Afadin is required for maintenance of dendritic structure and excitatory tone

Abstract

The dendritic field of a neuron, which is determined by both dendritic architecture and synaptic strength, defines the synaptic input of a cell. Once established, a neuron's dendritic field is thought to remain relatively stable throughout a cell's lifetime. Perturbations in a dendritic structure or excitatory tone of a cell and thus its dendritic field are cellular alterations thought to be correlated with a number of psychiatric disorders. Although several proteins are known to regulate the development of dendritic arborization, much less is known about the mechanisms that maintain dendritic morphology and synaptic strength. In this study, we find that afadin, a component of N-cadherin·β-catenin·α-N-catenin adhesion complexes, is required for the maintenance of established dendritic arborization and synapse number. We further demonstrate that afadin directly interacts with AMPA receptors and that loss of this protein reduces the surface expression of GluA1- and GluA2-AMPA receptor subunits. Collectively, these data suggest that afadin is required for the maintenance of dendritic structure and excitatory tone.

FIGURE 1.

Localization of afadin at excitatory synapses. A, structure of afadin. Both isoforms of afadin contain two Ras/Rap binding domains and a type II PDZ domain. Only L-afadin contains an F-actin binding domain. B, localization of afadin in DIV 25 cortical neurons. Shown is double immunofluorescence with antibodies against synaptic proteins PSD-95 and VGlut1 or staining with phalloidin to label F-actin and L/S-afadin. Shown is quantification of the percentage of colocalization of afadin and synaptic proteins PSD-95 and VGlut1 or F-actin. Yellow arrows indicate colocalization. n = 8–11 cells from three experiments. Scale bar, 5 μm. Error bars, S.E.

FIGURE 2.

Afadin is required for the maintenance of dendritic architecture and excitatory inputs. A, representative binary images of cortical neurons (DIV 28) expressing pGSuper, control-shRNA, afadin-shRNA, or afadin-shRNA + rescue. B, quantification of TDL and TDN of cells in A (n = 11; ***, p < 0.001). C, quantification of primary, secondary, and tertiary dendritic length and branch number in cells expressing pGSuper, mut-shRNA, afadin-shRNA, or afadin-shRNA + rescue (n = 11; *, p < 0.05; ***, p < 0.001). D, Sholl analysis of dendritic complexity of neurons in A. Expression of afadin-shRNA reduces dendritic complexity compared with control conditions, but co-expression of rescue restores dendritic complexity to control levels. E, representative AMPAR-mediated mEPSC traces of cells expressing control-shRNA or afadin-shRNA. F, quantification of mEPSC amplitude and frequency (n = 16 or 15, respectively; ***, p < 0.001). Scale bar, 50 μm. Error bars, S.E.

FIGURE 3.

Coordinated regulation of synaptic structure and function by afadin. A, representative high magnification images of neurons expressing pGSuper, mut-shRNA, afadin-shRNA, or afadin-shRNA + rescue. B and C, quantification of dendritic spines in A, either overall (B) or as a function of dendritic branch order (C) (n = 11; *, p < 0.05; ***, p < 0.001). D, example high magnification images of surface GluA2 (n-GluA2) and overlay with GFP, in cells expressing pGSuper, control-shRNA, afadin-shRNA, or afadin-shRNA + rescue. E and F, quantification of surface-GluA2 puncta number, either overall (E) or as a function of dendritic branch order (F) in D (n = 11; *, p < 0.05; ***, p < 0.001). G, example high magnification images of surface GluA1 (n-GluA1) and overlay with GFP in cells expressing pGSuper, control-shRNA, or afadin-shRNA. H and I, quantification of surface-GluA1 puncta number (H) and cluster intensity (I) in G (n = 12, p < 0.001). Scale bars, 5 μm. Error bars, S.E.

FIGURE 4.

Interaction of afadin with GluA2-containing AMPARs. A, quantification of the percentage of colocalization of afadin and GluA2 in DIV 26 cortical neurons (right). B and C, reciprocal co-immunoprecipitation (IP) of afadin with GluA2 and GluA1 from mouse frontal cortex. D, co-immunoprecipitation of myc-L-afadin with GFP-GluA2, but not GFP-GluA1, from HEK293 cells. E, plots of TDL against spine density or surface GluA2; plot of surface-GluA2 against TDL and spine density in cells expressing pGSuper, control-shRNA, or afadin-shRNA (n = 11, r² and p values indicated in the plots). Scale bar, 5 μm. Error bars, S.E.