Table 1.
List of the primers used for qPCR analyses.
Figure 1.
Neural stem cells (NSCs) express high levels of CB1 receptor.
(A) Bright field photographs of murine NSCs forming neurospheres from single cell suspensions after one, two and three days in EGF and b-FGF. (B) Bar graph representing the clonogenic capacity of NSCs at the 2nd and 10th passage (n = 6). (C) Immunofluorescence analysis showing NSCs cultured in differentiating conditions (devoid of EGF and b-FGF, and supplemented with 1% FCS) and stained with the neuronal marker β-III tubulin (left panel), the oligodendrocyte marker O4 (middle panel), and co-stained with the astrocyte marker GFAP (red in the right panel) and with the neural progenitor marker Nestin (green in the right panel). Cells positive for both GFAP and Nestin were considered neural progenitors that are differentiating toward astrocytes (i.e. immature astrocytes). (D) qPCR analysis showing the levels of CB1 and CB2 receptor transcripts, normalized for levels of the housekeeping gene β-actin. (E) Western blot analysis showing the expression levels of CB1 and CB2 receptor proteins in extracts obtained from NSCs, brain and spleen.
Figure 2.
CB1 receptor activation promotes neuronal differentiation of NSCs in vitro.
(A) The bar graph represents quantitative data (expressed in % of cells) of the immunofluorescence analysis of the effect exerted by anandamide (AEA) on NSCs cultured in differentiating condition. Immunofluorescence analysis was performed as described in Figure 1C to detect neurons (β-III positive cells), oligodendrocytes (O4-positive cells), astrocytes (GFAP-positive cells) and progenitor cells (Nestin-positive cells) in control (black bars) and AEA-treated (grey bars) cells. Data represents the mean ± standard deviation (SD) of 4 experiments. Statistical analysis was performed using the paired t-test; p value of statistically significant differences is reported above the bars. (B) The bar graph represents quantitative data (expressed in % of β-III tubulin-positive cells) of the immunofluorescence of neuronal differentiation of NSCs cultured in differentiating condition for 3 days in the presence or absence of AEA (1 µM), with or without the CB1 (AM251, 1 µM) or CB2 (AM630, 1 µM) antagonist, ACEA (1 µM) with or without the CB1 antagonist (AM251, 1 µM), and JWH133 (1 µM). Data represent the mean ± SD of 4 experiments. Statistical analysis of control versus treatment was performed using the paired t-test; * = p≤0.05, the p value of direct comparison of AEA+AM251 is indicated above the bars grouped by brackets. Where not indicated, samples did not show statistically significant changes.
Figure 3.
Selective activation of CB1 receptor is sufficient to promote neuronal differentiation of NSCs.
(A) Dose response of the effect of ACEA (0.1–1 µM) on the neuronal differentiation of NSCs in vitro. (B) Quantitative analysis of the effect of 1 µM ACEA on the differentiation of NSCs in neurons (β-III tubulin-positive cells), oligodendrocytes (O4-positive cells), astrocytes (GFAP-positive cells) and progenitor cells (Nestin-positive cells) in control (black bars) and AEA-treated (grey bars) cells. (C) Time course analysis (1–6 days) of the effect of ACEA on the neuronal differentiation of NSCs (expressed as % of β-III tubulin-positive cells). (D) Quantitative analysis of the effect of ACEA on apoptosis of NSCs undergoing differentiation. Apoptosis was measured by immunofluorescence analysis of DNA fragmentation (TUNEL assay) in control (black bars) and AEA-treated (grey bars) cells. Data of all the experiments represent the mean ± SD of at least 3 independent experiments. Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples and the bars grouped by brackets.
Figure 4.
CB1 receptor stimulation enhances maturation of the in vitro differentiated neurons.
(A) Representative images of the immunofluorescence analysis of β-III tubulin in cells differentiated from NSCs after three days of culture in 1% FBS in the presence or absence of 1 µM ACEA. (B–D) Bar graphs represent quantitative data of morphometric analyses of number (B), length (C) and branching level of neurites (D) in cells treated as described in (A). Data represent the mean ± SD of 4 experiments. Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples.
Figure 5.
Reduced activity of ERK1/2 kinases promotes neuronal differentiation of NSCs.
(A) Western blot analysis of the phosphorylation levels of ERK1/2 (p-ERK1/2) and rpS6 (p-rpS6), a downstream target of the mTOR pathway. Cell extracts (30 µg) from NSCs treated with or without ACEA (1 µM) and the CB1 antagonist AM251 (1 µM) in the presence of 1% FBS for the indicated time were analysed. (B) Representative images of the immunofluorescence analysis of β-III tubulin in cells differentiated from NSCs after three days of culture in 1% FBS in the presence or absence of 10 µM U0126, a selective inhibitor of the ERK1/2 pathway. (C) The bar graph represents quantitative data (expressed in % of β-III tubulin-positive cells) of the immunofluorescence analysis of neuronal differentiation of NSCs cultured in differentiating condition in the presence or absence of 10 µM U0126. (D–F) Bar graphs represent quantitative data of morphometric analyses of the number (D), the length (E), and branching level of neurites (F) in cells treated as described in (C). Data represent the mean ± SD of 6 experiments. Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples.
Figure 6.
CB1 receptor activation during neuronal differentiation of NSCs modulates gene expression.
(A–B) Results of the Neurogenesis and Neural Stem Cell PCR array. List of the genes up-regulated (A) and down-regulated (B) in NSCs undergoing differentiation for 24 hours in the presence of 1% FBS and 1 µM ACEA, obtained from the PCR Array analysis. (C) Validation of the results of the PCR array by qPCR analysis of seven genes (Drd2, Pax5, Slit2, Gdnf, Shh, Dll1 and Pax6) selected for their relevance for neuronal differentiation or NSC stemness. Data (mean ± SD of 5 experiments) are expressed as fold increase of ACEA-treated versus control samples, using Gapdh expression as standard control (see Materials and Methods). Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples.
Table 2.
Description of selected genes modulated by ACEA.