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

(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 (n = 5–6 independent culture experiments). All graphs represent mean values ± SEM *p < 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.

(A) 用于检测Rac1活性的Raichu-Rac1荧光共振能量转移（FRET）生物传感器的示意图。(B) 经大麻素1型受体（CB1）激动剂ACEA（100 nM）与CB1反向激动剂AM251（600 nM）处理后，发育中小鼠皮层神经元内Rac1活性变化的实时成像图。FRET信号强度以伪彩色热图形式呈现，内嵌小图展示生长锥的放大视图。白色比例尺对应20 μm，内嵌图中的黄色比例尺对应5 μm。(C) 本部分为定量汇总结果：以野生型小鼠来源的发育神经元在添加药理学试剂前同一区域的平均FRET比值作为归一化参照，统计添加ACEA、AM251、神经生长因子（NGF，100 ng/ml）或溶剂后，各时间点生长锥区域内的归一化FRET比值。(D) 在CB1基因敲除（CB1-/-）小鼠来源的发育皮层神经元中，NGF对Rac1活性的调控作用得以保留，而ACEA与AM251的调控作用消失。C、D两面板中的数值以平均值±标准误（SEM）表示，数据来源于每组10~16个神经元的至少3次独立培养实验分析。(E, F) 免疫印迹分析结果显示：分别对未预处理的野生型小鼠皮层神经元（E）、经百日咳毒素（PTX，100 ng/ml）过夜预处理的野生型小鼠皮层神经元，以及CB1-/-小鼠皮层神经元施加ACEA（100 nM）、AM251（600 nM）或溶剂处理后，WAVE1蛋白丝氨酸397位点（pSer397）的磷酸化状态变化。(G) 本部分为上述各组中，大麻素类物质对pSer397修饰的WAVE1蛋白水平的调控效应的定量汇总，该蛋白水平以βIII微管蛋白作为内参进行归一化处理（独立培养实验次数n=5~6）。所有图表均以平均值±标准误展示，*p < 0.05；C、D面板采用重复测量双因素方差分析，G面板采用单因素方差分析后辅以Tukey事后检验。N.s.代表无显著性差异。