Fig. 3: Stimulation with GDNF enhances WT axonal transport.

A Schematic representation of GDNF and GFRα1 transport experiments in three-compartment MFCs. Primary ventral horn neurons were cultured in the central (somatic) compartment of 3-channel MFCs, with axons projecting to two lateral fluidically isolated axonal chambers. 100 ng/ml GDNF, GFRα1, or both together were added to one axonal compartment, with PBS added to the other as an internal control. B–D Average endosome speeds following the addition of GDNF (B), GFRα1 (C), and GDNF + GFRα1 (D) in the right compartment and PBS in the left compartment (B–D). Each point represents a biological replicate. Connecting lines represent speeds observed in control and experimental condition-treated axons from the same cultures. Speeds were significantly increased with GDNF alone (*p = 0.02) and GDNF with GFRα1 (*p = 0.04), but not GFRα1 alone (p = 0.6799), as determined by two-tailed paired t tests. E Representative kymographs showing signalling endosome transport in axons of WT primary MN cultures following treatment with PBS, GDNF, GFRα1 or GDNF together with GFRα1. N = 3–5 biological replicates. Bars represent mean ± SEM. PBS results were collected from 443 endosomes in 42 videos for a total of 45 569 single movements (n = 5). GDNF results were collected from 218 endosomes in 22 videos for a total of 17 938 single movements (n = 4). GFRα1 results were obtained from 216 endosomes in 24 videos for a total of 21 981 single movements (n = 3). GDNF and GFRα1 results were collected from 400 endosomes in 29 axons for a total of 31 809 single movements (n = 5). F Representative western blot of primary MN lysates following starvation in Neurobasal and Glutamax, and treatment with 50 ng/ml BDNF, or 100 ng/ml GDNF, and GDNF and GFRα1 (both 100 ng/ml). G–I Western blot quantification of RET, AKT and ERK1/2 activation after starvation and stimulation with BDNF, GDNF and GFRα1. Starvation significantly decreased activation of AKT (****p < 0.0001) and ERK (****p < 0.0001), but not RET (one-way ANOVA with Dunnett’s multiple comparison test).