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Integrins are heterodimeric extracellular matrix receptors that are essential for the proper development of the vertebrate nervous system. We report here that selective loss of integrin β1 in excitatory neurons leads to reductions in the size and complexity of hippocampal dendritic arbors, hippocampal synapse loss, impaired hippocampus-dependent learning, and exaggerated psychomotor sensitivity to cocaine in mice. Our biochemical and genetic experiments demonstrate that the intracellular tail of integrin β1 binds directly to Arg kinase, and that this interaction stimulates activity of the Arg substrate p190RhoGAP, an inactivator of the RhoA GTPase. Moreover, genetic manipulations that reduce integrin β1 signaling through Arg recapitulate the integrin β1 knockout phenotype in a gene-dose sensitive manner. Together, these results describe a novel integrin β1-Arg-p190RhoGAP pathway that regulates dendritic arbor size, promotes synapse maintenance, supports proper hippocampal function, and mitigates the behavioral consequences of cocaine exposure.
Integrins are heterodimeric (αβ) extracellular matrix (ECM) receptors that are widely expressed throughout the mammalian nervous system (Pinkstaff et al., 1999), where they are essential for several processes in neuronal development, including neural stem cell proliferation (Blaess et al., 2004; Leone et al., 2005), neuronal migration (Anton et al., 1999; Graus-Porta et al., 2001; Schmid et al., 2004; Huang et al., 2006; Belvindrah et al., 2007), cortical basal lamina formation (Graus-Porta et al., 2001), and synapse maturation and plasticity (Benson et al., 2000; Chavis and Westbrook, 2001; Huang et al., 2006; Webb et al., 2007). Integrin-mediated adhesion provides structural support for developing neurons, and culturing neurons on ligands for the β1 family of integrins increases the length and complexity of dendritic arbors (Moresco et al., 2005; Schlomann et al., 2009). Conversely, inhibiting integrin β1 signaling in developing retinal ganglion cells leads to dendrite retraction (Marrs et al., 2006), suggesting that β1-containing integrins may influence circuit function by controlling dendritic arbor formation and/or stability. Recent studies have also reported changes in expression of integrin β1 following chronic cocaine exposure (Wiggins et al., 2009), suggesting that integrin-mediated adhesion may play a protective role to stabilize neuronal structures after psychostimulant exposure (Gourley et al., 2011).
In non-neuronal cells, integrin-mediated adhesion activates the Arg nonreceptor tyrosine kinase, which phosphorylates cortactin and p190RhoGAP to promote actin rearrangements (Hernandez et al., 2004a; Bradley et al., 2006; Lapetina et al., 2009). Arg is highly expressed in the brain, where it is enriched in dendritic spines (Koleske et al., 1998; Moresco et al., 2003). Although axons, dendrites, and synapses develop normally in arg−/− mice, these animals subsequently exhibit significant synapse loss and dendritic arbor atrophy in both the cortex and hippocampus by early adulthood (Moresco et al., 2005; Sfakianos et al., 2007). After synapse and dendrite loss, arg−/− mice display a number of behavioral defects, including impaired hippocampus-dependent learning and memory (Sfakianos et al., 2007), exaggerated psychomotor sensitivity to cocaine, and increased vulnerability to cocaine-induced cognitive deficits (Gourley et al., 2009).
We demonstrate here for the first time that integrin β1 signals through Arg to regulate neuronal structure in the postnatal mouse hippocampus. Conditional ablation of integrin β1 from excitatory neurons causes reductions in hippocampal synapse density and dendritic arbor size during late adolescence, coinciding with deficits in hippocampus-dependent memory tasks. In addition, we show that loss of integrin β1 from excitatory neurons in the hippocampus and cortex causes increased psychomotor sensitivity to cocaine. Using complementary biochemical and genetic approaches, we demonstrate that Arg binds directly to the β1 integrin intracellular tail and that integrin β1-Arg signaling regulates the activity of p190RhoGAP, which stabilizes dendritic arbors by inactivating the RhoA GTPase (Sfakianos et al., 2007). Together, these results identify a new role for integrin β1 in vivo and describe a novel integrin β1-Arg-p190RhoGAP signaling cascade that protects against synapse and dendrite loss.
Cortical and hippocampal homogenates were fractionated by the method of Jones and Matus (Jones and Matus, 1974) with modifications (Biederer et al., 2002). Briefly, tissue was dissected from mice under deep Nembutal sedation and homogenized in ice-cold 320 mM sucrose with 10 mM HEPES (pH 7.4) in the presence of phosphatase and protease inhibitors using a glass-Teflon homogenizer. Homogenates were spun at 800 × g for 10 minutes at 4°C to clear debris and the supernatant was collected and spun at 10,000 × g for 15 minutes at 4°C. The resulting pellet was resuspended and spun again to collect washed synaptosomes. A portion of the synaptosome fraction was separated, hypotonically lysed, and centrifuged at 24,000 × g for 20 minutes at 4°C to isolate synaptosomal membranes. Following SDS-PAGE, samples were immunoblotted with antibodies to integrin β1 (BD Biosciences) and Arg (a generous gift from Peter Davies, Albert Einstein Medical College) and detected using chemiluminescence. Quantification was performed on scanned films using Quantity One software (Bio-Rad Laboratories).
400 µm-thick hippocampal slices from male mice were maintained in a standard interface chamber at 33°C. Individual live CA1 pyramidal neurons were injected with 4% biocytin in a 2 M sodium acetate solution, pH 7.5 using 300 ms current injections of 5 nA at 1 Hz for 17 min. After a 10 min recovery, injected neurons were fixed in 4% paraformaldehyde overnight, cryoprotected in 30% sucrose, resectioned at 40 µm, and visualized using standard avidin–horseradish peroxidase (HRP) staining (Vectastain Elite ABC; Vector Laboratories). Biocytin-labeled neurons were then traced under 100× magnification using a light microscope outfitted with a Z drive and reconstructed using Neurolucida software by a single experimenter blinded to genotype (MicroBrightField). Sholl analysis, total dendritic length, and branchpoint number were determined using NeuroExplorer software (MicroBrightField).
Male mice were perfused transcardially under deep Nembutal anesthesia. A short (1 min) perfusion with PBS + heparin, pH 7.4, was followed by a perfusion with 4% paraformaldehyde/0.2% glutaraldehyde in 0.1 M PBS, pH 7.4. After fixation, brains were dissected and postfixed in the same solution overnight at 4°C. Vibratome sections of the hippocampus were cut and processed in 1% OsO4 at 4°C for 1 h (Schikorski and Stevens, 1997). After dehydration in ethanol, the sections were contrasted en bloc in 0.5% uranyl acetate in 95% ethanol for 1 h and flat-embedded in Epon. Embedded vibratome sections were further cut to 70 nm thickness for electron microscopy. Sections were photographed with a Jeol 100 CX II electron microscope as 12000× magnification. Excitatory synapses were identified in electron micrographs by the presence of a dendritic spine containing a clear electron-dense postsynaptic density apposed to an asymmetrical presynaptic compartment containing at least three synaptic vesicles (Harris and Stevens, 1989; Sfakianos et al., 2007). All measurements were made by a single experimenter blinded to the sample identity, and each mutant was matched with a WT littermate control. Dendritic spine head area was measured using ImageJ in spines with a clearly visible neck, and measured from the thinnest part of the neck.
The cytoplasmic tail of integrin β1 (amino acids 752–798) was subcloned from cDNA (ATCC, accession number BC020057) into pGEX-4T1 (GE Healthcare). An α-helical linker to separate the globular GST moiety from the β1 tail was subcloned from the Bcr protein (amino acids 1–68).
His-tagged recombinant full length Arg, ArgΔC (amino acids 1–557), Arg557-C (amino acids 557–1182), and Arg kinase domain (amino acids 281–557) were produced in baculovirally-infected Hi5 insect cells (Invitrogen) as previously described (Lapetina et al., 2009). GST-tagged recombinant Arg SH3SH2 (amino acids 112–254) was produced in Rosetta cells by inoculating culture, then inducing protein expression with the addition of 0.25 mM IPTG overnight. Cells were centrifuged, resuspended in 50 mM Tris pH 8, 150 mM NaCl, 10% glycerol, 1 mM DTT, 5 mM EDTA, and protease inhibitors, passed through French Press, and lysed with 1% Triton X-100. Following clarification, lysates were incubated with glutathione-agarose beads (Sigma) for 1 hour, applied to column, washed with 10 volumes Wash A (50 mM Tris pH 8, 500 mM NaCl, 5% glycerol, 1 mM DTT, 0.01% Triton X-100, 1 mM PMSF), then 10 volumes Wash B (Wash A with 100 mM NaCl), then eluted in Wash B plus 10 mM glutathione. The GST tag was removed with PreScission Protease (GE Healthcare) following the manufacturer’s protocol. GST-linker and GST-β1 integrin tails were purified as above, without GST tag cleavage. Full length Arg, ArgΔC, Arg557-C, Arg kinase domain, and SH3SH2 were dialyzed into 36.5 mM phosphate buffer pH 7.4, 500 mM NaCl and coupled to AminoLink beads (Pierce) according to the manufacturer’s protocol at a final concentration of 100 µM. Beads were blocked with BSA and 1 M Tris, then washed, and stored in 50% glycerol.
1 µM Arg or Arg fragments coupled to beads were incubated with varying concentrations of GST-β1 tail or GST-linker control in binding buffer (25 mM HEPES, pH 7.25, 100 mM NaCl 5% glycerol, 0.01% Triton X-100, 1 mM DTT) for 1 hour at 4° C with gentle mixing. Reactions were centrifuged at 16,000 × g, pellets were washed twice with binding buffer, before resuspension in sample buffer. Samples were separated on 10% BIS-Tris gels using MOPS running buffer, stained with Coomassie R-250 (Pierce), then scanned and quantitated using ImageQuant software (BioRad).
Primary neuronal cultures were prepared from postnatal day 0 mouse hippocampi. Neurons were dissociated and plated at density of 0.3 × 106 cells/ml on glass coverslips coated with poly-(D)-lysine (20 µg/ml; BD Biosciences). Cultures were maintained in serum free media containing 1% penicillin:streptomycin, 1% L-glutamine, and 2% B27-supplement (Gibco) in neural basal media (Invitrogen). Neurons were transfected with Arg-RFP and β1-integrin-GFP at 12 DIV, fixed, and immunostained at 15 DIV. Images were taken using an inverted fluorescent microscope (Nikon) at 40× objective.
Integrin β1 was immunoprecipitated from Triton X-100 (1%) solubilized brain membrane extracts in the presence of protease and phosphatase inhibitors (Dulabon et al., 2000). Protein extract (0.5 mg, standardized to 1 mg/ml) was precleared with 40 µl of protein G Plus/A agarose beads (Calbiochem) for 20 min at 4°C. The precleared supernatant was then incubated with 40 µl of beads that had been incubated for 12 hrs with anti-integrin β1 antibody (BD Biosciences) for 1 hour at 4°C with gentle mixing. Immunoprecipitates were washed three times with 1 ml of lysis buffer, suspended in 40 µl sample buffer, and separated by SDS-PAGE for immunoblot analysis. Coimmunoprecipitated proteins and loading controls were detected by stripping and reprobing the blot with anti-integrin β1 and anti-Arg antibodies.
For p190RhoGAP immunoprecipitations, hippocampi were dissected from male mice in ice-cold PBS and homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease and phosphatase inhibitors). Protein extract (0.5 mg, standardized to 1 mg/ml) was precleared with 40 µl of protein G Plus/A agarose beads (Calbiochem) for 20 min at 4°C. The precleared supernatant was then incubated with two micrograms of anti-p190RhoGAP monoclonal antibody (Upstate) overnight at 4°C with gentle mixing. 40 µl of protein G Plus/A agarose beads (pre-equilibrated) was then added, and immune complexes were incubated for 1 h at 4°C with gentle mixing. Immunoprecipitates were washed three times with 1 ml of lysis buffer, suspended in 40 µl sample buffer, and separated by SDS-PAGE for immunoblot analysis. Coimmunoprecipitated proteins and loading controls were detected by stripping and reprobing the blot with anti-120RasGAP (Upstate) and anti-p190RhoGAP (BD Biosciences) antibodies. Bands were quantified using a densitometer and ImageQuant software, and the relative amount of co-immunoprecipitated protein was standardized to WT littermate controls run on the same blot.
Rho activity assays were performed from hippocampal samples using an ELISA-based assay (Cytoskeleton) as described previously (Bradley et al., 2006).
Object recognition tests were conducted in a lit, quiet room using 7-week-old male mice as previously described (Sfakianos et al., 2007). Briefly, mice were placed in a large, clean field (41×20 cm) and allowed to explore 2 identical objects placed at opposite ends of the field for a total of 30 s. Forty-eight hours later, mice were placed in the same field, and time spent exploring 1 familiar and 1 novel object was quantified until animals had accumulated 30 s of exploration time. Mice that did not explore the objects for a total of 30 s within 5 min were excluded. Exploration was defined as nasal or oral contact with the objects, and raters blinded to mouse genotype scored all sessions.
Male adult mice were injected with cocaine hydrochloride (10 mg/kg, i.p., 1 ml/100 g; generously provided by NIDA) after 1-h habituation to a large, clean cage normally used to house rats (41×20×20 cm) that was placed in Omnitech Digiscan Micromonitor frames equipped with 16 photocells. The number of photobeam breaks generated during the 30 minutes after cocaine administration was normalized to each animal's own baseline.
A spatial reversal learning task sensitive to hippocampal lesions was conducted using methods similar to those previously described (Gourley et al., 2010); food-restricted male mice were trained to nose poke for food reinforcement (20 mg grain pellets; Bioserv) in standard Med-Associates conditioning chambers equipped with 3 stationary nose poke recesses. Nose poke training was initiated with training sessions during which mice were reinforced for entering the nose into a single designated recess, and each response was reinforced until animals earned 30 pellets or 135 min elapsed. All mice acquired the response within 5 daily sessions, with no differences in response acquisition. Then, the location of the reinforced aperture within the chamber was “reversed,” such that a mouse trained to respond on the leftmost aperture was now reinforced for responding on the rightmost aperture or vice versa. Again, the session ended when mice acquired 30 pellets or 135 min elapsed, and errors (responses made on the previously reinforced aperture) were quantified during 2 sessions.
All data are reported as mean ± SEM. Comparisons between WT and Nex-β1−/− mice were performed with two-tailed Student's t-tests. For all comparisons with multiple mutant genotypes, a one-way ANOVA was performed to determine if the genotype had a statistically significant overall effect, and post-hoc statistical comparisons were performed with two-tailed Student's t-tests. For RhoA activity assays, experimental samples were standardized to WT littermate samples run during the same assay to reduce inter-assay error, and datasets were analyzed using a one-sample t-test comparing the mean to a value of 1. Datasets were also analyzed by two-factor (Sholl analysis, cocaine sensitivity, reversal learning) or three-factor (novel object recognition) ANOVA. Statistics were performed with GraphPad Prism (GraphPad Software).
We inactivated integrin β1 expression by crossing mice containing a floxed allele of the gene encoding β1 (itgb1) (Graus-Porta et al., 2001) with Nex-Cre transgenic mice that express Cre recombinase selectively in excitatory forebrain neurons in the forebrain (Goebbels et al., 2006). This combination efficiently inactivates itgb1 in excitatory neurons of the cortex and hippocampus beginning at approximately embryonic day 11.5 (E11.5) (Belvindrah et al., 2007). For simplicity, mice expressing one copy of the floxed itgb1 allele and Nex-Cre (itgb1+/flox Nex-Cre+) will be referred to as Nex-β1+/−, while mice carrying two copies of the floxed itgb1 allele and Nex-Cre (itgb1flox/flox Nex-Cre+) will be referred to as Nex-β1−/−. Quantitative immunoblotting revealed that integrin β1 levels were reduced by over 80% in hippocampal synaptic membrane fractions from Nex-β1−/− mice (Figure 1a). The residual integrin β1 in the synaptic membrane fractions of Nex-β1−/− mice may reflect inadequate integrin β1 recombination in a limited subset of neurons or contamination of the hippocampal synaptic membrane fraction by integrin β1 present in inhibitory neurons and glia. Both Nex-β1+/− and Nex-β1−/− mice were visually indistinguishable from wild type (WT) littermates (data not shown), and we did not detect any differences in hippocampal ultrastructure (Figure 1b) or cortical lamination in Nex-β1−/− mice compared to wild type (WT) mice, in agreement with previously published results (Belvindrah et al., 2007).
We used live-cell labeling of hippocampal CA1 pyramidal neurons in acute slices followed by 3D reconstruction and morphometric analysis to quantitatively assess dendritic structure in Nex-β1−/− mice (Figure 1c). Apical dendritic arbors developed normally in Nex-β1−/− mice through postnatal day 21 (P21), but at P42 were reduced by 21% in length (Figure 1d) and had a 26% reduction in the number of branch points (Figure 1e) compared to WT littermates. Sholl analysis (Sholl, 1953) revealed that these reductions were distributed throughout the dendritic arbor (Figure 1f). The length and branch point number of basal dendritic arbors were not significantly different from WT in Nex-β1−/− mice at either P21 or P42 (Figure 1d,e).
We next examined whether the density of Schaffer collateral-CA1 (SC-CA1) synapses (Figure 2a), which form on the apical arbors of CA1 pyramidal neurons, was altered in Nex-β1−/− mice. As with dendritic arbors, SC-CA1 synapse density was similar to WT in Nex-β1−/− mice at P21 but was reduced by 23% at P42 (Figure 2b). As a measure of dendritic spine morphology, we measured spine head area and postsynaptic density (PSD) length in spines from WT and Nex-β1−/− mice. In agreement with previously published results (Sfakianos et al., 2007), we observed a broad distribution of spine head sizes at P21, which become reduced in size by P42. No significant differences were observed in head size or PSD length in Nex-β1−/− mice at either age.
Given the hippocampal synapse and dendrite loss observed in Nex-β1−/− mice, we tested whether hippocampal function was compromised by measuring performance in a hippocampus-dependent novel object recognition task which we have previously used to characterize behavioral deficits in arg−/− mice15. Forty-eight hours after exposure to two identical objects, P42 mice were placed into a test cage with one object identical to the previously explored objects and one novel object. WT mice showed a significant preference for the novel object, indicating memory for the familiar one (Figure 2e). By contrast, Nex-β1−/− mice failed to discriminate between the novel and familiar objects (Figure 2e). Thus, the reduction in synapses and dendritic arbors are accompanied by impairments in a hippocampus-dependent task.
Cocaine exposure alters integrin β1 expression (Wiggins et al., 2009), but the effect of integrin signaling on cocaine sensitivity in excitatory hippocampal and cortical neurons has not been directly tested. Therefore, we compared the psychomotor response to a low dose (10 mg/kg i.p.) of cocaine in adult WT and Nex-β1−/− mice. In agreement with previous reports (Gourley et al., 2009), this dose did not affect locomotion in WT mice; however, it significantly heightened locomotor activity in Nex-β1−/− mice (Figure 2f), indicating that these mice are more sensitive to cocaine's stimulant properties.
The Nex-β1−/− phenotype closely resembles that previously described in arg−/− mice, in which hippocampal synapses and dendrites develop normally through P21, but synapses and apical dendritic arbors are reduced at P42 (Sfakianos et al., 2007). Adhesion of fibroblasts and cultured cortical neurons to extracellular matrix proteins triggers Arg-mediated phosphorylation of p190RhoGAP (Hernandez et al., 2004b; Bradley et al., 2006; Peacock et al., 2007) and cortactin (Lapetina et al., 2009), suggesting that integrins stimulate Arg activity.
Integrin β intracellular tails bind directly to and activate several signaling partners, including Src family kinases (Arias-Salgado et al., 2003b; Harburger and Calderwood, 2009). To test whether the integrin β1 tail also binds directly to Arg, we covalently coupled purified recombinant Arg or Arg fragments (Figure 3a, b) to beads and measured their ability to bind to a protein fusion comprised of glutathione-S-transferase, a short linker, and the integrin β1 tail (GST-β1). Full length Arg bound to the GST-β1 tail in a concentration-dependent saturable manner, while a GST-linker control protein did not exhibit significant binding to Arg (Figure 3a–e). Both Src and Abl family kinases contain Src homology (SH) 3, SH2, and kinase domains, and Src employs its SH3 domain to bind the integrin β3 cytoplasmic tail (Arias-Salgado et al., 2003a). Therefore, we tested whether Arg uses its SH3 domain to engage integrin β1. Surprisingly, a construct containing only the SH3 and SH2 domains did not bind to GST-β1 appreciably above background (Figure 3c), while the Arg kinase domain alone was sufficient for high-affinity binding to β1 (Figure 3a–e). The kinase domain bound to GST-β1 with similar affinity to full length Arg (Figure 3e, Arg FL Kd = 1.29 ± 0.20 µM, Arg kinase domain Kd = 1.23 ± 0.26 µM). We also tested the specificity of the interaction by measuring whether the Arg kinase domain also binds to the tail of integrin β3. The kinase domain did not bind appreciably to GST-β3 (Figure 3d, estimated Kd > 10 µM), indicating that the integrin β1-Arg interaction is specific.
To test whether the proteins have the potential to interact in neurons, we co-expressed functional fluorescently protein-tagged versions of Arg (Arg-RFP) and integrin β1 (β1-GFP) in cultured hippocampal neurons and examined their localization at 15 days in vitro (DIV). Arg-RFP was significantly enriched in dendritic spines, as identified by staining for the postsynaptic protein PSD95 (Figure 3f), in agreement with previous immunolocalization studies (Koleske et al., 1998; Moresco et al., 2003). β1-GFP localized throughout the dendritic arbor, but was also present in dendritic spines, where a portion colocalized with Arg-RFP and PSD95 (Figure 3f). Expression of β1-GFP in WT neurons did not affect overall dendritic length (n = 15–16 neurons, p = .83), dendritic branch number (n = 15–16 neurons, p = .26), or dendritic spine density (n = 13–17 neurons, p = .91). Similarly, expression of Arg-RFP does not alter dendritic morphology or spine density (Y.C.L. and A.J.K., manuscript in preparation). Arg also co-immunoprecipitated with integrin β1 from cortical and hippocampal tissue (Figure 3g). Together, these binding, localization, and co-immunoprecipitation studies demonstrate that β1 integrin interacts with Arg and that both proteins localize to dendritic spines, suggesting that β1 integrin may signal directly through Arg in dendritic spines in vivo.
p190RhoGAP is a major inactivator of the RhoA GTPase and is a substrate of Arg in the brain, where Arg and p190RhoGAP interact genetically to regulate dendritic arbor size (Hernandez et al., 2004a; Sfakianos et al., 2007). The relative amounts of Arg and its downstream signaling partners p190RhoGAP and p120RasGAP were unchanged in synaptoneurosomal fractions from Nex-β1−/− mice relative to WT mice, indicating that integrin β1 was not essential for localization of these signaling components to synapses (Figure 4a). Arg-mediated phosphorylation of p190RhoGAP promotes its binding to p120RasGAP, which is required for its recruitment to the membrane where it can target active RhoA (Parsons, 1996; Arthur et al., 2000; Hernandez et al., 2004a; Bradley et al., 2006; Peacock et al., 2007). Therefore, the relative amount of p120RasGAP bound to p190RhoGAP is used as a measure of p190RhoGAP activity (Hernandez et al., 2004a; Bradley et al., 2006; Sfakianos et al., 2007). We used immunoprecipitation to measure p120RasGAP:p190RhoGAP complex levels and p190RhoGAP phosphorylation in hippocampal extracts (Figure 4b–e). In P21 animals, the amount of p120RasGAP bound to p190RhoGAP was similar in WT, arg+/−, Nex-β1+/−, arg+/−Nex-β1+/−, and Nex-β1−/− mice. At p42 however, the level of p120RasGAP:p190RhoGAP complex was significantly reduced in arg+/−Nex-β1+/−, and Nex-β1−/− mice relative to WT, indicative of reduced p190RhoGAP activity (Figure 4b, c). Consistent with this result, p190RhoGAP phosphorylation levels were also reduced in arg+/−Nex-β1+/−, and Nex-β1−/− mice at P42, but not at P21 (Figure 4d, e). These results demonstrate that integrin β1 is required for optimal p190RhoGAP activation and that itgb1 and arg interact in a gene-dose dependent manner to regulate p190RhoGAP activity. Together with the binding experiments showing a physical interaction between integrin β1 and Arg, these results strongly suggest that integrin β1 and Arg interact functionally to regulate p190RhoGAP activity in vivo.
To confirm that the decrease in p120RasGAP:p190RhoGAP complex formation represented decreased p190RhoGAP activity, we measured hippocampal RhoA activity in WT, arg+/−Nex-β1+/−, and Nex-β1−/− mice. Consistent with the reduced levels of p120RasGAP:p190RhoGAP complex, hippocampal RhoA activity was significantly increased in arg+/−Nex-β1+/− and Nex-β1−/− mice at P42 relative to WT littermate controls (Figure 4f), but were not significantly different from WT at P21. Elevated RhoA signaling antagonizes dendrite development and stability in a wide range of contexts (Threadgill et al., 1997; Van Aelst and Cline, 2004). Importantly, both the decrease in p120RasGAP:p190RhoGAP complex formation and resultant increase in RhoA activity were age-dependent and followed the same timeline as the reductions in synapse number and dendrite size seen in arg−/− (Sfakianos et al., 2007) and Nex-β1−/− mice (Figure 1d–f, Figure 2b).
Having established that integrin β1 and Arg interact functionally to regulate p190RhoGAP signaling, we used dose-sensitive interactions to test whether this interaction regulates dendritic arbor size and synapse number. Hippocampal CA1 pyramidal neurons from arg+/− and Nex-β1+/− mice had apical dendritic arbors that were indistinguishable in length (Figure 5a) and branch point number (Figure 5b) from control WT littermates at both P21 and P42. In contrast, although similar to WT at P21, apical dendritic arbors in arg+/−Nex-β1+/− double heterozygotes were 18% shorter in length and had 24% fewer branch points than WT at P42 (Figure 5a,b). Importantly, the time course, magnitude, and distribution (Figure 5c) of these reductions were similar to those observed in arg−/− (Sfakianos et al., 2007) and Nex-β1−/− mice (Figure 1c–f). Together with the interaction data presented above, these data strongly suggest that integrin β1 signals through Arg to stabilize hippocampal dendritic arbors. arg+/− mice were also crossed with mice harboring a heterozygous deletion of itgb3 (integrin β3) as an additional control. No reduction in dendritic arbor size or branch point number was observed in mice doubly heterozygous for germline mutations in arg and integrin β3 (arg+/−β3+/−) (Figure 5a,b), suggesting that Arg may interact selectively with integrin β1.
We also measured the density of SC-CA1 synapses in arg+/−Nex-β1+/− mice via electron microscopy. As in Nex-β1−/− mice, SC-CA1 synapse density in arg+/−Nex-β1+/− mice was identical to WT at P21, but was reduced by 28% at P42 (Figure 5d). These reductions are similar in magnitude to the synapse loss observed in arg−/− mice at P42 (Sfakianos et al., 2007).
We tested the behavioral consequence of reduced integrin β1:Arg signaling using the novel object recognition task. While WT, arg+/−, and Nex-β1+/− P42 mice all exhibited a significant preference for the novel object (Figure 5e), arg+/−Nex-β1+/− mice failed to discriminate between the novel and familiar objects (Figure 5e), a similar deficit to that observed in both arg−/− and Nex-β1−/− mice (Figure 2e). As an additional test of hippocampal function, mice were tested in a spatial reversal learning task (Gourley et al., 2010). Mice initially trained to enter their noses into a specific recess to earn food rewards were required to redirect responding to a new reinforced recess. WT, arg+/−, and Nex-β1+/− mice did not differ in the number of errors committed. As in the novel object recognition task, however, arg+/−Nex-β1+/− mice were significantly impaired, committing substantially more errors than all other groups (Figure 5f). Thus, arg+/−Nex-β1+/− mice are impaired in two distinct behavioral tasks that are sensitive to hippocampal defects.
Both arg−/− (Gourley et al., 2009) and Nex-β1−/− (Figure 2f) mice display heightened psychomotor sensitivity to cocaine. To determine whether the integrin β1-Arg interaction affects cocaine sensitivity, we also measured the psychomotor response to low-dose cocaine (10 mg/kg i.p.) in adult mice with differing arg or itgb1 gene dosage. This dose of cocaine had no effect on locomotion in WT, arg+/−, or Nex-β1+/− mice, but arg+/−Nex-β1+/− mice exhibited greatly increased locomotion after exposure (Figure 5g), demonstrating a functional interaction between integrin β1 and Arg that mitigates against the stimulant properties of cocaine.
We report here that loss of integrin β1 function in excitatory hippocampal neurons leads to reductions of apical dendritic arbor size in the CA1 stratum radiatum, synapse loss, and behavioral deficits. Our biochemical and genetic experiments indicate that integrin β1 and Arg interact both physically and functionally to activate p190RhoGAP and attenuate RhoA activity in the mouse brain. Moreover, genetic manipulations that reduce integrin β1 signaling through Arg recapitulate the hippocampal phenotypes observed in both arg−/− and conditional itgb1 knockout mice. Together, these results link integrin β1 with an Arg-p190RhoGAP pathway that promotes synapse maintenance, regulates dendritic arbor size, supports proper hippocampal function, and reduces the effects of cocaine in mice.
Previous work has strongly suggested a role for integrin β1 in the development of dendritic arbors. Application of integrin inhibitory peptides or expression of dominant-negative chimeric integrin β1 can each block dendritic growth in developing retinal ganglion neurons (Lilienbaum et al., 1995), and plating cortical neurons on integrin substrates such as laminin promotes dendritic branching and extension (Moresco et al., 2005). In addition, application of peptides that mimic the talin-binding domain of β1 or transfection of dominant-negative β1 tail constructs during development of chick retinal ganglion cells prior to synapse formation causes dendritic retraction (Marrs et al., 2006). However, we did not observe deficits in the initial formation of dendritic arbors; itgb1 knockout neurons developed normally through p21, but apical arbors were significantly reduced at P42. This result demonstrates a role for integrin β1 signaling in the late elaboration and maintenance, rather than initial formation, of hippocampal neuron structure.
Interestingly, the reduction in dendritic arbor size and complexity at P42 was limited to the apical arbor, similar to the phenotype observed in arg−/− neurons at this age (Sfakianos et al., 2007). This selectivity may be caused by the distribution of the relevant α integrin subunit or its upstream ligand, as some α integrins primarily localize to apical dendrites in the hippocampus (Bi et al., 2001). An alternative possibility is that consequences of Arg-deficiency takes place on different timescales in the distinct arbors in vivo. In support of this, although normal at P42, the basal dendritic arbors of hippocampal CA1 pyramidal neurons are significantly reduced at P120 in arg−/− mice (A.J.K., unpublished data).
One obstacle in previous studies of integrin β1 functions in the forebrain has been the major defects in neuronal positioning associated with the loss of integrin β1 function in radial glia (Graus-Porta et al., 2001; Huang et al., 2006; Belvindrah et al., 2007). Here, we used a Nex-Cre line (Goebbels et al., 2006) to eliminate integrin β1 selectively in excitatory neurons. Previous work demonstrated that integrin β1 localizes to hippocampal synapses (Schuster et al., 2001) and that conditional genetic ablation of integrin β1 using CaMKII-Cre or emx1-Cre lines leads to reduced hippocampal synaptic efficacy and impaired synaptic plasticity (Chan et al., 2006; Huang et al., 2006). Our results demonstrate that integrin β1 is largely dispensable for the early formation of synapses and dendrites through P21, but that loss of integrin β1 function leads to hippocampal synapse loss by P42. These results suggest that integrin β1 plays a role in maintenance of established synapses. Interestingly, no reduction in hippocampal synapse density was reported following inactivation of integrin β1 using CamKII-Cre (Chan et al., 2006). As CamKII-Cre is expressed significantly later than the Nex-Cre transgene used in these studies (Tsien et al., 1996), these observations raise the possibility that integrin β1 may play a critical role between embryonic day 11.5 when Nex-Cre expression begins (Goebbels et al., 2006) and ~P25 when CamKII-Cre is active, (Tsien et al., 1996) and that inactivating integrin β1 after this period may not affect synapse maintenance and dendritic arborization. Although defects in age-dependent maturation of dendritic spine head size are observed in arg−/− mice (Sfakianos et al., 2007), our results demonstrate that knocking out itgb1 does not affect spine morphogenesis, suggesting another upstream regulator may act through Arg to control spine shape refinement.
We report that conditional ablation of integrin β1 leads to deficits in a novel object recognition task. This behavioral defect is consistent with diminished hippocampal connectivity (Baker and Kim, 2002), and hippocampal lesions produce similar deficits (Broadbent et al., 2004). We also demonstrate that genetic ablation of integrin β1 from forebrain neurons leads to an increase in the hypermotor effects of cocaine. While studies have previously shown that cocaine changes the levels of integrin β1 signaling components (Wiggins et al., 2009; Chen et al., 2010), these results are the first to demonstrate that reducing integrin β1 expression can modulate the effects of cocaine. Although the anatomical studies in this report focus on the hippocampus, it is important to note that the effects of integrin β1 on cocaine sensitivity likely reflect contributions from other brain structures as well, since the Nex-Cre transgene knocks out expression in excitatory cortical neurons (Goebbels et al., 2006). In addition, arg knockout also causes synapse and dendrite destabilization in the cortex (Koleske et al., 1998; Moresco et al., 2005; Gourley et al., 2009), and pharmacological inhibition of Arg selectively in prefrontal cortex leads to heightened cocaine sensitivity (Gourley et al., 2009). Therefore, defects in cortical structure and function also likely contribute to the arg+/−Nex-β1+/− reversal learning and cocaine phenotypes (Gourley et al., 2010).
Previous studies in non-neuronal cells have demonstrated that adhesion to the integrin ligand fibronectin stimulates p190RhoGAP activity, which inactivates RhoA (Ren et al., 1999; Arthur et al., 2000; Bradley et al., 2006). Here, we provide the first evidence of a direct interaction between Arg and β1 and demonstrate that integrin β1 acts through Arg to regulate p190RhoGAP in neurons. Based on these results, along with previous studies on Arg-p190RhoGAP signaling in the nervous system (Hernandez et al., 2004a; Sfakianos et al., 2007; Gourley et al., 2009), we propose a model wherein integrin β1-mediated adhesion stimulates Arg kinase activity, which leads to p120RasGAP:p190RhoGAP complex formation and RhoA inhibition (Figure 6). Together this cascade promotes neuronal stability, as excessive RhoA signaling leads to reductions in dendritic complexity and length (Threadgill et al., 1997; Ruchhoeft et al., 1999; Li et al., 2000; Nakayama et al., 2000; Sfakianos et al., 2007). In support of this model, we have previously demonstrated that reduced p190RhoGAP signaling leads to dendritic atrophy and that reducing RhoA activity rescues the dendrite loss seen in arg−/− mice (Sfakianos et al., 2007). Interestingly, reducing RhoA signaling does not prevent synapse loss in arg−/− mice (Sfakianos et al., 2007), suggesting that Arg may protect against synapse loss and dendritic retraction via distinct mechanisms.
What activates β1-containing integrins to promote synapse stabilization and increase dendrite arbor size? Extracellular space in the brain is filled with numerous ECM components that are integrin ligands (Sanes, 1989; Reichardt and Tomaselli, 1991), and acute application of peptides containing an arginine-glycine-aspartate (RGD) integrin β1 binding motif induce filopodia extensions and synaptic remodeling in cultured hippocampal neurons (Shi and Ethell, 2006). Of the integrin β1 ligands that are present at or near synapses and may regulate the β1-Arg-p190RhoGAP signaling cascade, laminin is a particularly promising candidate, as cultured arg−/− hippocampal neurons do not increase dendritic branching when plated on laminin compared to wild type controls (Moresco et al., 2005). Importantly, changes in hippocampal laminin expression have been reported in human cocaine addicts (Mash et al., 2007), suggesting that integrin-mediated adhesion to laminin may affect vulnerability to cocaine addiction by regulating sensitivity. Alternatively, integrin β1-Arg signaling may be regulated by an unconventional integrin ligand such as Semaphorin7A (Pasterkamp et al., 2003; Moresco et al., 2005). Identification of the relevant upstream ligand and characterization of its developmental expression profile may also explain why the integrin β1-Arg signaling axis is essential for the late elaboration, but not initial formation of synapses and dendrites. Interestingly, the period of synapse and dendrite loss seen in both arg−/− and Nex-β1−/− mice coincides with late adolescence, a period marked by an increased vulnerability to addiction (Chambers et al., 2003; Gourley et al., 2009). However, it is currently unclear whether these reductions in neuronal connectivity are directly responsible for the behavioral abnormalities seen in arg−/− and Nex-β1−/− mice. Future studies should determine which ligands activate the β1-Arg-p190RhoGAP signaling cascade, and what effect they have on neuronal connectivity and function during this critical period in the cortico-striatal and limbic regions known to be involved in addiction (Jentsch and Taylor, 1999).
We are grateful to T. Biederer and E. Robbins for advice on synaptic plasma membrane isolation, to C. Kaliszewski and X. Ye for expert technical assistance, to P. Davies for providing the anti-Arg antibody, and to D. Calderwood for critical discussions. Supported by predoctoral NRSA awards AG039124 (M.S.W.) and NS058086 (W.D.B.) and PHS grants DC00210 (C.A.G.), DA011717 and AA017537 (J.R.T.), NS19090 (L.F.R.), NS39475 and CA133346 (A.J.K), and an Established Investigator Award from the American Heart Association (A.J. K.).
Author Contributions:M.S.W. enumerated synapses (with assistance from C.A.G.), performed β1/p190RhoGAP immunoprecipitations, and determined RhoA activity. W.D.B. performed all in vitro binding experiments, including development of constructs. S.L.G. ran behavioral experiments (with assistance from J.R.T. and M.S.W.). Y.L. performed imaging of Arg and integrin β1 in cultured cells. M.A.S. performed the β3:Arg binding experiment. L.F.R. helped plan experiments and provided key reagents. A.J.K. performed dendritic reconstructions. M.S.W., W.D.B., and A.J.K. wrote the manuscript, with substantial input and editing from all other authors.