Role of GPR55 during Axon Growth and Target Innervation

GPR55 protein expression in retinal explants and primary cortical neurons. A, Expression of GPR55 in the mouse cortex and (B) retina at different developmental stages. C, Expression of GPR55 in the retina of gpr55^+/+ and gpr55^-/- mouse embryo (E14/15). D–F, Expression of GPR55 in retinal explants from gpr55^+/+ and gpr55^-/- mice, and in the presence of the specific blocking peptide (BP). G–I, E14/15 axons and growth cones of retinal explants from gpr55^+/+ and gpr55^-/- mice, and in the presence of the specific BP. J–L, The expression of GPR55 in dendrites (MAP2) and (M–O) axons (NFM) of RGCs. P, Expression of GPR55 in primary cortical neuron using GPR55 antibody, (Q) using 3 µm of specific GPR55 fluorescent Tocrifluor ligand T-1117 and (R) using GPR55 antibody in a purified RGC culture. Scale bars: D–F, 100 μm; G–I, P–R, 10 μm; J–O. 5 μm.

GPR55 ligands reorganize the morphology of the GC and modulate axon growth via a cannabinoid independent pathway. A–C, Growth cone surface area and filopodia number of retinal projection GCs after a 60 min treatment with GPR55 agonists LPI (1 µm) and O-1602 (300 nm) or antagonist CBD (300 nm) in WT, cnr1^-/- , and cnr2^-/- mice. D, E, Total retinal neurite growth of retinal explants cultured for 1 DIV and treated for 15 h with LPI (1 µm), O-1602 (300 nm), and CBD (300 nm) in WT, cnr1^-/- , and cnr2^-/- mice. Scale bars: A, 5 μm; D, 100 μm. Values are presented as mean ± SEM. *Indicates significant changes between LPI, O-1602, or CBD compared with control in B, C, and E; p < 0.004.

GPR55 mediates the reorganization of the GC morphology and the modulation of axon growth. A–D, Basal growth cone surface area, filopodia number and total neurite outgrowth in gpr55^+/+ and gpr55^-/- retinal explants. E–G, Growth cone surface area and filopodia number in gpr55^+/+ and gpr55^-/- retinal explants treated for 1 h with LPI (1 µm), O-1602 (300 nm), or CBD (300 nm). H, I, Total neurite outgrowth in gpr55^+/+ and gpr55^-/- retinal explant treated for 15 h with LPI, O-1602, or CBD at the same previously cited concentrations. Scale bars: A, E, 5μm for GC; A, H, 100 μm for explants. Values are presented as mean ± SEM. *Indicates a significant change induced by LPI, O-1602, or CBD compared with the control in F, G, and I; p < 0.0001; #Indicates a significant change between LPI, O-1602, or CBD and the control in gpr55^+/+ compared to gpr55^-/- in F, G, I, and B–D; p < 0.004.

At low concentration, GPR55 ligand modulates GC morphology and axon growth via the ERK1/2 pathway. A, Expression of P-ERK-1/2, ERK-1/2 and β-actin in primary cortical neurons incubated with one of the following: 1 µm LPI, 300 nm O-1602, or 300 nm CBD at 37°C for 2, 5, and 20 min. The antibody β-actin was used to verify (and correct for) equal loading in all lanes. B, Histogram illustrating the quantification of ERK-phosphorylation. C, ERK phosphorylation state following 15 min pretreatment with CI-1040, an ERK1/2 inhibitor, before the incubation with or without 1 µm LPI, 300 nm O-1602 or 300 nm CBD. D, E, AKT and PKA phosphorylation states, no significant variations were observed in the presence of GPR55 ligands for the times indicated. F–H, Growth cone surface area and filopodia number of retinal explant treated with GPR55 agonists in the presence or the absence of the ERK inhibitor. I–J, Total projection growth of retinal explant cultures treated with GPR55 agonists LPI and O-1602 in the presence or the absence of CI-1040. Scale bars: F, 10 μm; I, 100 μm. Values are presented as mean ± SEM. *Indicates a significant change compared with the control group in B, G, H, and J; p < 0.0001. #Indicates a significant change induced by the ERK inhibitor in G, H, and J; p < 0.004.

At a higher concentration, LPI activates RhoA pathway and produces growth cone collapse and neurite repulsion. A, B, Using p-RhoA and RhoA antibodies, RhoA activation was determined analyzing the respective phosphorylation state by Western blotting following treatment with 1 or 10 µm LPI. C–E, Growth cone surface area and filopodia number of retinal explants after treatment with GPR55 agonist in the presence or the absence of the ROCK1 inhibitor, Y-27632. F, G, Total projection length of retinal explant treated with GPR55 agonist LPI (1 or 10 µm) in the presence or the absence of the Y-27632. Scale bars: C, 5 μm; F, 100 μm. Values are presented as mean ± SEM. *indicates a significant change compared to the control group; #indicates a significant change induced by CBD (300 nm) or Y-27632 (20 µm) in D, E, and G; p < 0.0001 and p < 0.001 in B.

Pharmacological modulation of GPR55 affects RGC turning in vitro. A, Photomicrographs of time-lapse microscopy from 1 DIV mouse retinal explant growth cone taken at t = 0 min and t = 60 min during GC turning assay experiments. Black arrows indicate the direction of the microgradient, whereas blue arrowheads indicate initial GC position. Green arrowheads show the GC position following neurite attraction and red arrowheads indicate the GC position after repulsion. B, C, Superimposed RGC axon trajectories over the 60 min observation period for vehicle and LPI; no significant changes were observed on growth cone behavior in the presence of the vehicle, whereas LPI increased axon growth and turning toward the pipette tip. Black arrows indicate the direction of the gradient. D, Turning angle cumulative frequency curves of RGC growth cones. The turning angle of each growth cone was plotted against the percentage of growth cones turning that angle or less. E, Quantification of neurite elongation and GC turning responses following drug stimulation. F, Representative photomicrograph of the effect of repulsion and GC collapse created during 60 min stimulation with 10 µm LPI. Scale bars: A, F, 40 μm. Values are presented as means ± SEM; *indicates significant change compared with the vehicle in E; p < 0.0001.