
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synaptic strength followed by glutamate receptor mediated effects on the GTPases. Alternatively, NMDA receptor stimulation might increase the numbers of trkB receptors at postsynaptic sites. This, in turn, could increase BDNF signaling and activity of GTPasemediated dendrite growth. The importance of these signaling pathways for human brain function is demonstrated by a recent finding in which a mutation in BDNF in the human population prevents BDNF secretion and leads to poor episodic memory and deficient dendritic arbor development of hippocampal neurons [34]. Numerous studies have followed those of McAllister and Katz. Because cortical neurons have been used to examine the function of the Rho GTPases in dendritic arbor development, it is possible to map the effects of the neurotrophins on those of Rho GTPase-mediated changes in dendritic arbor structure. For instance, in cortical neurons, BDNF is likely to increase arbor complexity by increasing the rate of branch additions and branch stabilizations, which is comparable to the morphological changes seen with expression of Rac [4]. Similarly, insulin-like growth factor appears to promote dendritic arbor development in cortical neurons through a mechanism that might require Cdc42 [35]. The EphB2 receptor kinase is a receptor for B-type ephrins, which are membrane-bound ligands found at excitatory synapses in developing and mature neurons [36]. Recent data suggest that the EphB receptor plays a key part in synaptic development, plasticity, and spine morphogenesis [37–39]. The EphB receptor associates with the NMDA receptor, which might lead to clustering of the receptors at synaptic sites and enhanced NMDA receptor activity [40]. Two recent studies have implicated the Rho GTPases as crucial mediators of the effect of EphB2 on spine morphology. In one study, Penzes et al. [41] demonstrated that trans-synaptic EphrinB–EphB receptor activation resulted in the phosphorylation and recruitment of Kalirin-7 to synapses, as well as the phosphorylation of the serine/threonine kinase, PAK, a downstream effector of Rac and Cdc42. Kalirin-7 is the most prevalent isoform of the dual Rac1/RhoA-GEF Kalirin in the adult rat brain, containing the first Rac1–GEF domain, but lacking the second RhoA GEF domain. Indeed, Kalirin-7 was found to activate Rac1 [42]. Interfering with the function of the EphB receptor, Kalirin, Rac, or Pak eliminated EphrinB-induced spine development of hippocampal neurons [41]. These findings support a model in which activation of the EphB2 receptor induces the recruitment of Kalirin-7 to the synapse, which results in a local activation of Rac1 and its downstream effector Pak. The local activation of these proteins consequently triggers remodeling of the actin cytoskeleton in spines. Notably, ectopic expression of Kalirin-7 in cortical and hippocampal cultured neurons induces the formation of spine-like structures, whereas reducing levels of Current Opinion in Neurobiology 2004, 14:297–304
Kalirin by means of an antisense approach causes a reduction in spine density followed by simplification of the dendritic tree [42,43]. Signaling through the EphB receptor on spine structure (combined with NMDA receptor function) ensures a coordination of structural and functional aspects of synaptic plasticity. In an independent study, Irie and Yamaguchi [44] provided evidence for the involvement of Cdc42 in mediating the effect of EphB2 on dendritic spine development. They isolated intersectin-1, a GEF for Cdc42, together with the Cdc42 effector, N-WASP, in a complex with EphB2. Both N-WASP (by directly interacting with intersectin-1) and EphB2 were found to enhance the GEF activity of intersectin-1, which resulted in increased Cdc42–GTP levels. Notably, the finding that an effector of Rho GTPases can influence the activity of a regulator of these GTPases is not that uncommon (see Soderling et al. [45]). They also observed that dominant negative mutant forms of intersectin-1, N-WASP, and Cdc42 interfered with spine formation in cultured hippocampal neurons [44]. Whether or not these dominant negative forms interfere with EphrinB-induced spine morphology remains to be seen. On the basis of these findings, a model was proposed in which the EphB receptor in a complex with intersectin-1 and N-WASP triggers the activation of Cdc42, which in turn promotes actin polymerization through N-WASP and the Arp2/3 (actin related protein) complex, leading to spherical expansion of spine heads. Such an expansion of spine head size might be coordinated with recruitment of glutamate receptors to the spine and addition of other postsynaptic machinery during synaptic development and plasticity. A recent study by Yu and Malenka [46] has tied together signaling pathways in the regulation of dendritic arbor growth through experiments with the soluble factor Wnt (Wingless), the N-cadherin/b-catenin complex, Rho GTPases, and neuronal activity. Overexpression of bcatenin increased dendritic arbor growth by a mechanism that required the interaction of its binding partner, the transmembrane adhesion protein N-cadherin, and neuronal activity. Wnts are soluble factors that regulate cell proliferation and cell fate [47]. Wnt7b has recently been shown to affect synaptogenesis in the cerebellum [48]. Wnt signaling through the receptor, frizzled, leads to the dissociation of b-catenin from N-cadherin and subsequent enhancement of dendritic arbor growth. The arbor growth mediated by this pathway is positively regulated by neuronal activity, which also leads to a release of Wnt from the neurons, indicating positive feedback between neuronal activity- and Wnt-mediated arbor growth and synapse formation, which in turn results in further Wnt release and enhanced neuronal activity. The groups of Malenka [4,6] and Salinas (pers comm) have begun to investigate the role of Rho GTPases downstream from this multifaceted signaling sequence. It will be www.sciencedirect.com
Rho GTPases and activity-dependent dendrite development Van Aelst and Cline 303
interesting to determine the mechanisms that integrate signals from neuronal activity, N-cadherin-mediated association with other synaptic partners, and Wnt/frizzled activity with the Rho GTPases.
Conclusions Taken together, the above studies reveal molecular links among neuronal activity, Rho GTPases, and signaling through neurotrophins and other extracellular molecules in the control of dendritic arbor development. Future experiments will be essential to define the signal transduction cascades by which these molecules control dendritic growth. As suggested above, these experiments are likely to be guided by the more detailed knowledge available on these pathways in axon growth cone guidance (see Figure 2 and the reviews cited here [14,26,49,50,51]). A second area of important research will be to determine the genetic link among Rho GTPases, behavior [52], and mental retardation [53,54].
Acknowledgements This work was supported by an endowment from the Charles Robertson family (to HT Cline), the National Institutes of Health (to HT Cline, L Van Aelst), and the Dana Foundation (to HT Cline, L Van Aelst).
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