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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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Effects of Rac mutants on spine morphology and synaptic density. (A) Effects of Rac mutants on spine morphology and synaptic density. Hippocampal neurons were transfected with myc-tagged RacV12 or RacN17 at day 10 in culture and stained for myc and either rhodamine-conjugated phalloidin (left column) or synapsin1 (right column) at day 12 in culture. Nearby untransfected cells (control) were stained for phalloidin or synapsin1. Note the increase in dendritic protrusions in RacV12-expressing neurons and the decrease in the number of spines in RacN17-expressing neurons. Synaptic density is decreased in both cases. Bar, 2 μm. (B) Quantification of synaptic linear density in neurons transfected with the Rac mutants. Synaptic density is significantly decreased in both RacV12- and RacN17-transfected neurons, *P < 0.0001 (n > 50 for each construct) compared with nearby untransfected neurons (control). (C) Quantification of the number of spines and dendritic protrusions in neurons transfected with the Rac mutants. (D) RacN17 blocks the SLD phenotype. Hippocampal neurons were transfected with either GFP-SLD alone (top) or GFP-SLD and RacN17 (bottom). Note that the dendritic protrusions induced by SLD were completely inhibited by RacN17. Bar, 5 μm.
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fig7: Effects of Rac mutants on spine morphology and synaptic density. (A) Effects of Rac mutants on spine morphology and synaptic density. Hippocampal neurons were transfected with myc-tagged RacV12 or RacN17 at day 10 in culture and stained for myc and either rhodamine-conjugated phalloidin (left column) or synapsin1 (right column) at day 12 in culture. Nearby untransfected cells (control) were stained for phalloidin or synapsin1. Note the increase in dendritic protrusions in RacV12-expressing neurons and the decrease in the number of spines in RacN17-expressing neurons. Synaptic density is decreased in both cases. Bar, 2 μm. (B) Quantification of synaptic linear density in neurons transfected with the Rac mutants. Synaptic density is significantly decreased in both RacV12- and RacN17-transfected neurons, *P < 0.0001 (n > 50 for each construct) compared with nearby untransfected neurons (control). (C) Quantification of the number of spines and dendritic protrusions in neurons transfected with the Rac mutants. (D) RacN17 blocks the SLD phenotype. Hippocampal neurons were transfected with either GFP-SLD alone (top) or GFP-SLD and RacN17 (bottom). Note that the dendritic protrusions induced by SLD were completely inhibited by RacN17. Bar, 5 μm.

Mentions: 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, A–C; 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.


Synapse formation is regulated by the signaling adaptor GIT1.

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

Effects of Rac mutants on spine morphology and synaptic density. (A) Effects of Rac mutants on spine morphology and synaptic density. Hippocampal neurons were transfected with myc-tagged RacV12 or RacN17 at day 10 in culture and stained for myc and either rhodamine-conjugated phalloidin (left column) or synapsin1 (right column) at day 12 in culture. Nearby untransfected cells (control) were stained for phalloidin or synapsin1. Note the increase in dendritic protrusions in RacV12-expressing neurons and the decrease in the number of spines in RacN17-expressing neurons. Synaptic density is decreased in both cases. Bar, 2 μm. (B) Quantification of synaptic linear density in neurons transfected with the Rac mutants. Synaptic density is significantly decreased in both RacV12- and RacN17-transfected neurons, *P < 0.0001 (n > 50 for each construct) compared with nearby untransfected neurons (control). (C) Quantification of the number of spines and dendritic protrusions in neurons transfected with the Rac mutants. (D) RacN17 blocks the SLD phenotype. Hippocampal neurons were transfected with either GFP-SLD alone (top) or GFP-SLD and RacN17 (bottom). Note that the dendritic protrusions induced by SLD were completely inhibited by RacN17. Bar, 5 μm.
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fig7: Effects of Rac mutants on spine morphology and synaptic density. (A) Effects of Rac mutants on spine morphology and synaptic density. Hippocampal neurons were transfected with myc-tagged RacV12 or RacN17 at day 10 in culture and stained for myc and either rhodamine-conjugated phalloidin (left column) or synapsin1 (right column) at day 12 in culture. Nearby untransfected cells (control) were stained for phalloidin or synapsin1. Note the increase in dendritic protrusions in RacV12-expressing neurons and the decrease in the number of spines in RacN17-expressing neurons. Synaptic density is decreased in both cases. Bar, 2 μm. (B) Quantification of synaptic linear density in neurons transfected with the Rac mutants. Synaptic density is significantly decreased in both RacV12- and RacN17-transfected neurons, *P < 0.0001 (n > 50 for each construct) compared with nearby untransfected neurons (control). (C) Quantification of the number of spines and dendritic protrusions in neurons transfected with the Rac mutants. (D) RacN17 blocks the SLD phenotype. Hippocampal neurons were transfected with either GFP-SLD alone (top) or GFP-SLD and RacN17 (bottom). Note that the dendritic protrusions induced by SLD were completely inhibited by RacN17. Bar, 5 μm.
Mentions: 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, A–C; 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.

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