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Synapse formation is regulated by the signaling adaptor GIT1.

Zhang H, Webb DJ, Asmussen H, Horwitz AF - J. Cell Biol. (2003)

Bottom Line: Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines.The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac.These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908-0732, USA.

ABSTRACT
Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

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Overexpression of the SLD from GIT1 regulates spine morphology and synaptic density. (A) Hippocampal neurons were cotransfected with GFP-SLD and GIT1-FLAG and stained for synapsin1. Note the localization of GFP-SLD to the synapses in relatively low expressing cells causes a decreased localization of GIT1 in the synapses (arrows). Bar, 20 μm. (B) Hippocampal neurons were transfected with various GIT1 constructs at 1 wk in culture and stained for SV2 at 2 wk in culture. Note the increase in dendritic protrusions (left column) and the decrease in synaptic density (right column) in neurons expressing high levels of GFP-SLD. Bar, 20 μm. (C) Quantification of the number of spines and dendritic protrusions in hippocampal neurons transfected with either GFP-GIT1 or GFP-SLD. 80–100 dendrites from independent transfections were quantified for each construct. The definitions of spines and dendritic protrusions are provided in Materials and methods. (D) Quantification of synaptic density in hippocampal neurons transfected with GIT1, nGIT1, CD-GIT1, or SLD. 85–110 dendrites from independent transfections were quantified for each construct (as described in Materials and methods). The difference between SLD and other GIT1 constructs was statistically significant as determined by Student's t test (*P < 0.0001). Note that even though nGIT1 contains SHD1, it has a decreased affinity for PIX, as assayed by coimmunoprecipitation, when compared with wild-type GIT1 (Zhao et al., 2000; unpublished data).
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fig3: Overexpression of the SLD from GIT1 regulates spine morphology and synaptic density. (A) Hippocampal neurons were cotransfected with GFP-SLD and GIT1-FLAG and stained for synapsin1. Note the localization of GFP-SLD to the synapses in relatively low expressing cells causes a decreased localization of GIT1 in the synapses (arrows). Bar, 20 μm. (B) Hippocampal neurons were transfected with various GIT1 constructs at 1 wk in culture and stained for SV2 at 2 wk in culture. Note the increase in dendritic protrusions (left column) and the decrease in synaptic density (right column) in neurons expressing high levels of GFP-SLD. Bar, 20 μm. (C) Quantification of the number of spines and dendritic protrusions in hippocampal neurons transfected with either GFP-GIT1 or GFP-SLD. 80–100 dendrites from independent transfections were quantified for each construct. The definitions of spines and dendritic protrusions are provided in Materials and methods. (D) Quantification of synaptic density in hippocampal neurons transfected with GIT1, nGIT1, CD-GIT1, or SLD. 85–110 dendrites from independent transfections were quantified for each construct (as described in Materials and methods). The difference between SLD and other GIT1 constructs was statistically significant as determined by Student's t test (*P < 0.0001). Note that even though nGIT1 contains SHD1, it has a decreased affinity for PIX, as assayed by coimmunoprecipitation, when compared with wild-type GIT1 (Zhao et al., 2000; unpublished data).

Mentions: 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 4–7 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.


Synapse formation is regulated by the signaling adaptor GIT1.

Zhang H, Webb DJ, Asmussen H, Horwitz AF - J. Cell Biol. (2003)

Overexpression of the SLD from GIT1 regulates spine morphology and synaptic density. (A) Hippocampal neurons were cotransfected with GFP-SLD and GIT1-FLAG and stained for synapsin1. Note the localization of GFP-SLD to the synapses in relatively low expressing cells causes a decreased localization of GIT1 in the synapses (arrows). Bar, 20 μm. (B) Hippocampal neurons were transfected with various GIT1 constructs at 1 wk in culture and stained for SV2 at 2 wk in culture. Note the increase in dendritic protrusions (left column) and the decrease in synaptic density (right column) in neurons expressing high levels of GFP-SLD. Bar, 20 μm. (C) Quantification of the number of spines and dendritic protrusions in hippocampal neurons transfected with either GFP-GIT1 or GFP-SLD. 80–100 dendrites from independent transfections were quantified for each construct. The definitions of spines and dendritic protrusions are provided in Materials and methods. (D) Quantification of synaptic density in hippocampal neurons transfected with GIT1, nGIT1, CD-GIT1, or SLD. 85–110 dendrites from independent transfections were quantified for each construct (as described in Materials and methods). The difference between SLD and other GIT1 constructs was statistically significant as determined by Student's t test (*P < 0.0001). Note that even though nGIT1 contains SHD1, it has a decreased affinity for PIX, as assayed by coimmunoprecipitation, when compared with wild-type GIT1 (Zhao et al., 2000; unpublished data).
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Related In: Results  -  Collection

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fig3: Overexpression of the SLD from GIT1 regulates spine morphology and synaptic density. (A) Hippocampal neurons were cotransfected with GFP-SLD and GIT1-FLAG and stained for synapsin1. Note the localization of GFP-SLD to the synapses in relatively low expressing cells causes a decreased localization of GIT1 in the synapses (arrows). Bar, 20 μm. (B) Hippocampal neurons were transfected with various GIT1 constructs at 1 wk in culture and stained for SV2 at 2 wk in culture. Note the increase in dendritic protrusions (left column) and the decrease in synaptic density (right column) in neurons expressing high levels of GFP-SLD. Bar, 20 μm. (C) Quantification of the number of spines and dendritic protrusions in hippocampal neurons transfected with either GFP-GIT1 or GFP-SLD. 80–100 dendrites from independent transfections were quantified for each construct. The definitions of spines and dendritic protrusions are provided in Materials and methods. (D) Quantification of synaptic density in hippocampal neurons transfected with GIT1, nGIT1, CD-GIT1, or SLD. 85–110 dendrites from independent transfections were quantified for each construct (as described in Materials and methods). The difference between SLD and other GIT1 constructs was statistically significant as determined by Student's t test (*P < 0.0001). Note that even though nGIT1 contains SHD1, it has a decreased affinity for PIX, as assayed by coimmunoprecipitation, when compared with wild-type GIT1 (Zhao et al., 2000; unpublished data).
Mentions: 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 4–7 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.

Bottom Line: Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines.The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac.These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908-0732, USA.

ABSTRACT
Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

Show MeSH
Related in: MedlinePlus