The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity

Regulation of CD2AP expression is growth-mode specific. A, Regulation of CD2AP mRNA levels after sciatic nerve crush. Samples are L4/L5 whole DRG homogenates. Control values were set to 1. Contra, Contralateral; Ipsi, ipsilateral. *Statistically significant compared with contra (multivariate ANOVA, post hoc t test). B, CD2AP mRNA expression during postnatal development. By convention, the lowest expression condition for each tissue sample was set to 1. Samples are whole tissue homogenates. *Statistically significant changes (ANOVA and post hoc t test).

CD2AP is expressed in neurons. A, Western blot assessment of CD2AP protein in tissue lysates. CB, Cerebellum; CX, cortex; Liv, liver; Kid, kidney. Allen Brain Atlas demonstrates CD2AP mRNA in cerebellum (B) and hippocampus (C). D–F, CD2AP protein expression in rat cerebellum. Note high expression levels in neurons. G, CD2AP protein in DRG. Note elevated levels of punctate staining in small diameter soma (closed arrowheads) and axons (open arrowheads).

CD2AP is expressed in axons in superficial skin. Whole naive adult mouse dorsal skin was immunostained for CD2AP (green) and neuron-specific tubulin (TUBB3, magenta). Skin was cleared using organic solvent to allow confocal imaging throughout the tissue in whole mount. A–C, Maximum-intensity projection of confocal slices acquired from 10 μm through 30 μm into the tissue from the epidermal surface. A subpopulation of epidermal axons are CD2AP-positive (filled arrowheads). D–F, Maximum-intensity projection of slices from 40 μm through 80 μm from the epidermal surface (therefore dermis). CD2AP is largely undetectable in axons in the dermis (open arrowheads). In merged images: b, Blood vessel; e, epidermis; h, hair follicle.

Automated screening using cultured postnatal rat cerebellar granule neurons reveals that CD2AP is a positive regulator of axon outgrowth. A, Electroporation of expression plasmids for the indicated genes into CGNs reveals effects on axon outgrowth. *Statistically significant changes compared with GFP (ANOVA and post hoc t test). B, Reduction of CD2AP protein levels using four different shRNA plasmids (1–4) with differing efficiencies reduced outgrowth in accordance with CD2AP levels. Inset (over neurite-length bar graph), Example Western blot of CD2AP protein expression after transfection of PC12 cells with siRNA plasmids (1–4) or scrambled-sequence control (Sc.) plasmid. *Statistically significant changes compared with scrambled-sequence plasmid (ANOVA followed by post hoc t test).

NGF treatment upregulates CD2AP, which is a positive regulator of PC12 neurite growth and complexity. A, PC12 cells were treated with NGF for 24 h before harvesting and processing for Western blot analysis. Ai, Representative blot. Aii, Densitometric quantification. *Significant difference (Student's t test). The effect of increasing the levels of CD2AP on neurite morphology was examined with PC12 cells transfected with plasmids for GFP (control) or GFP-CD2AP fusion protein for 24 h and then treated with NGF for 48 h. Examples of neurite morphology are provided for GFP-transfected (B) and GFP-CD2AP-transfected (C) PC12 cells. Neurite morphology of transfected populations was characterized by Sholl analysis (D) and by total neurite length per cell (E). *Statistically significant difference between groups (repeated-measures ANOVA with post hoc t test).

CD2AP locates to the growing tips of PC12 neurites. PC12 cells were treated with NGF for 48 h, fixed, and costained to visualize the following: A, CD2AP (green); B, C, α-tubulin (red); and D, E, F-actin (using phalloidin; blue). CD2AP locates to tubulin⁻/F-actin⁺ neurite tips (F, G). *CD2AP-positive neurite tip (growth cone).

CD2AP is a positive regulator of the number of filopodia on growth cones. PC12 cells were transfected with GFP (A–C), GFP-CD2AP fusion protein (D–F), control siRNA (G–I), or CD2AP siRNA (J–L) for 24 h before NGF treatment for 48 h. Cells were fixed and stained to visualize F-actin (using phalloidin; red; C, F, I, L), α-tubulin (blue; B, E, H, K), and either GFP (green; A, D) or CD2AP (green; G, J). Growth cones were imaged by confocal microscopy using 100× objective with 5× digital zoom. Images are representative deconvolved maximum-intensity projections. Line histograms were generated for lines exactly 20 μm in length applied exactly 1 μm outside of the tubulin-positive boundary (gray lines). Registration marks (yellow dots) indicate how the histogram relates to the line (x axes = distance, y axes = log of actin fluorescence intensity). M, Mean number of filopodia per condition. *Significant difference (t test). Error bars indicate SEM.

CD2AP forms complexes with the NGF receptor TrkA. Representative maximum-intensity projection of a representative PC12 growth cone stained to visualize CD2AP (green; A) and TrkA (red; B) with associated colocalization heat map (C) and intensity scatter plot from the outlined area (D). E, Mean Pearson's correlation (R) and Mander's coefficients (M1 = percentage of CD2AP signal associated with TrkA; M2 = percentage of TrkA signal associated with CD2AP) in PC12 growth cones. F, Protein samples from NGF-treated PC12 cells were immunoprecipitated with anti-CD2AP and assessed by Western blot for CD2AP, TrkA, IG heavy chain (H.C.; this serves as a loading control for the bound fraction and shows that there is no antibody remaining in the unbound fraction), and GAPDH. n = antibody control lane.

CD2AP is a positive regulator of AKT phosphorylation and increases p85 recruitment to TrkA complexes. A, Western blot assay of protein samples from NGF-treated PC12 cells assessed for phospho-AKT (pAKT), total-AKT (tAKT), phospho-ERK (pERK), total-ERK (tERK), and loading control (GAPDH) following transfection with scramble control (Sc) or CD2AP siRNA. B, Densitometric quantification of pAKT and pERK, relative to tAKT and tERK, respectively. C, Western blot assay of p85 and TrkA immunoprecipitates following transfection with scramble control (Sc) or CD2AP (C) siRNA. CD2AP, TrkA, and p85 protein concentrations are shown in input (In) and bound fractions. D, Quantification of TrkA:p85 colocalization in growth cones following transfection with GFP (E–G), GFP-CD2AP (H–J), scramble control (K–M), or CD2AP siRNA (N–P). G, J, M, P, Representative images from the 3D colocalization analyses are shown for each condition. *Statistically significant changes compared with the respective control (ANOVA, followed by t test compared with control).

CD2AP colocalizes with RAB5 in growth cones and is a positive regulator of TrkA recruitment into endosomes. Maximum-intensity projection of a representative growth cone stained to visualize CD2AP (A) and RAB5 (B) and a 3D colocalization heat map (C). D, CD2AP effect on TrkA recruitment to RAB5⁺ endosomes was quantified in growth cones following transfection either with GFP-CD2AP or with GFP control or following transfection either with CD2AP siRNA or with scramble control. Quantification was done on the ratio of GFP-CD2AP:GFP (Over Exp.) and on the ratio of scramble:siCD2AP (siRNA). *Statistically significant changes compared with the respective control (t test). E–H, Representative images from the 3D colocalization analyses from the overexpression experiment.

CD2AP is present in growing axons in reinnervated skin. Representative maximum-intensity projection through the epidermis of denervated (A–C), and adjacent collaterally reinnervated (D–F) adult mouse dorsal skin, stained to visualize CD2AP (green) and neuron-specific tubulin (TUBB3; magenta). Skin was cleared using organic solvent to allow confocal imaging throughout the tissue in whole mount. Note the CD2AP⁺ axons in reinnervating skin (white arrowheads). F, Boxed area is shown in D′–F′. Colored dots indicate registration marks.