Article |
Address correspondence to Alan F. Horwitz, Dept. of Cell Biology, UVA School of Medicine, P.O. Box 800732, Charlottesville, VA 22908-0732 (for express mail add 1300 Jefferson Park Ave.). Tel.: (434) 243-6813. Fax: 434-982-3912. E-mail: horwitz{at}virginia.edu
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Abstract |
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Key Words: synapse formation; GIT1; PIX; Rac; spine morphology
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Introduction |
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Organization of the actin cytoskeleton is regulated by members of the Rho family of small GTPases, including Rho, Rac, and Cdc42, that cycle between an inactive (GDP-bound) and an active (GTP-bound) state (Hall, 1998; Ridley, 2001). The activation of these molecules is tightly controlled by guanine nucleotide exchange factors (GEFs),* which facilitate the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) that promote GTP hydrolysis. Recent studies have shown that regulation of Rho family signaling plays a key role in normal cognitive functions. Three proteins that interact directly with Rho GTPases are mutated in patients with nonsyndromic mental retardation (MR). These proteins are oligophrenin1, a GAP for Rho-GTPases, PIX, a GEF for Rac and Cdc42, and p21-activated kinase (PAK)3, a Rac and Cdc42 effector (Allen et al., 1998; Billuart et al., 1998; Kutsche et al., 2000; Barnes and Milgram, 2002; Ramakers, 2002). It is not yet known how alterations in Rho signaling result in MR. An attractive hypothesis is that abnormalities in the organization of the actin cytoskeleton give rise to decreased neuronal connectivity and thus impaired cognitive function.
Global activation or shutdown of Rho family signaling usually leads to defects in cellular functions. Thus, one emerging theme is that these proteins are likely to be activated in specific locations within the cell. This could be accomplished by spatial targeting of actin regulators. Adaptor proteins that bind GEFs, GAPs, and their effectors, such as the G proteincoupled receptor kinaseinteracting protein (GIT)1, could potentially function in this capacity by concentrating these molecules at sites of actin organization. In epithelial cells and fibroblasts, GIT1 localizes to distinct subcellular compartments, including adhesions, membrane protrusions, and cytoplasmic complexes. GIT1 regulates migration and protrusive activity by assembling and targeting multiprotein signaling complexes, which contain important actin regulators including PIX, a Rac GEF, and PAK, a Rac effector, between the subcellular compartments (Di Cesare et al., 2000; de Curtis, 2001; Manabe et al., 2002).
In epithelial cells and fibroblasts, the function and localization of GIT1 is mediated by a series of domains including the NH2-terminal ADP-ribosylation factor (ARF)GAP domain that regulates receptor endocytosis (Premont et al., 1998; Claing et al., 2000), ankyrin repeats, Spa2 homology domain (SHD)1 that binds PIX (Zhao et al., 2000), and a COOH-terminal paxillin-binding domain (West et al., 2001; Manabe et al., 2002). In fibroblasts, the COOH-terminal 140 residues of GIT1 (cGIT1), which contain the paxillin binding domain, target it to adhesions, whereas the central domain that contains the ankyrin repeats and the PIX-binding domain is necessary for localization to the cytoplasmic complexes (Manabe et al., 2002).
In this study, we show that GIT1 is enriched in synapses on cultured hippocampal neurons. GIT1 is targeted to the synapse by a novel domain that differs from its adhesion targeting in fibroblasts. Disruption of the postsynaptic localization of GIT1 by a dominant interfering GIT1 mutant that competes for the synaptic binding site results in altered spine morphology and a significant decrease in the density of synapses. These effects result, at least in part, from the mislocalization of PIX. Recruitment of PIX to synapses by GIT1 and the GEF activity of PIX are both necessary for the formation and stabilization of synapse-bearing spines. These results suggest that spatially localized, regulated Rac activity is essential for this process. Thus, GIT1 serves as a key regulator of spine morphology and synapse formation through assembling and targeting multimolecular complexes to synapses where they locally regulate actin dynamics.
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Results |
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The localization of GIT1 to synapses was confirmed by transfecting full-length GFP-tagged and FLAG-tagged GIT1 into hippocampal neurons. In previous studies using fibroblasts, these constructs localized indistinguishably from endogenous GIT1 (Manabe et al., 2002). In hippocampal neurons, the localization of both constructs was also identical to the endogenous GIT1. Both GFP-GIT1 and GIT1-FLAG colocalize in clusters with synapsin1, which, like SV2, labels presynaptic terminals and serves as a synaptic marker (Fig. 1 C). The GIT1 clusters in the dendrites were in close apposition to the presynaptic marker synapsin1 and almost completely merged with the postsynaptic marker PSD-95 (Fig. 1 D). This shows that GIT1 is postsynaptic. We also observed GIT1 in clusters along the axons of the transfected neurons. These clusters completely merged with the presynaptic marker SV2 and were in close apposition to PSD-95 (Fig. 1 D), indicating that GIT1 is not only postsynaptic but also presynaptic. Consistent with this, Kim et al. (2003) recently showed an interaction of GIT1 with the presynaptic protein Piccolo.
Synaptic targeting of GIT1
To identify the region that targets GIT1 to synapses, we expressed various domains of GIT1 as GFP fusion proteins in hippocampal neurons (Fig. 2 A). Their synaptic localization was assayed by GFP fluorescence and coimmunostaining with synaptic markers. We began by examining the localization of cGIT1, since it contains the binding site for paxillin and is responsible for the adhesion targeting of GIT1 in fibroblasts (Manabe et al., 2002). However, in the neurons, cGIT1 did not concentrate in synapses but distributed diffusely throughout the cytoplasm of the soma and processes (Fig. 2 D). Thus, the domain that targets GIT1 to synapses is different than the adhesion localization domain in fibroblasts. As expected, GIT1c, which lacks the COOH-terminal paxillin binding domain, localized in synapses (Fig. 2 B). To further localize the targeting domain within GIT1
c, we first examined whether the NH2-terminal ARF-GAP domain is necessary for synaptic targeting. A central domain deletion mutant, GIT1
CD, which contains the ARF-GAP domain and the paxillin binding domain, did not accumulate in synapses (Fig. 2 D). Another mutant that comprises the central domain, CD-GIT1, localized in synapses (Fig. 2 B). Therefore, neither the ARF-GAP nor the paxillin binding domain is necessary for synaptic targeting, pointing to the central domain of GIT1 as the targeting domain.
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The smallest localizing domain that we identified (CDAS) is located between SHD1 and the paxillin binding domain. Since it is responsible for the synaptic targeting of GIT1, we named it SLD for synaptic localization domain. To test if the intact SLD is necessary for synaptic targeting, we prepared a mutant in which the NH2-terminal 32 aa in SLD were deleted (SLD
32). This mutant showed significantly decreased localization in synapses (Fig. 2 D), suggesting that these 32 aa contribute to efficient synaptic localization. We also prepared two smaller FLAG-tagged constructs, N-SLD and C-SLD, which contain the NH2-terminal and COOH-terminal half of SLD, respectively. Subcellular localization of these constructs was determined by FLAG and synapsin coimmunostaining. N-SLD showed partial localization to synapses (Fig. 2 C), whereas C-SLD localized diffusely in the cytoplasm (Fig. 2 D). These results suggest that the localization domain resides at least in part in the NH2-terminal half of SLD; however, the COOH terminus of SLD may also contribute to efficient synaptic localization. Finally, an internal deletion mutant with the SLD deleted (GIT1
SLD) did not localize to synapses (Fig. 2 D), further confirming that SLD is the localizing domain.
Disruption of the synaptic localization of GIT1 affects spine morphology and synapse formation
To examine the function of GIT1 in synapses, we took a dominant-interfering approach and expressed the smallest localizing construct, SLD, in the neurons. We hypothesized that this construct would prevent GIT1 from localizing to synapses by competing for the synaptic binding sites. This approach has been successful in fibroblasts where the adhesion targeting domain of GIT1 effectively prevents endogenous GIT1 from localizing to adhesions (Manabe et al., 2002). To test if this approach is also effective in reducing the synaptic localization of GIT1, we coexpressed GFP-SLD and GIT1 in the neurons. Indeed, ectopic expression of SLD in neurons showed a dose-dependent effect. In neurons expressing relatively low levels of SLD (<2-fold relative to endogenous GIT1), the synaptic localization of GIT1 was reduced (Fig. 3 A). However, no apparent changes in spine morphology were observed. In neurons expressing high levels of SLD (fivefold relative to endogenous GIT1), GIT1 was distributed diffusely, the number of normal, mushroom-shaped spines was significantly decreased, and the number of long, thin dendritic protrusions was dramatically increased (Fig. 3, B and C). In addition, in the SLD-expressing neurons, the linear density of synapses (number of synapses per 100-µm dendrite) decreased significantly compared with neurons expressing comparable levels of GIT1, nGIT1, and CD-GIT1 (Fig. 3, B and D). When the synaptic density was quantified by another method (the number of synapses per unit area [µm2]), a similar decrease was observed in the SLD-expressing neurons. This effect on synaptic density was observed when SLD was expressed at day 47 in culture and the number of synapses quantified at day 14. When we expressed SLD at day 10 in culture and quantified at day 14, the effect on synaptic density is less dramatic. This suggests that SLD affects synapse formation; however, we cannot exclude the possibility that it is also affecting the maintenance of synapses. The effect of SLD on synaptic density is unlikely to be due to neurite outgrowth defects since it does not affect hippocampal neurite extension on poly-L-lysine on which we grow these neurons (unpublished data). Thus, these data suggest that perturbing GIT1 localization results in defects in spine morphology and synapse formation.
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To further confirm that PIX is targeted to the synapse by GIT1, we constructed a PIX mutant that is deficient in GIT1 binding (PIXGBD) (Koh et al., 2001). Localization of this PIX mutant in hippocampal neurons was examined by transfecting the mutant into the neurons and coimmunostaining with synapsin1. Unlike wild-type PIX, which exhibits synaptic localization, PIX
GBD showed a diffuse staining pattern with no accumulation in synapses (Fig. 5 C). This shows that GIT1 binding of PIX is necessary for its targeting to synapses.
Effects of PIX mutants on spine morphology and synapse formation
If the SLD phenotype results from mistargeting of the GIT1PIX complex, increasing the diffuse, nonsynaptic distribution of PIX should give a similar phenotype. To test this hypothesis, we transfected neurons with either wild-type PIX or PIXGBD. Wild-type PIX, when expressed at high levels, showed a diffuse labeling pattern. Likewise, PIX
GBD, which did not localize to synapses, also distributed diffusely. Thus, overexpression of either construct should effectively increase the nonsynaptic distribution of PIX and give a phenotype similar to SLD-expressing neurons. Indeed, in cells expressing high levels of either construct multiple dendritic protrusions were observed with a concomitant decrease in spine and synaptic density (Fig. 6). This suggests that precise targeting of PIX to synapses by GIT1 is necessary for dendritic spine and synapse formation and that mislocalization of PIX perturbs this process.
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Effects of Rac mutants on spine morphology and synapse formation
Since active Rac has been reported to induce multiple dendritic protrusions in hippocampal slices (Nakayama et al., 2000), a phenotype like that produced by SLD, altered Rac activation appears a likely mediator of the effects of SLD overexpression. To address this question, we transfected hippocampal neurons with a myc-tagged constitutively active Rac, RacV12, at day 10 in culture. The phenotype was examined 48 h after transfection. RacV12-transfected neurons form numerous dendritic protrusions with a significant decrease in the number of normal spines (Fig. 7, A and C). This is consistent with the constitutively active Rac phenotype previously reported in hippocampal slices (Nakayama et al., 2000) and in the mouse cerebellar Purkinje cells expressing a RacV12 transgene (Luo et al., 1996). To see if RacV12 has a similar effect on synaptic density as SLD, we immunostained RacV12-transfected neurons with synapsin1 and anti-myc antibodies. RacV12-expressing neurons form significantly fewer synapses than the adjacent untransfected neurons (Fig. 7, A and B; P < 0.0001), suggesting that overactivation of Rac disrupts synapse formation. To further elucidate how Rac activity affects synapse formation, we transfected a myc-tagged dominant-negative version of Rac, RacN17, into the neurons. RacN17-expressing neurons exhibited very smooth dendrites with a drastic reduction in the number of spines compared with the adjacent untransfected neurons. The synaptic density was also significantly reduced (Fig. 7, AC; P < 0.0001). The effects of Rac mutants on synapse formation are unlikely due to neurite outgrowth defects for two reasons. First, the Rac mutants were transfected at day 10 when the neurites have reached sufficient length for synapses to form. Second, Rac mutants have been shown to affect only axonal growth but not dendritic growth (Luo et al., 1997), whereas the effects of Rac mutants on synaptic density were observed on the dendrites of the transfected neurons.
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Discussion |
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Interestingly, both RacV12 and RacN17 cause a decrease in synaptic density. One explanation is that synapse formation requires properly localized, critical levels of Rac activation. In the case of RacV12-expressing cells, Rac is constitutively activated and distributed diffusely throughout the processes, which results in aberrant actin organization and an inhibition of synapse formation. In neurons expressing RacN17, the level of active Rac is insufficient to support synapse formation. Another explanation is that cycling of Rac between active and inactive forms is necessary for the formation and/or stabilization of synapses. Expression of either RacV12 or RacN17 blocks the cycling and thus the formation of synapses. The observation that both the constitutively active and dominant-negative mutants of Rac have similar effects has been made in other systems as well. In Drosophila, for example, expression of either mutant results in defects in axonal growth (Luo et al., 1997; Song and Poo, 1999).
Several genes that are involved in the regulation of Rho family signaling are mutated in patients with nonsyndromic MR (Allen et al., 1998; Billuart et al., 1998; Kutsche et al., 2000; Barnes and Milgram, 2002; Ramakers, 2002). However, the mechanisms by which these mutations lead to cognitive defects are not understood. One likely possibility is a decreased neuronal connectivity that results from aberrant actin organization (Marin-Padilla, 1972; Huttenlocher, 1974; Purpura, 1974; Kaufmann and Moser, 2000). Indeed, some children with nonsyndromic MR show abnormalities in dendritic spine morphology in their cerebral cortex, i.e., numerous very long and thin spines and a reduction in the number of stubby and mushroom-shaped spines (Purpura, 1974). In our study, the GIT1, PIX, and Rac mutants that produce mislocalized Rac activity recapitulate this phenotype in cultured neurons. These mutants also cause a decrease in synaptic density. Thus, our results suggest a potential mechanism by which aberrant Rho family signaling can lead to decreased neuronal connectivity and eventually impaired cognitive functions. Interestingly, one of the MR mutants has a large deletion in a PIX isoform, which includes the GIT1 binding domain (Koh et al., 2001) that could result in mislocalized PIX (Kutsche et al., 2000).
Our studies point to several important immediate avenues of investigation. For example, GIT1 is targeted to synapses by a novel site, but the molecule that targets GIT1 to synapses is unknown (Fig. 8). Since a poorly characterized G proteincoupled receptor kinase binding domain resides in the vicinity of the SLD (Premont et al., 2000), G proteincoupled receptors and G proteincoupled receptor kinases may contribute to this process (Pitcher et al., 1998). Further investigation into events downstream of GIT1 is also an important challenge. For example, it would be very interesting to visualize changes in Rac activity during synapse formation and synaptic activity. In addition, since PAK3, a downstream effector of Rac, is mutated in some patients with MR (Allen et al., 1998), it is important to determine the role of PAK3 in this process. Finally, it is possible that other domains of GIT1 besides the SLD also play a role in synaptic activity by interacting with proteins from other pathways. Since GIT1 also localizes to the presynaptic terminals, it is tempting to speculate that it serves additional presynaptic functions.
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Materials and methods |
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Plasmids
Full-length human GIT1 cDNA, which includes the 9 aa insertion (nt 774800) found in rat GIT1, GIT1-FLAG, GFP-tagged full-length GIT1, GIT1C, cGIT1, and GIT1
CD were cloned as described (Manabe et al., 2002). GFPCD-GIT1 was obtained by subcloning a HindIII (nt 3691802) fragment into the pEGFP-C3 vector (CLONTECH Laboratories, Inc.). The following GFP-tagged deletion constructs of GIT1 were obtained by subcloning the corresponding GIT1 fragment into the pEGFP-C1 vector (CLONTECH Laboratories, Inc.): CD
Ank (BglII [nt 774]-HindIII [nt 1802]); SLD
32 (FspI [nt 1233]-HindIII [nt 1802]); and nGIT1 ([nt 1]-FspI[nt 1233]). GFP-SLD was obtained by subcloning a PCR fragment (nt 1137-nt 1802) into pEGFP-C1. GFP-GIT1
SLD was obtained by subcloning two PCR fragments, nt 1-nt 1163 and nt 1803-nt 2324, into pEGFP-C1. N-SLD and C-SLD were prepared by PCR amplification of nt 1137-nt 1442 and nt 1443-nt 1802, respectively. The PCR products were then subcloned into pcDNA3 vector with a built-in NH2-terminal FLAG tag. GFP-GIT1
SHD was obtained by subcloning the appropriate GIT1 fragments (nt1-nt774 and nt1137-nt2324) into pEGFP-C1. HA-tagged mouse ßPix was a gift from Chris Turner (SUNY Upstate Medical University, Syracuse, NY). HA-PIX-LL was provided by Lorraine Santy and Jim Casanova (University of Virginia). The L238R, L239S mutations of HA-PIX-LL were introduced by the Quickchange kit (Stratagene) using HA-mouse ßPix as the template. HA-PIX
GBD was generated by subcloning two PCR fragments of ßPix (nt1-nt1474 and nt1763-nt2038) into the pEBB-HA vector. Myc-tagged V12-Rac1 and N17-Rac1 were provided by Alan Hall (University College London, London, UK).
Cell culture and transfection
Hippocampal low density cultures were prepared as described previously (Goslin et al., 1998). Neurons were plated at an approximate density of 70 cells/mm2 and were transfected using a modified calcium phosphate precipitation method (Kohrmann et al., 1999). Briefly, for transfection of a 6-cm dish, 6 µg of plasmid DNA was mixed with 120 µl of 250 mM CaCl2 in a polypropylene tube. 120 µl of 2x HBS (274 mM NaCl, 9.5 mM KCl, 15 mM glucose, 42 mM Hepes, 1.4 mM Na2HPO4, pH 7.107.15) was then added dropwise to the mixture with aeration. This mixture was added immediately to a 6-cm dish of the neurons with 4 ml of 24-h glia-conditioned medium. When complex formation was observed (typically 3060 min), the cells were washed twice with HBS (135 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 20 mM Hepes, pH 7.35), and then glia-conditioned medium with 0.5 mM kynurenic acid was added. Using this method, the transfection efficiency for the hippocampal neurons ranged from 10 to 30%.
Western blot analysis
For Western blot analysis, hippocampal neurons were plated at a density of 280 cells/mm2. At day 10 in culture, neurons were harvested, subjected to SDS-PAGE on a 10% slab, transferred to PVDF, and probed with the appropriate antibodies.
Immunocytochemistry and image analysis
Neurons were fixed in PBS containing 4% PFA with 4% sucrose and permeabilized with 0.2% Triton X-100. Alternatively, they were simultaneously fixed and permeabilized in cold methanol. After blocking with 20% goat serum/PBS, neurons were incubated with the appropriate antibodies in 5% goat serum/PBS. Images were acquired using a cooled CCD camera (Hamamatsu OrcaII) attached to a Nikon TE-300 inverted microscope with a 60x objective (NA1.4; Nikon). To estimate expression levels of the GIT1 constructs, GFP-GIT1expressing neurons were coimmunostained for total GIT1. The fluorescent intensity levels of GIT1 staining in GFP-GIT1expressing neurons and untransfected neurons were measured and compared. For low expression the difference is less than 2x and for high expression it is approximately 5x. Neurons expressing high levels of the constructs were chosen for quantification as follows. 85110 dendrites from independent transfections were randomly selected for each construct. The number of synapses per unit length or per unit area was quantified using NIH Image. We quantified the number of spines and dendritic protrusions by examining 80100 separate dendrites from independent transfections. Spines are defined as stubby or mushroom-shaped protrusions with associated synapses. Dendritic protrusions are defined as protrusions on the dendrites without associated synapses.
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Acknowledgments |
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This work was supported by National Institutes of Health grant GM23244. D.J. Webb was supported by National Institutes of Health postdoctoral training grant HD07528-01.
Submitted: 1 November 2002
Revised: 6 March 2003
Accepted: 6 March 2003
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