Figure 5

Proneurotrophins abolish retinal axon branching via p75^NTR. The outgrowth/branching assay was performed as described [6]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro. The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both proBDNF and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75^NTR, retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75^NTR, with an RNAi vector not affecting p75^NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75^NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc. ***P < 0.001; n.s., not significant, the error bars represent S.E, M.