Cyclase-associated protein (CAP) inhibits inverted formin 2 (INF2) to induce dendritic spine maturation

Abstract

The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.

Keywords: Acetylated actin; Actin acetylation; Spinogenesis; Synapse formation; Synaptogenesis.

Fig. 1

CAP2 inactivation does not alter dendritic spine density or size. A Immunoblots showing CAP1 and CAP2 expression throughout postnatal hippocampus development. Total protein staining was performed to confirm equal loading. B Expression levels of CAP1, CAP2 and INF2 determined by mass spectrometry on hippocampal lysates from E18.5, P10 and P40 mice. Graph includes values of individual protein samples (N = 4), mean values (MV) and standard error of the means (SEM). Statistical comparison of CAP1 and CAP2 expression levels were performed using Student’s t-test and corrected for multiple comparison with Bonferroni method. Violin plots to the right show expression level distribution of all proteins in these lysates. Norm. log2 LFQ intensity of zero indicates median expression level of all proteins detected within respective condition. C Immunoblots showing CAP1 and CAP2 expression in isolated cortical neurons. Total protein staining was performed to confirm equal loading. D Micrograph of a representative DIV16 hippocampal neuron expressing dsRed (red) together with myc-CAP2 (magenta). Box indicate area shown with higher magnification. E Fluorescence intensity profiles for dsRed and myc-CAP2 immunoreactivity along white line in D. F Graph showing ratio of spine head/dendritic shaft for dsRed and myc-CAP2. Graph includes values of individual spines, MV and SEM. 45 spines from 15 neurons and 3 biological replicates have been investigated (N = 45/15/3). Student’s t-test was performed to test for statistical significance. G Representative micrographs of GFP-expressing DIV16 hippocampal neurons from littermates of various genotype. Graphs showing H spine density and I spine volume in neurons from wildtype mice (CAP2^+/+), heterozygous CAP2 mutants (CAP2^+/-) and systemic CAP2 mutants (CAP2^−/−). Scale bars: 20 µm. ns: P > 0.05, ***: P < 0.001, ****: P < 0.0001

Fig. 2

Spine morphological changes in DIV16 dKO neurons lacking CAP1 and CAP2. A Micrographs of representative DIV16 neurons expressing GFP. Boxes indicate areas shown at higher magnification. Graphs showing B spine density, C spine volume, D spine length, E head length, F head width and G fraction of spine types. Scale bar (µm): 20. ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Fig. 3

Expression of CAP1 or CAP2 is sufficient to rescue spine morphological changes in DIV16 dKO neurons. Micrographs of representative dendritic shafts from A CTR neurons or B dKO neurons expressing either dsRed only or dsRed together with either CAP1-GFP or CAP2-GFP or CAP1-GFP and CAP2-GFP. Graphs showing C-D spine length, E–F head length, G-H head width and I-J fraction of spine types in CTR and dKO neurons upon expression of either CAP1-GFP, CAP2-GFP or both. Scale bar (µm): 2. ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Fig. 4

Overall normal spine morphology in DIV11 dKO neurons. A Micrographs of representative DIV11 CTR and dKO neurons expressing GFP. Boxes indicate areas shown at higher magnification. Graphs showing B spine density, C spine volume, D spine length, E head length, F head width and G fraction of spine types in both groups. H Log2 values for DIV16/DIV11 ratio of spine types in CTR and dKO neurons. Scale bar (µm): 20. ns: P ≥ 0.05, ***: P < 0.001

Fig. 5

INF2 is located in spines and interacts with CAP1 and CAP2 in neuronal cells. Immunoblots showing INF2 expression A in postnatal hippocampus and B cultured cortical neurons. Total protein staining was performed to control equal loading. C DIV16 hippocampal neuron expressing dsRed (red) together with INF2-GFP (green). Box indicate area shown with higher magnification. D Fluorescence intensity profiles for dsRed and INF2-GFP along white line in C. E Graph showing ratio of spine head/dendritic shaft for dsRed and INF2-GFP. Graph includes values of individual spines and MV ± SEM, N = 45/15/3. Student’s t-test was performed to test for statistical significance. F Immunoblots with antibody against myc and β-actin in lysates from HT-22 cells expressing either myc-CAP1 and GFP or myc-CAP1 and INF2-GFP. *: lower bands correspond to the heavy chain of IgG. G Immunoblots with antibody against myc in lysates from HT-22 cells expressing either myc-CAP2 and GFP or myc-CAP2 and INF2-GFP. Scale bar (µm): 20. ****: P < 0.0001

Fig. 6

INF2 overactivation phenocopied spine defects of dKO neurons. A Micrographs of CTR neurons expressing GFP (green) together with either CTR-sh or INF2-sh. Boxes indicate areas shown at higher magnification. Graphs showing B spine density, C spine volume, D spine length, E head length, F head width and G fraction of spine types in CTR neurons expressing either CTR-sh or INF2-sh. H Micrographs of representative CTR neurons expressing dsRed (red) together with either GFP or INF2-GFP (green). Boxes indicate areas shown at higher magnification. Graphs showing I spine density, J spine volume, K spine length, L head length, M head width and N fraction of spine types in CTR neurons expressing either GFP or INF2-GFP. Scale bars (µm): 20. ns: P ≥ 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Fig. 7

INF2 inhibition rescues spine maturation defect in dKO neurons. A Micrographs of representative dKO neurons expressing dsRed (red) together with either GFP or INF2-GFP (green). Boxes indicate areas shown at higher magnification. Graphs showing B spine density, C spine volume, D spine length, E head length, F head width and G fraction of spine types in dKO neurons expressing either GFP or INF2-GFP. H Micrographs of representative dKO neurons expressing GFP (green) together with either CTR-sh or INF2-sh. Boxes indicate areas shown at higher magnification. Graphs showing I spine density, J spine volume, K spine length, L head length, M head width and N fraction of spine types in dKO neurons upon transfection of either CTR-sh or INF2-sh. Scale bars (µm): 20. ns: P ≥ 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001