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Spine architecture and synaptic plasticity
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TRENDS in Neurosciences Vol.28 No.4 April 2005
Synaptic Connectivity series
Spine architecture and synaptic plasticity Holly J. Carlisle and Mary B. Kennedy California Institute of Technology, Division of Biology 216-76, Pasadena, CA 91125, USA
Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using liveimaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.
Introduction Most excitatory synapses in the brain are located on dendritic spines – highly specialized subcellular structures, w0.5 mm in diameter and 0.5–2.0 mm in length, that are believed to compartmentalize biochemical responses to activation of individual synapses [1]. The shape of a spine head can change within a few seconds [2] and is determined by the architecture of its actin cytoskeleton [3,4]. The functional significance of rapid regulation of spine shape and the underlying actin scaffold has become evident from the results of many lines of research. It has long been known that several types of mental retardation and cognitive disorders are associated with abnormalities in spine density and morphology [5–7]. However, the fundamental importance of molecules controlling spine morphology for normal cognition was highlighted by Ger Ramakers in a review of genes mutated in nonsyndromic X-linked mental retardation (nsXLMR), which is defined as XLMR accompanied by grossly normal brain development and few or no other symptoms [8]. The pathology of nsXLMR is believed to be limited to mechanisms specifically involved in cognition. Three of the genes discussed by Ramakers encode proteins that regulate Rho family GTPase pathways, key regulators of actin dynamics [8,9]: oligophrenin 1, a RhoA GTPase-activating protein [10]; a-Pix (also known as Cool-2, encoded by ARHGEF6), Corresponding author: Kennedy, M.B. ([email protected]).
a Rho guanine nucleotide exchange factor (GEF); and p21-activated kinase 3 (PAK3). A fourth protein, tetraspanin (also known as TALLA-1, encoded by TM4SF2), is a membrane-spanning protein that interacts with b1 integrins and could thus influence dynamics of the actin cytoskeleton. A more recent review describes additional mutations in genes implicated in XLMR and related to actin regulation [11]. Why is regulation of spine shape and size crucial for processing of information at synapses? Spine formation during development involves the extension of numerous filopodia from dendritic shafts. Some of these filopodia are eventually contacted by a searching growth cone, and the contact matures into a spine synapse [12]. Similarly, stimuli that produce various forms of long-term potentiation (LTP) cause a rapid local increase in extension of filopodia and formation of new spines at the site of stimulation [13,14]. The increase requires activation of NMDA receptors [13] and can be induced by focal application of Ca2C [14]. These studies suggest that formation of new spines, which would require extensive reshaping of the actin cytoskeleton, is a mechanism for some forms of LTP. Persistent changes in spine shape also accompany increased synaptic strength. Many investigators have documented that the volume of a spine head is proportional to its synaptic area, the number of postsynaptic receptors, and the number of presynaptic docked vesicles [15–17]. Thus, the different parts of a spine synapse must grow or shrink in a coordinated manner. One likely mechanism proposed for some forms of LTP is the addition of new AMPA receptors at the postsynaptic site [18]. It follows that such potentiation would require an increase in size of the spine head. Yasunori Hayashi and colleagues recently provided support for this idea using a fluorescence resonance energy transfer (FRET)-based technique to image changes in actin polymerization in response to different patterns of synaptic stimulation [19]. They found that tetanic stimulation that typically induces LTP increased the formation of actin filaments in spines, whereas low-frequency stimulation, which is typically used to induce long-term depression (LTD), increased actin depolymerization. In short, the increase in synaptic strength that occurs during LTP is likely to involve structural changes in spines, whether through expansion of existing spines or through increased connectivity by addition of new spines.
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Regulation of the actin cytoskeleton in spines The actin cytoskeleton is regulated by transmembrane receptors through their actions on Rho family GTPbinding proteins, including RhoA, RhoB, Rac and Cdc42 [20]. Although many pathways converge on these common regulators, evidence that one pathway plays an especially prominent role in spines has emerged in the past few years (Figure 1). The pathway begins with activation of EphB receptors, which activate the Rho GEF kalirin. Kalirin catalyzes activation of Rac, which activates p21-activated kinase (PAK) family members, which in turn activate LIM kinase 1 (LIMK1). LIMK1 phosphorylates cofilin, which inhibits its actin-severing activity and reduces depolymerization of actin filaments. The latest evidence for each step in this pathway will now be considered in turn. EphB receptors EphB receptors are protein tyrosine kinase-linked receptors located at postsynaptic sites [21]. They are activated by contact with membrane-bound ligands termed ephrins (in this case, of the ephrin-B subtype), which are located on axons [22]. Two recent papers have established that signaling through EphB receptors is essential for normal spine formation in the hippocampus. Henkemeyer et al. [23] examined spine formation in EphB1, EphB2 and EphB3 double and triple mutant mice. In the absence of all
Figure 1. EphB and NMDA receptor signaling pathways that regulate actin-binding proteins. Pathways marked with solid lines have been shown to exist in neurons. Dashed lines indicate pathways in which intermediate reactions in neurons are not yet known. The EphB/Rac/PAK pathway is highlighted in yellow and discussed in the text. Receptors, small GTPases, and actin-binding proteins are grouped within the horizontal gray bars. Acronyms: ADF, actin-depolymerizing factor; ARP2/3, actin-related protein 2/3; CaMKII, Ca2C/calmodulin-dependent protein kinase II; LIMK, LIM kinase 1; PAK, p21-activated protein kinase; N-Wasp, neuronal Wiscott– Aldrich syndrome protein; RasGRF, Ras guanine nucleotide-releasing factor. www.sciencedirect.com
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three receptors, spines failed to form in cultured hippocampal neurons and were grossly abnormal in vivo. Interestingly, these triple homozygous mutant mice were nonetheless viable and able to breed. In culture, the neurons did not form postsynaptic specializations, and expression of AMPA-type and NMDA-type glutamate receptor subunits was substantially reduced. In vivo, spine density was reduced to w25% of wild-type levels, although dendritic arborization was normal. Spine heads were reduced in size, suggesting that the average strength of individual synapses was lower than in wild-type neurons. Actin polymerization was not disrupted in vitro or in vivo; however, filamentous actin (F-actin) accumulated in dendritic shafts rather than in spines, indicating that EphB receptors influence targeting of actin to spines. The disruption of normal glutamate receptor accumulation and clustering in these mutants suggests that proper sculpting of the actin cytoskeleton is required for appropriate targeting of glutamate receptors to the synapse. Kalirin-7 Work from Penzes et al. [24,25] sheds light on the mechanisms by which EphB receptors influence actin filaments. Treatment of neurons with clustered ephrin-B1 causes clustering and activation of postsynaptic EphB receptors. This, in turn, catalyzes tyrosine phosphorylation of the Rho GEF kalirin-7 and its redistribution into synaptic puncta [25]. Kalirin-7 is an isoform of the kalirin family of Rho GEFs that contains a single Rac1 GEF domain. When kalirin-7 is brought into contact with Rac1-GDP, it catalyzes exchange of the GDP for GTP, which activates Rac1. Kalirin-7 is concentrated in the postsynaptic density (PSD), where it can bind to the PDZ domains of several scaffold proteins, including PSD-95 and multiple-PDZ-domain-containing protein 1 (MUPP-1) [24,26]. Overexpression of either a GEF-inactive mutant of kalirin-7 or a dominant-negative form of Rac1 blocked the effects of ephrin-B ligands on spine formation [25]. Thus, activation of Rac1 by kalirin-7, triggered by activation of EphB receptors, is necessary for spine formation in cultured hippocampal neurons. PAKs Penzes et al. [25] went on to identify PAK proteins as major effectors in spines that are activated by Rac1 following stimulation of EphB receptors. PAKs are a family of closely related serine/threonine protein kinases that were discovered relatively recently in a screen for Rho-GTP binding partners [27,28]. In the inactive state, PAKs form homodimers in which the regulatory domain of one PAK molecule inhibits the catalytic domain of another. Binding of GTP-bound Cdc42 or Rac1 to PAK causes conformational changes that destabilize folding of the inhibitory switch domain and facilitate catalytic activation [28]. Phosphorylation of Thr423 in the PAK catalytic domain is required to suppress autoinhibition and achieve full catalytic function. Studies by King et al. [29] indicate that Thr423 is probably phosphorylated by an exogenous kinase, such as 3-phosphoinositide-dependent kinase 1 (PDK1), because autophosphorylation at
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this site is inefficient. In fact, PAKs appear to be activated by several mechanisms, some of which are still being investigated [28]. It is clear that PAKs have a crucial role in regulation of spine formation. PAKs phosphorylated at Thr423 (pPAKs), in particular pPAK1 and pPAK3, are concentrated in dendritic spines [25,30] and enriched in the PSD fraction [30]. Penzes et al. [25] treated cultured hippocampal neurons with an inhibitory peptide consisting of the PAK1 inhibitory domain fused to the cell-penetrating peptide TAT, and found that the treatment reduced the effect of ephrin-B ligands on spine formation. More recently, Hayashi et al. [30] produced transgenic mice expressing the peptide inhibitor of PAK in forebrain neurons under the control of the a Ca2C/calmodulindependent protein kinase II (a-CaMKII) promoter. By four weeks after birth, the level of pPAK in the PSD fractions from both cortex and hippocampus of transgenic mice was reduced to w60% of wild type. Spine morphology was significantly altered in these animals; spine density was reduced and the average size of spine heads increased. However, effects on spine morphology were observed only in cortical neurons, not in hippocampal neurons. The authors suggest that this difference results from the fact that the amount of pPAK in PSDs from wildtype mice is twofold lower in cortex than in hippocampus. Thus, the PAK activity associated with PSDs in the hippocampus of the transgenic mice is reduced to approximately the wild-type cortical level. Therefore, PAK activity in the PSDs in hippocampus might not have fallen below a critical threshold level. In any case, the study supports an important role for PAKs in determining spine morphology in the cortex. Furthermore, the mice were deficient in consolidation of memories, as would be expected if synaptic plasticity in the cortex was disrupted. LIMK1 How do the PAKs influence spine morphology? PAKs have many potential downstream regulatory targets. One target likely to mediate effects on the actin cytoskeleton is the enzyme LIMK, which phosphorylates and inactivates ADF/cofilin (Figure 1). LIMK1 is a serine/threonine kinase that contains two LIM domains and a PDZ domain [31] and is enriched in hippocampal pyramidal neurons [32]. Activated PAK forms a complex with LIMK1 and activates it by phosphorylating Thr508 in the catalytic domain [33]. The activated LIMK1 then phosphorylates Ser3 of ADF/cofilin, inhibiting its activity [34,35], and completing the link between EphB and the actin cytoskeleton (Figure 1). LIMK signaling has received a tremendous amount of attention since it was identified as being encoded by one of the genes heterozygously deleted in Williams syndrome (WS). Patients with this syndrome suffer from a variety of abnormalities, including cognitive deficits resulting in a low IQ (ranging from 40 to 79) [36]. Identification of patients with partial deletions of the WS chromosomal region that result in a partial set of WS features has allowed researchers to assign functions to some of the deleted genes. In one report, two patients with minor www.sciencedirect.com
hemizygous deletions that included LIMK1 suffered from cognitive deficits [37] (but see Ref. [38]). Interestingly, mice with a homozygous deletion of LIMK1 have behavioral abnormalities that are perhaps analogous to those observed in WS patients. LIMK1K/K mice display heightened locomotor activity and impaired spatial learning [39]. They also demonstrate a role for LIMK1 in the regulation of spine morphology through cofilin. Golgi-staining of neurons in brain sections from LIMK1K/K mice reveal abnormal spine morphology compared with wild type: spine head size is dramatically decreased and spine necks are thicker. Furthermore, cofilin is significantly less phosphorylated in the mutant than in wild-type brains. These data are consistent with a model in which phosphorylation of cofilin by LIMK1 stabilizes F-actin in the spine, permitting formation of larger spine heads. Remodeling of the actin cytoskeleton by activity Observation of green fluorescent protein (GFP)-labeled actin at synapses [40] immediately following stimulation revealed that clusters of actin undergo a rapid expansion within the spine followed either by return to their original size or by long-lasting expansion, depending on the number of tetani delivered to the neuron. Fukazawa et al. [41] reported that LTP-inducing stimuli in the intact hippocampus increase the F-actin content in spines as early as 20 min after stimulation. The simplest way to visualize remodeling of spine heads involves an initial disassembly of existing stable actin filament networks, followed by assembly and stabilization of a new, reshaped network. However, a recent paper suggests a somewhat different model. Star et al. [42], using fluorescence recovery after photobleaching, made the surprising discovery that 85% of actin in spines of cultured neurons is rapidly cycling onto and off filaments, with an average turnover time of 44 s. They found that the proportion of treadmilling actin does not correlate with the size or shape of spines, and that low-frequency stimulation that activated NMDA receptors stabilized about half of the treadmilling actin. Thus, it appears that spines in which NMDA receptors have not been recently activated contain rapidly treadmilling actin. Activation of NMDA receptors by lowfrequency or high-frequency stimulation leads to decreased treadmilling and stabilization of actin filaments. Activity-induced alteration in actin dynamics is at least partly controlled by regulation of the actin binding proteins (Box 1). For example, mutation of the Ca2Cactivated actin-capping protein gelsolin impairs the stabilization observed by Star et al. [42]. Fukazawa et al. [41] showed enhanced phosphorylation and inactivation of the filament-severing protein ADF/cofilin after their stimulation paradigm. Finally, Ackermann and Matus [43] showed that newly expressed GFP-labeled profilin, which catalyzes the exchange of ADP for ATP on actin monomers, redistributed from being diffusely localized throughout dendrites to highly concentrated within spines as soon as 5 min after activation of NMDA receptors. They found that a peptide that blocks association of profilin with polyproline-rich docking sites on proteins nearly completely blocks the redistribution of profilin to spines.
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Box 1. Regulation of actin dynamics Filamentous actin (F-actin) is a double-stranded chain-like structure of monomeric actin subunits (G-actin) that grows in a polarized fashion (Figure I). F-actin is in a continuous state of turnover, with new subunits added to the barbed end of the filament and older subunits removed from the pointed end in a treadmill process [61]. Growth of filaments depends on the availability of actin monomers with bound ATP (actin-ATP). When subunits are incorporated into filaments, slow hydrolysis of actin-ATP to actin-ADP is catalyzed, which stabilizes the filamentous form. A variety of actin-binding proteins regulate and shape the treadmilling process, and thereby the cytoskeleton. The F-actin-severing protein ADF/cofilin binds to actin monomers with a higher affinity for actin-ADP than for actin-ATP. Binding of ADF/cofilin to actin-ADP changes the twist in the actin helix and promotes severing [62–64]. In this manner, ADF/cofilin catalyzes disassembly of the older, pointed ends of actin filaments. Phosphorylation of ADF/cofilin at Ser3 inhibits its ability to bind to actin [65]. Thus, depolymerization of actin can be regulated by cytosolic signaling pathways. Profilin stabilizes actin filaments by catalyzing exchange of ADP for ATP on actin monomers, maintaining a pool of actin-ATP monomers that can be added to the growing end of filaments [66]. In addition, profilin potentiates treadmilling by ‘sequestering’ actin monomers in a form that can bind to the barbed end, but not the pointed end, of a filament [67]. The rate of filament growth is also controlled by capping proteins such as gelsolin, which sever existing filaments and block addition of actin monomers to the barbed end [68,69]. Nucleation of new actin filaments, formation of branches on actin filaments, and cross-linking of filaments into networks are achieved through the highly regulated ARP2/3 complex. Activated ARP2/3 acts as a template for assembly of new filaments and caps the pointed ends of F-actin, allowing elongation of the barbed end. It can also initiate branching points on existing filaments. ARP2/3 can be activated by SCAR/WAVE, WASP, VASP or cortactin [54]. Binding of these proteins to ARP2/3 might also anchor the actin cytoskeleton at specific cellular locations [70,71].
Figure I. Control of actin assembly and disassembly (based on a figure from Ref. [61]). The growing (or ‘barbed’) end of the filament is extended by addition of actin-ATP (T, red) to the existing filament; cofilin severs actin-ADP (D, light blue) from the older ‘pointed’ end.
This finding implies that the redistribution requires association of profilin with polyproline-containing proteins, such as VASP or other scaffold proteins, within the spines. Much remains to be learned about how these complex changes in the spine cytoskeleton are coordinated after synaptic stimulation and how they contribute to changes in synaptic efficacy. A complete understanding will undoubtedly require quantitative methods for studying shifts in cytoskeletal equilibria in dendritic spines. Synaptic activity can increase the formation of new spines, in addition to changing the shape of existing spines [44]. Jourdain et al. found that CaMKII has a role in formation of new spines concomitant with induction of some forms of LTP. They used time-lapse two-photon confocal microscopy of organotypic slice cultures to monitor spines on CA1 hippocampal pyramidal neurons filled with fluorescent dye. When they also filled the neurons with activated CaMKII, they observed an order of magnitude increase in the number of new spines formed over a period of 60 min compared with the number in control neurons filled with heat-inactivated CaMKII. At the same time, they measured an increase in the amplitude of excitatory postsynaptic potentials in the www.sciencedirect.com
neurons that contained active CaMKII, consistent with previous studies [45]. They were able to block formation of new spines with either an NMDA receptor antagonist or the CaMKII inhibitor KN93. Role of scaffold proteins in shaping the actin cytoskeleton in spines Scaffold proteins that localize signaling components in discrete locations have significant roles in organizing specialized functions of all cells, including neurons. Several scaffold proteins interact with actin or actinbinding proteins in spines. However, we are only beginning to understand their importance in coordinating and shaping spine architecture. GIT1 Work in motile fibroblasts indicates that, although inactive Rac is diffusely distributed throughout the cell, activated Rac is discretely localized [46], suggesting that Rac activators are tethered to certain locations. G-proteincoupled receptor kinase-interacting protein (GIT1) targets key regulators of the actin cytoskeleton to spines. Two important GIT1 binding partners are PIX (a Rac GEF) and
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PAK proteins [47–49]. When Zhang et al. [50] disrupted the synaptic localization of GIT1, by overexpressing dominant-negative mutant GIT1, they observed a significant increase in the number of dendritic protrusions and a decrease in the number of synapses and mushroomshaped spines. They recapitulated this phenotype by overexpressing constitutively active Rac, constitutively active PIX, or PIX that is unable to bind GIT. They hypothesize that untethered PIX can activate Rac throughout the cell, causing formation of aberrant protrusions. Likewise, overexpression of constitutively activated PIX or activated Rac would cause a similar phenotype owing to promiscuous Rac activity along the dendritic shaft. Intersectin Intersectin is a multi-domain scaffold protein that interacts with several signaling proteins. A long splice variant of intersectin that contains a Dbl domain with GEF activity for Cdc42 is expressed specifically in neurons [51,52]. Activated EphB receptors physically associate with intersectin, resulting in formation of a complex containing intersectin, Cdc42 and N-WASP, an ARP2/3 activator [53]. Within the complex, N-WASP upregulates the GEF activity of intersectin for Cdc42 [52], catalyzing the formation of Cdc42-GTP. Cdc42-GTP in turn activates N-WASP, which then increases the affinity of ARP2/3 for ATP [54]. The ability of ARP2/3 to nucleate new filaments or initiate branching of an existing filament is widely believed to require ATP hydrolysis [54]. Proteins such as GIT1 and intersectin control local formation and branching of actin filaments. Other PSD scaffold proteins, such as shank [55] and densin [56], that associate with actin and actin-filament-binding proteins might also have a role in shaping the interaction of the spine membrane with the actin cytoskeleton, but we have only begun to discern how this might be orchestrated. Outstanding questions Activation of Ras and its signaling pathways by Ca2C flux through NMDA receptors, and activation of Rac and its pathways through Eph receptors, are well documented. However, very little is known about how NMDA receptors influence Rho family GTPases, including Rac, Cdc42 and RhoA, to regulate activity-dependent spine morphogenesis. Additional studies will undoubtedly shed light on whether the Rho family can be activated directly by Ca2C influx or whether it is activated by crosstalk from Ras, as has been described in other systems [57,58]. A recent study by Vazquez et al. [59] shows that homozygous knockout of synGAP, a synaptic Ras and Rap [60] GTPaseactivating protein that is regulated by NMDA-receptordependent phosphorylation by CaMKII, results in the precocious formation of abnormally large spines in cultured hippocampal neurons. This could be an example in which indirect activation of Rho family GTPases is triggered by abnormally high Ras or Rap activity. Clearly, the size and morphology of spines, and the arrangement of signaling molecules within the spine head, are tightly regulated and crucial for correct information processing and storage in neural networks. www.sciencedirect.com
Studies of the regulating mechanisms will generate a wealth of interesting new information about brain function and could shed light on the etiology of mental retardation. Acknowledgements We thank members of the Kennedy laboratory, in particular Holly Beale, Edoardo Marcora and Luis Vazquez for helpful discussions. This work was supported by National Institutes of Health Grants 1F32 NS047894 (H.J.C.) and R01 NS28710 (M.B.K).
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47 Manabe, R. et al. (2002) GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration. J. Cell Sci. 115, 1497–1510 48 Di Cesare, A. et al. (2000) p95-APP1 links membrane transport to Racmediated reorganization of actin. Nat. Cell Biol. 2, 521–530 49 de Curtis, I. (2001) Cell migration: GAPs between membrane traffic and the cytoskeleton. EMBO Rep. 2, 277–281 50 Zhang, H. et al. (2003) Synapse formation is regulated by the signaling adaptor GIT1. J. Cell Biol. 161, 131–142 51 Hussain, N.K. et al. (1999) Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells. J. Biol. Chem. 274, 15671–15677 52 Hussain, N.K. et al. (2001) Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat. Cell Biol. 3, 927–932 53 Irie, F. and Yamaguchi, Y. (2002) EphB receptors regulate dendritic spine development via intersectin, Cdc42 and N-WASP. Nat. Neurosci. 5, 1117–1118 54 Millard, T.H. et al. (2004) Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex. Biochem. J. 380, 1–17 55 Sheng, M. and Kim, E. (2000) The Shank family of scaffold proteins. J. Cell Sci. 113, 1851–1856 56 Walikonis, R.S. et al. (2001) Densin-180 forms a ternary complex with the a-subunit of CaMKII and a-actinin. J. Neurosci. 21, 423–433 57 Hall, A. (1992) Ras-related GTPases and the cytoskeleton. Mol. Biol. Cell 3, 475–479 58 Nimnual, A.S. et al. (1998) Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 279, 560–563 59 Vazquez, L.E. et al. (2004) SynGAP regulates spine formation. J. Neurosci. 24, 8796–8805 60 Krapivinsky, G. et al. (2004) SynGAP–MUPP1–CaMKII synaptic complexes regulate p38 MAP kinase activity and NMDA receptordependent synaptic AMPA receptor potentiation. Neuron 43, 563–574 61 Pollard, T.D. and Borisy, G.G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 62 Maciver, S.K. (1998) How ADF/cofilin depolymerizes actin filaments. Curr. Opin. Cell Biol. 10, 140–144 63 McGough, A. et al. (1997) Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function. J. Cell Biol. 138, 771–781 64 Blanchoin, L. and Pollard, T.D. (1999) Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments. J. Biol. Chem. 274, 15538–15546 65 Agnew, B.J. et al. (1995) Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. J. Biol. Chem. 270, 17582–17587 66 Wolven, A.K. et al. (2000) In vivo importance of actin nucleotide exchange catalyzed by profilin. J. Cell Biol. 150, 895–904 67 Didry, D. et al. (1998) Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover. J. Biol. Chem. 273, 25602–25611 68 Wear, M.A. et al. (2003) How capping protein binds the barbed end of the actin filament. Curr. Biol. 13, 1531–1537 69 McGough, A.M. et al. (2003) The gelsolin family of actin regulatory proteins: modular structures, versatile functions. FEBS Lett. 552, 75–81 70 Martinez, M.C. et al. (2003) Dual regulation of neuronal morphogenesis by a d-catenin–cortactin complex and Rho. J. Cell Biol. 162, 99–111 71 Hering, H. and Sheng, M. (2003) Activity-dependent redistribution and essential role of cortactin in dendritic spine morphogenesis. J. Neurosci. 23, 11759–11769
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Synaptic Connectivity series
Spine architecture and synaptic plasticity Holly J. Carlisle and Mary B. Kennedy California Institute of Technology, Division of Biology 216-76, Pasadena, CA 91125, USA
Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using liveimaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.
Introduction Most excitatory synapses in the brain are located on dendritic spines – highly specialized subcellular structures, w0.5 mm in diameter and 0.5–2.0 mm in length, that are believed to compartmentalize biochemical responses to activation of individual synapses [1]. The shape of a spine head can change within a few seconds [2] and is determined by the architecture of its actin cytoskeleton [3,4]. The functional significance of rapid regulation of spine shape and the underlying actin scaffold has become evident from the results of many lines of research. It has long been known that several types of mental retardation and cognitive disorders are associated with abnormalities in spine density and morphology [5–7]. However, the fundamental importance of molecules controlling spine morphology for normal cognition was highlighted by Ger Ramakers in a review of genes mutated in nonsyndromic X-linked mental retardation (nsXLMR), which is defined as XLMR accompanied by grossly normal brain development and few or no other symptoms [8]. The pathology of nsXLMR is believed to be limited to mechanisms specifically involved in cognition. Three of the genes discussed by Ramakers encode proteins that regulate Rho family GTPase pathways, key regulators of actin dynamics [8,9]: oligophrenin 1, a RhoA GTPase-activating protein [10]; a-Pix (also known as Cool-2, encoded by ARHGEF6), Corresponding author: Kennedy, M.B. ([email protected]).
a Rho guanine nucleotide exchange factor (GEF); and p21-activated kinase 3 (PAK3). A fourth protein, tetraspanin (also known as TALLA-1, encoded by TM4SF2), is a membrane-spanning protein that interacts with b1 integrins and could thus influence dynamics of the actin cytoskeleton. A more recent review describes additional mutations in genes implicated in XLMR and related to actin regulation [11]. Why is regulation of spine shape and size crucial for processing of information at synapses? Spine formation during development involves the extension of numerous filopodia from dendritic shafts. Some of these filopodia are eventually contacted by a searching growth cone, and the contact matures into a spine synapse [12]. Similarly, stimuli that produce various forms of long-term potentiation (LTP) cause a rapid local increase in extension of filopodia and formation of new spines at the site of stimulation [13,14]. The increase requires activation of NMDA receptors [13] and can be induced by focal application of Ca2C [14]. These studies suggest that formation of new spines, which would require extensive reshaping of the actin cytoskeleton, is a mechanism for some forms of LTP. Persistent changes in spine shape also accompany increased synaptic strength. Many investigators have documented that the volume of a spine head is proportional to its synaptic area, the number of postsynaptic receptors, and the number of presynaptic docked vesicles [15–17]. Thus, the different parts of a spine synapse must grow or shrink in a coordinated manner. One likely mechanism proposed for some forms of LTP is the addition of new AMPA receptors at the postsynaptic site [18]. It follows that such potentiation would require an increase in size of the spine head. Yasunori Hayashi and colleagues recently provided support for this idea using a fluorescence resonance energy transfer (FRET)-based technique to image changes in actin polymerization in response to different patterns of synaptic stimulation [19]. They found that tetanic stimulation that typically induces LTP increased the formation of actin filaments in spines, whereas low-frequency stimulation, which is typically used to induce long-term depression (LTD), increased actin depolymerization. In short, the increase in synaptic strength that occurs during LTP is likely to involve structural changes in spines, whether through expansion of existing spines or through increased connectivity by addition of new spines.
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Regulation of the actin cytoskeleton in spines The actin cytoskeleton is regulated by transmembrane receptors through their actions on Rho family GTPbinding proteins, including RhoA, RhoB, Rac and Cdc42 [20]. Although many pathways converge on these common regulators, evidence that one pathway plays an especially prominent role in spines has emerged in the past few years (Figure 1). The pathway begins with activation of EphB receptors, which activate the Rho GEF kalirin. Kalirin catalyzes activation of Rac, which activates p21-activated kinase (PAK) family members, which in turn activate LIM kinase 1 (LIMK1). LIMK1 phosphorylates cofilin, which inhibits its actin-severing activity and reduces depolymerization of actin filaments. The latest evidence for each step in this pathway will now be considered in turn. EphB receptors EphB receptors are protein tyrosine kinase-linked receptors located at postsynaptic sites [21]. They are activated by contact with membrane-bound ligands termed ephrins (in this case, of the ephrin-B subtype), which are located on axons [22]. Two recent papers have established that signaling through EphB receptors is essential for normal spine formation in the hippocampus. Henkemeyer et al. [23] examined spine formation in EphB1, EphB2 and EphB3 double and triple mutant mice. In the absence of all
Figure 1. EphB and NMDA receptor signaling pathways that regulate actin-binding proteins. Pathways marked with solid lines have been shown to exist in neurons. Dashed lines indicate pathways in which intermediate reactions in neurons are not yet known. The EphB/Rac/PAK pathway is highlighted in yellow and discussed in the text. Receptors, small GTPases, and actin-binding proteins are grouped within the horizontal gray bars. Acronyms: ADF, actin-depolymerizing factor; ARP2/3, actin-related protein 2/3; CaMKII, Ca2C/calmodulin-dependent protein kinase II; LIMK, LIM kinase 1; PAK, p21-activated protein kinase; N-Wasp, neuronal Wiscott– Aldrich syndrome protein; RasGRF, Ras guanine nucleotide-releasing factor. www.sciencedirect.com
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three receptors, spines failed to form in cultured hippocampal neurons and were grossly abnormal in vivo. Interestingly, these triple homozygous mutant mice were nonetheless viable and able to breed. In culture, the neurons did not form postsynaptic specializations, and expression of AMPA-type and NMDA-type glutamate receptor subunits was substantially reduced. In vivo, spine density was reduced to w25% of wild-type levels, although dendritic arborization was normal. Spine heads were reduced in size, suggesting that the average strength of individual synapses was lower than in wild-type neurons. Actin polymerization was not disrupted in vitro or in vivo; however, filamentous actin (F-actin) accumulated in dendritic shafts rather than in spines, indicating that EphB receptors influence targeting of actin to spines. The disruption of normal glutamate receptor accumulation and clustering in these mutants suggests that proper sculpting of the actin cytoskeleton is required for appropriate targeting of glutamate receptors to the synapse. Kalirin-7 Work from Penzes et al. [24,25] sheds light on the mechanisms by which EphB receptors influence actin filaments. Treatment of neurons with clustered ephrin-B1 causes clustering and activation of postsynaptic EphB receptors. This, in turn, catalyzes tyrosine phosphorylation of the Rho GEF kalirin-7 and its redistribution into synaptic puncta [25]. Kalirin-7 is an isoform of the kalirin family of Rho GEFs that contains a single Rac1 GEF domain. When kalirin-7 is brought into contact with Rac1-GDP, it catalyzes exchange of the GDP for GTP, which activates Rac1. Kalirin-7 is concentrated in the postsynaptic density (PSD), where it can bind to the PDZ domains of several scaffold proteins, including PSD-95 and multiple-PDZ-domain-containing protein 1 (MUPP-1) [24,26]. Overexpression of either a GEF-inactive mutant of kalirin-7 or a dominant-negative form of Rac1 blocked the effects of ephrin-B ligands on spine formation [25]. Thus, activation of Rac1 by kalirin-7, triggered by activation of EphB receptors, is necessary for spine formation in cultured hippocampal neurons. PAKs Penzes et al. [25] went on to identify PAK proteins as major effectors in spines that are activated by Rac1 following stimulation of EphB receptors. PAKs are a family of closely related serine/threonine protein kinases that were discovered relatively recently in a screen for Rho-GTP binding partners [27,28]. In the inactive state, PAKs form homodimers in which the regulatory domain of one PAK molecule inhibits the catalytic domain of another. Binding of GTP-bound Cdc42 or Rac1 to PAK causes conformational changes that destabilize folding of the inhibitory switch domain and facilitate catalytic activation [28]. Phosphorylation of Thr423 in the PAK catalytic domain is required to suppress autoinhibition and achieve full catalytic function. Studies by King et al. [29] indicate that Thr423 is probably phosphorylated by an exogenous kinase, such as 3-phosphoinositide-dependent kinase 1 (PDK1), because autophosphorylation at
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this site is inefficient. In fact, PAKs appear to be activated by several mechanisms, some of which are still being investigated [28]. It is clear that PAKs have a crucial role in regulation of spine formation. PAKs phosphorylated at Thr423 (pPAKs), in particular pPAK1 and pPAK3, are concentrated in dendritic spines [25,30] and enriched in the PSD fraction [30]. Penzes et al. [25] treated cultured hippocampal neurons with an inhibitory peptide consisting of the PAK1 inhibitory domain fused to the cell-penetrating peptide TAT, and found that the treatment reduced the effect of ephrin-B ligands on spine formation. More recently, Hayashi et al. [30] produced transgenic mice expressing the peptide inhibitor of PAK in forebrain neurons under the control of the a Ca2C/calmodulindependent protein kinase II (a-CaMKII) promoter. By four weeks after birth, the level of pPAK in the PSD fractions from both cortex and hippocampus of transgenic mice was reduced to w60% of wild type. Spine morphology was significantly altered in these animals; spine density was reduced and the average size of spine heads increased. However, effects on spine morphology were observed only in cortical neurons, not in hippocampal neurons. The authors suggest that this difference results from the fact that the amount of pPAK in PSDs from wildtype mice is twofold lower in cortex than in hippocampus. Thus, the PAK activity associated with PSDs in the hippocampus of the transgenic mice is reduced to approximately the wild-type cortical level. Therefore, PAK activity in the PSDs in hippocampus might not have fallen below a critical threshold level. In any case, the study supports an important role for PAKs in determining spine morphology in the cortex. Furthermore, the mice were deficient in consolidation of memories, as would be expected if synaptic plasticity in the cortex was disrupted. LIMK1 How do the PAKs influence spine morphology? PAKs have many potential downstream regulatory targets. One target likely to mediate effects on the actin cytoskeleton is the enzyme LIMK, which phosphorylates and inactivates ADF/cofilin (Figure 1). LIMK1 is a serine/threonine kinase that contains two LIM domains and a PDZ domain [31] and is enriched in hippocampal pyramidal neurons [32]. Activated PAK forms a complex with LIMK1 and activates it by phosphorylating Thr508 in the catalytic domain [33]. The activated LIMK1 then phosphorylates Ser3 of ADF/cofilin, inhibiting its activity [34,35], and completing the link between EphB and the actin cytoskeleton (Figure 1). LIMK signaling has received a tremendous amount of attention since it was identified as being encoded by one of the genes heterozygously deleted in Williams syndrome (WS). Patients with this syndrome suffer from a variety of abnormalities, including cognitive deficits resulting in a low IQ (ranging from 40 to 79) [36]. Identification of patients with partial deletions of the WS chromosomal region that result in a partial set of WS features has allowed researchers to assign functions to some of the deleted genes. In one report, two patients with minor www.sciencedirect.com
hemizygous deletions that included LIMK1 suffered from cognitive deficits [37] (but see Ref. [38]). Interestingly, mice with a homozygous deletion of LIMK1 have behavioral abnormalities that are perhaps analogous to those observed in WS patients. LIMK1K/K mice display heightened locomotor activity and impaired spatial learning [39]. They also demonstrate a role for LIMK1 in the regulation of spine morphology through cofilin. Golgi-staining of neurons in brain sections from LIMK1K/K mice reveal abnormal spine morphology compared with wild type: spine head size is dramatically decreased and spine necks are thicker. Furthermore, cofilin is significantly less phosphorylated in the mutant than in wild-type brains. These data are consistent with a model in which phosphorylation of cofilin by LIMK1 stabilizes F-actin in the spine, permitting formation of larger spine heads. Remodeling of the actin cytoskeleton by activity Observation of green fluorescent protein (GFP)-labeled actin at synapses [40] immediately following stimulation revealed that clusters of actin undergo a rapid expansion within the spine followed either by return to their original size or by long-lasting expansion, depending on the number of tetani delivered to the neuron. Fukazawa et al. [41] reported that LTP-inducing stimuli in the intact hippocampus increase the F-actin content in spines as early as 20 min after stimulation. The simplest way to visualize remodeling of spine heads involves an initial disassembly of existing stable actin filament networks, followed by assembly and stabilization of a new, reshaped network. However, a recent paper suggests a somewhat different model. Star et al. [42], using fluorescence recovery after photobleaching, made the surprising discovery that 85% of actin in spines of cultured neurons is rapidly cycling onto and off filaments, with an average turnover time of 44 s. They found that the proportion of treadmilling actin does not correlate with the size or shape of spines, and that low-frequency stimulation that activated NMDA receptors stabilized about half of the treadmilling actin. Thus, it appears that spines in which NMDA receptors have not been recently activated contain rapidly treadmilling actin. Activation of NMDA receptors by lowfrequency or high-frequency stimulation leads to decreased treadmilling and stabilization of actin filaments. Activity-induced alteration in actin dynamics is at least partly controlled by regulation of the actin binding proteins (Box 1). For example, mutation of the Ca2Cactivated actin-capping protein gelsolin impairs the stabilization observed by Star et al. [42]. Fukazawa et al. [41] showed enhanced phosphorylation and inactivation of the filament-severing protein ADF/cofilin after their stimulation paradigm. Finally, Ackermann and Matus [43] showed that newly expressed GFP-labeled profilin, which catalyzes the exchange of ADP for ATP on actin monomers, redistributed from being diffusely localized throughout dendrites to highly concentrated within spines as soon as 5 min after activation of NMDA receptors. They found that a peptide that blocks association of profilin with polyproline-rich docking sites on proteins nearly completely blocks the redistribution of profilin to spines.
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Box 1. Regulation of actin dynamics Filamentous actin (F-actin) is a double-stranded chain-like structure of monomeric actin subunits (G-actin) that grows in a polarized fashion (Figure I). F-actin is in a continuous state of turnover, with new subunits added to the barbed end of the filament and older subunits removed from the pointed end in a treadmill process [61]. Growth of filaments depends on the availability of actin monomers with bound ATP (actin-ATP). When subunits are incorporated into filaments, slow hydrolysis of actin-ATP to actin-ADP is catalyzed, which stabilizes the filamentous form. A variety of actin-binding proteins regulate and shape the treadmilling process, and thereby the cytoskeleton. The F-actin-severing protein ADF/cofilin binds to actin monomers with a higher affinity for actin-ADP than for actin-ATP. Binding of ADF/cofilin to actin-ADP changes the twist in the actin helix and promotes severing [62–64]. In this manner, ADF/cofilin catalyzes disassembly of the older, pointed ends of actin filaments. Phosphorylation of ADF/cofilin at Ser3 inhibits its ability to bind to actin [65]. Thus, depolymerization of actin can be regulated by cytosolic signaling pathways. Profilin stabilizes actin filaments by catalyzing exchange of ADP for ATP on actin monomers, maintaining a pool of actin-ATP monomers that can be added to the growing end of filaments [66]. In addition, profilin potentiates treadmilling by ‘sequestering’ actin monomers in a form that can bind to the barbed end, but not the pointed end, of a filament [67]. The rate of filament growth is also controlled by capping proteins such as gelsolin, which sever existing filaments and block addition of actin monomers to the barbed end [68,69]. Nucleation of new actin filaments, formation of branches on actin filaments, and cross-linking of filaments into networks are achieved through the highly regulated ARP2/3 complex. Activated ARP2/3 acts as a template for assembly of new filaments and caps the pointed ends of F-actin, allowing elongation of the barbed end. It can also initiate branching points on existing filaments. ARP2/3 can be activated by SCAR/WAVE, WASP, VASP or cortactin [54]. Binding of these proteins to ARP2/3 might also anchor the actin cytoskeleton at specific cellular locations [70,71].
Figure I. Control of actin assembly and disassembly (based on a figure from Ref. [61]). The growing (or ‘barbed’) end of the filament is extended by addition of actin-ATP (T, red) to the existing filament; cofilin severs actin-ADP (D, light blue) from the older ‘pointed’ end.
This finding implies that the redistribution requires association of profilin with polyproline-containing proteins, such as VASP or other scaffold proteins, within the spines. Much remains to be learned about how these complex changes in the spine cytoskeleton are coordinated after synaptic stimulation and how they contribute to changes in synaptic efficacy. A complete understanding will undoubtedly require quantitative methods for studying shifts in cytoskeletal equilibria in dendritic spines. Synaptic activity can increase the formation of new spines, in addition to changing the shape of existing spines [44]. Jourdain et al. found that CaMKII has a role in formation of new spines concomitant with induction of some forms of LTP. They used time-lapse two-photon confocal microscopy of organotypic slice cultures to monitor spines on CA1 hippocampal pyramidal neurons filled with fluorescent dye. When they also filled the neurons with activated CaMKII, they observed an order of magnitude increase in the number of new spines formed over a period of 60 min compared with the number in control neurons filled with heat-inactivated CaMKII. At the same time, they measured an increase in the amplitude of excitatory postsynaptic potentials in the www.sciencedirect.com
neurons that contained active CaMKII, consistent with previous studies [45]. They were able to block formation of new spines with either an NMDA receptor antagonist or the CaMKII inhibitor KN93. Role of scaffold proteins in shaping the actin cytoskeleton in spines Scaffold proteins that localize signaling components in discrete locations have significant roles in organizing specialized functions of all cells, including neurons. Several scaffold proteins interact with actin or actinbinding proteins in spines. However, we are only beginning to understand their importance in coordinating and shaping spine architecture. GIT1 Work in motile fibroblasts indicates that, although inactive Rac is diffusely distributed throughout the cell, activated Rac is discretely localized [46], suggesting that Rac activators are tethered to certain locations. G-proteincoupled receptor kinase-interacting protein (GIT1) targets key regulators of the actin cytoskeleton to spines. Two important GIT1 binding partners are PIX (a Rac GEF) and
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PAK proteins [47–49]. When Zhang et al. [50] disrupted the synaptic localization of GIT1, by overexpressing dominant-negative mutant GIT1, they observed a significant increase in the number of dendritic protrusions and a decrease in the number of synapses and mushroomshaped spines. They recapitulated this phenotype by overexpressing constitutively active Rac, constitutively active PIX, or PIX that is unable to bind GIT. They hypothesize that untethered PIX can activate Rac throughout the cell, causing formation of aberrant protrusions. Likewise, overexpression of constitutively activated PIX or activated Rac would cause a similar phenotype owing to promiscuous Rac activity along the dendritic shaft. Intersectin Intersectin is a multi-domain scaffold protein that interacts with several signaling proteins. A long splice variant of intersectin that contains a Dbl domain with GEF activity for Cdc42 is expressed specifically in neurons [51,52]. Activated EphB receptors physically associate with intersectin, resulting in formation of a complex containing intersectin, Cdc42 and N-WASP, an ARP2/3 activator [53]. Within the complex, N-WASP upregulates the GEF activity of intersectin for Cdc42 [52], catalyzing the formation of Cdc42-GTP. Cdc42-GTP in turn activates N-WASP, which then increases the affinity of ARP2/3 for ATP [54]. The ability of ARP2/3 to nucleate new filaments or initiate branching of an existing filament is widely believed to require ATP hydrolysis [54]. Proteins such as GIT1 and intersectin control local formation and branching of actin filaments. Other PSD scaffold proteins, such as shank [55] and densin [56], that associate with actin and actin-filament-binding proteins might also have a role in shaping the interaction of the spine membrane with the actin cytoskeleton, but we have only begun to discern how this might be orchestrated. Outstanding questions Activation of Ras and its signaling pathways by Ca2C flux through NMDA receptors, and activation of Rac and its pathways through Eph receptors, are well documented. However, very little is known about how NMDA receptors influence Rho family GTPases, including Rac, Cdc42 and RhoA, to regulate activity-dependent spine morphogenesis. Additional studies will undoubtedly shed light on whether the Rho family can be activated directly by Ca2C influx or whether it is activated by crosstalk from Ras, as has been described in other systems [57,58]. A recent study by Vazquez et al. [59] shows that homozygous knockout of synGAP, a synaptic Ras and Rap [60] GTPaseactivating protein that is regulated by NMDA-receptordependent phosphorylation by CaMKII, results in the precocious formation of abnormally large spines in cultured hippocampal neurons. This could be an example in which indirect activation of Rho family GTPases is triggered by abnormally high Ras or Rap activity. Clearly, the size and morphology of spines, and the arrangement of signaling molecules within the spine head, are tightly regulated and crucial for correct information processing and storage in neural networks. www.sciencedirect.com
Studies of the regulating mechanisms will generate a wealth of interesting new information about brain function and could shed light on the etiology of mental retardation. Acknowledgements We thank members of the Kennedy laboratory, in particular Holly Beale, Edoardo Marcora and Luis Vazquez for helpful discussions. This work was supported by National Institutes of Health Grants 1F32 NS047894 (H.J.C.) and R01 NS28710 (M.B.K).
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