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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 PIX mutants on spine morphology and synaptic density. (A) Hippocampal neurons were transfected with various PIX constructs at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in PIX- and PIXΔGBD- overexpressing neurons and the decrease in spines and dendritic protrusions in PIX-LL–expressing neurons. “Control” denotes GFP-expressing neurons. Bar, 5 μm. (B) Quantification of the number of spines and dendritic protrusions in various PIX constructs. (C) Quantification of the number of synapses in various PIX constructs. The difference between PIX constructs and the untransfected neurons (control) was statistically significant as determined by the Student's t test (*P < 0.0001).
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fig6: Effects of PIX mutants on spine morphology and synaptic density. (A) Hippocampal neurons were transfected with various PIX constructs at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in PIX- and PIXΔGBD- overexpressing neurons and the decrease in spines and dendritic protrusions in PIX-LL–expressing neurons. “Control” denotes GFP-expressing neurons. Bar, 5 μm. (B) Quantification of the number of spines and dendritic protrusions in various PIX constructs. (C) Quantification of the number of synapses in various PIX constructs. The difference between PIX constructs and the untransfected neurons (control) was statistically significant as determined by the Student's t test (*P < 0.0001).

Mentions: If the SLD phenotype results from mistargeting of the GIT1–PIX 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 PIXΔGBD. Wild-type PIX, when expressed at high levels, showed a diffuse labeling pattern. Likewise, PIXΔGBD, 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.


Synapse formation is regulated by the signaling adaptor GIT1.

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

Effects of PIX mutants on spine morphology and synaptic density. (A) Hippocampal neurons were transfected with various PIX constructs at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in PIX- and PIXΔGBD- overexpressing neurons and the decrease in spines and dendritic protrusions in PIX-LL–expressing neurons. “Control” denotes GFP-expressing neurons. Bar, 5 μm. (B) Quantification of the number of spines and dendritic protrusions in various PIX constructs. (C) Quantification of the number of synapses in various PIX constructs. The difference between PIX constructs and the untransfected neurons (control) was statistically significant as determined by the Student's t test (*P < 0.0001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172873&req=5

fig6: Effects of PIX mutants on spine morphology and synaptic density. (A) Hippocampal neurons were transfected with various PIX constructs at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in PIX- and PIXΔGBD- overexpressing neurons and the decrease in spines and dendritic protrusions in PIX-LL–expressing neurons. “Control” denotes GFP-expressing neurons. Bar, 5 μm. (B) Quantification of the number of spines and dendritic protrusions in various PIX constructs. (C) Quantification of the number of synapses in various PIX constructs. The difference between PIX constructs and the untransfected neurons (control) was statistically significant as determined by the Student's t test (*P < 0.0001).
Mentions: If the SLD phenotype results from mistargeting of the GIT1–PIX 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 PIXΔGBD. Wild-type PIX, when expressed at high levels, showed a diffuse labeling pattern. Likewise, PIXΔGBD, 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.

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