The Cannabinoid Receptor CB1 Interacts with the WAVE1 Complex and Plays a Role in Actin Dynamics and Structural Plasticity in Neurons

<div><p>The molecular composition of the cannabinoid type 1 (CB1) receptor complex beyond the classical G-protein signaling components is not known. Using proteomics on mouse cortex in vivo, we pulled down proteins interacting with CB1 in neurons and show that the CB1 receptor assembles with multiple members of the WAVE1 complex and the RhoGTPase Rac1 and modulates their activity. Activation levels of CB1 receptor directly impacted on actin polymerization and stability via WAVE1 in growth cones of developing neurons, leading to their collapse, as well as in synaptic spines of mature neurons, leading to their retraction. In adult mice, CB1 receptor agonists attenuated activity-dependent remodeling of dendritic spines in spinal cord neurons in vivo and suppressed inflammatory pain by regulating the WAVE1 complex. This study reports novel signaling mechanisms for cannabinoidergic modulation of the nervous system and demonstrates a previously unreported role for the WAVE1 complex in therapeutic applications of cannabinoids.</p></div

Cannabinoids directly modulate Rac1 activity and WAVE1 phosphorylation by Rac1 Activation and WAVE1 Phosphorylation via CB1.

<p>(A) Schematic representation of the Raichu-Rac1 FRET-biosensor employed to measure Rac1 activity. (B) Real-time images representing changes in Rac1 activity in developing mouse cortical neurons following treatment with a CB1 agonist (ACEA; 100 nM) and an inverse agonist at CB1 (AM251; 600 nM). The FRET signal intensity is represented as a pseudocoloured heat map, and insets show magnified view of growth cones. Scale bars (white) represent 20 μm, and scale bars in the inset (yellow) represent 5 μm. (C) Quantitative summary of normalized FRET ratios over the growth cone area at various time points after addition of ACEA, AM251, NGF (100 ng/ml), or vehicle normalized to the average FRET ratio value over the same area prior to addition of pharmacological agents in developing neurons derived from wild-type mice. (D) Preserved effect of NGF and loss of effects of ACEA as well as AM251 on Rac1 activity in developing cortical neurons derived from CB1^-/- mice. Values in panels C and D represent the mean ± SEM and are derived from analyses on 10–16 neurons per group over at least three independent culture experiments. (E, F) Immunoblot analyses showing changes in phosphorylation state of Serine 397 (pSer397) in WAVE1 upon treatment with ACEA (100 nM) or AM251 (600 nM) as compared to vehicle treatment in cortical neurons derived from wild-type mice without pretreatment (E), with overnight pertussis toxin (PTX) (100 ng/ml) pretreatment or from CB1^-/- mice (F). (G) Quantitative summary of cannabinoid-induced modulation of pSer397 WAVE1 levels normalized to βIII-tubulin in the above groups (<i>n</i> = 5–6 independent culture experiments). All graphs represent mean values ± SEM *<i>p</i> < 0.05, two-way ANOVA for repeated measures (C, D) or one-way (G) ANOVA followed by posthoc Tukey’s test. N.s. stands for not significant.</p

CB1 receptor is expressed in axonal as well as dendritic compartments of primary neurons matured over 4 wk in vitro.

<p>(A) Coimmunostaining against CB1 receptor and dendritic marker MAP2 on 28 days in vitro (DIV) cortical neurons derived from wild-type mouse embryos (upper panels) shows the distribution of CB1 receptor in the axonal compartments (MAP2-negative) as well as dendritic compartments (MAP2-positive). The rabbit anti-CB1 antibody used throughout this experiment did not yield substantial staining in cortical neurons cultured from CB1-deficient mouse embryos at 28 DIV (lower panels). (B) Magnified images represent white boxed areas from main images. (C) CB1 receptor colocalizes with CamKII-GFP and the synaptic protein PSD95 in dendritic compartments, including several spine heads (white arrow heads), indicating postsynaptic localization. (D) Heterologously-expressed CB1-EGFP in mature cultured cortical neurons shows axonal and dentritic localization upon costaining with MAP2. Phalloidin is used to counterstain the entire actin cytoskeleton. Phall. stands for Phalloidin. (E) Dendritic (arrows) ad axonal (arrowhead) localization of endogenous CB1 in cortical neurons cultured from wild-type mice and matured at 28 DIV. White scale bars represent 20 μm and yellow scale bars represent 5 μm.</p

Role of spinally-expressed WAVE1 in nociceptive activity-induced structural plasticity, inflammatory pain, and cannabinoidergic analgesia in vivo.

<p>(A, B) An immunoblot example and quantitative data from western blot analysis showing down-regulation of WAVE1 in spinal cord segments L3–L5 after intrathecal in vivo application of WAVE1 siRNA as compared to control siRNA. (C) Normal basal spine density but lack of CFA-induced spine remodeling in lamina II/V lumbar spinal neurons at 24 h post-CFA in mice intrathecally injected with WAVE1 siRNA as compared to control siRNA (<i>n</i> = 16–17 spines counted over four mice per group). (D) Frequency of paw withdrawal responses to mechanical force via plantar application of graded von Frey hairs recorded prior to (dashed lines) and 24 h after hind paw intraplantar injection of CFA (solid lines). Leftward shift in response curves following CFA (indicative of hypersensitivity) is diminished with intrathecal siRNA-mediated WAVE1 knockdown (red symbols) as compared to mice intrathecally injected with control siRNA (black symbols). (E) Mechanical hypersensitivity at 24 h post-CFA is significantly reduced by intrathecal ACEA injection over 24 h in mice intrathecally treated with control siRNA (black squares in E, upper graph), but not in mice intrathecally treated with WAVE1 siRNA (red squares in E, lower graph). (F) Quantitative summary of dendritic spine density in laminae II or V neurons in L3–L5 segments of mice which were tested behaviorally in panels animals in (E). Intrathecal ACEA reduces spine density in control siRNA-injected CFA-inflamed mice, but not in WAVE1 siRNA-treated CFA-inflamed mice (16–18 neurons counted from over four mice per group). All graphs represent mean values ± SEM, <i>n</i> = 7–9 mice per group in panels D & E. *<i>p</i> < 0.05 as compared to basal values within the group and †<i>p</i> < 0.05 as compared to corresponding control, two-way (D, E) and one-way (B, C and F) ANOVA for repeated measures followed by posthoc Tukey’s test.</p

Cannabinoids regulate growth cone morphology via CB1-Gi-Rac1-WAVE1 signaling.

<p>(A) Characterization of changes in growth cone morphology in developing cultured cortical neurons immunostained with anti-pGAP43 to recognize normal or enlarging growth cones and Phalloidin to label actin. Insets show normal, enlarged, or collapsed morphology in developing cortical neurons; scale bar represents 10 μm in all images and insets. (B) Bidirectional modulation of the proportion of growth cones with collapsed or enlarged morphology upon treatment with ACEA (100 nM), AM251 (600 nM) or vehicle (DMSO 1:30,000) for 1 h in neurons derived from wild-type mice (<i>n</i> = 10 independent culture experiments), but not in neurons from CB1^-/- mice (<i>n</i> = 4). (C–F) Lack of modulation of cannabinoidergic modulation of growth cone morphology upon treatment in the presence of PTX (100 ng/ml), Rac1 inhibitor, CAS 1090893-12-1 (50 μM) or in neurons with siRNA-mediated downregulation of WAVE1; control siRNA-transfected neurons showed significant cannabinoidergic modulation (<i>n</i> = 4). All graphs represent mean values ± SEM. *<i>p</i> < 0.05, one-way ANOVA followed by posthoc Tukey’s test. N.s. stands for not significant.</p

Increased localization of colocalization of WAVE1 and CB1-EGFP at the cell membrane in heterologously-transfected COS7 cells upon ACEA treatment.

<p>(A) Distribution of heterologously-transfected WAVE1 in cells cotransfected with either GFP (control, upper panels) or CB1-EGFP (lower panels) following treatment with vehicle (DMSO 1:30,000) or CB1 agonist (ACEA 100 nM) for 45 min. Scale bar represents 5 μm. (B) Quantitative summary of intensity of WAVE1-immunoreactivity relative to Phalloidin-stained actin at the plasma membrane in GFP- or CB1-EGFP-coexpressing COS7 cells treated with vehicle (white bars) or ACEA (red bars). (C) Quantitative summary of CB1-EGFP-WAVE1 colocalization in vehicle- or ACEA-treated cells calculated as a fraction of the total intensity of CB1-EGFP fluorescence. Values in panels B and C represent the mean ± SEM and are derived from analyses on at least 15 COS7 cells each over several independent culture experiments. *<i>p</i> < 0.05 one way ANOVA followed by posthoc Tukey’s test.</p

Visualization of cannabinoidergic modulation of actin dynamics in neuronal growth cones mediated by CB1 and WAVE1.

<p>(A) Real time images of wild-type developing cortical neurons transfected to express LifeAct-GFP, a biosensor for levels of F-actin prior to and at different time points following treatment with ACEA (100 nM), AM251 (600 nM), or vehicle. Scale bar represents 10 μm. (B, C) Analysis on the F-actin dynamics based on cannabinoid-induced changes in LifeAct-GFP intensity (B) and area (C) over time in growth cones of cultured cortical neurons derived from wild-type mice (<i>n</i> = 15–17 neurons per group from 5 independent culture experiments). (D, E) However, no changes in LifeAct intensity (D) nor area of the growth cones (E) were observed with cultured cortical neurons derived from CB1^-/- mice (<i>n</i> = at least 4 neurons per group from 2 independent culture experiments) after treatment. (F, G) Similarly, cultured cortical neurons with down-regulated WAVE1 showed no changes in LifeAct intensity (F) or area of growth cones (G) after cannabinoid treatment (<i>n</i> = at least 4 neurons per group). (H, I) Moreover, the abrogation of the effects after cannabinoid treatment can be contributed to the down-regulation state of WAVE1, since cultured cortical neurons nucleofected with scrambled siRNA, in turn show changes in LifeAct intensity (H) and area (I) of the growth cone following cannabinoid treatment (<i>n</i> = at least 4 neurons per group). (J, K) Immunoblot representation of WAVE1 down-regulation upon siRNA delivery in developing cortical neurons (J) and the corresponding quantification (K) (<i>n</i> = 6). All graphs represent mean values ± SEM *<i>p</i> < 0.05, two-way ANOVA (B-I) or one-way ANOVA (K) for random measures followed by posthoc Tukey’s test.</p