Figure 4

SRF-VP16 overcomes myelin- and astrocyte-associated neurite growth inhibition in vitro . Primary neurons were plated on Nogo, total myelin and CSPGs, followed by staining for expression of SRF via the VP16 domain and tubulin to visualize the entire neuron. (A-H) Primary cerebellar neurons were grown on either an inactive Nogo peptide (NogoΔ21; A, C, E, G) or the growth-inhibiting peptide NogoΔ20 (B, D, F, H). Neurons were expressing either the control protein SRFΔMADS-VP16 (A, B, E, F) or SRF-VP16 (C, D, G, H). Neurons were stained for SRF (green) and tubulin (red). The active Nogo peptide (B, F) reduced neurite growth of SRF-ΔMADS-VP16 expressing neurons compared to the permissive control substrate NogoΔ21 (A, E). Expression of SRF-VP16 stimulated neurite growth on the inhibitory Nogo substrate (D, H). Arrowheads point at individual VP16-positive neurons. (E-H) represent higher magnification images of individual neurons with nuclear SRF localization (in green). (I-N) Quantification of neurite growth on Nogo (I, L), myelin (J, M) and CSPGs (K, N). In (I, J, K) percentage of neurons with neurite growth is plotted for the various conditions. In (L, M, N) neurite length was quantified by taking only neurons into account with neurites grown longer than 50 μm. In each bar diagram, the condition reflecting control substrate and SRFΔMADS-VP16 was set to 100%. (O) SRF-VP16 requires actin dynamics to overcome Nogo-mediated neurite growth inhibition. In the presence of the actin depolymerizing agent Latrunculin B (Lat B), SRF-VP16 failed to elevate neurite outgrowth inhibited by Nogo. Numbers in bars depict independent cultures analyzed for each condition. Standard error is provided. Scale-bar (A-D) =100 μm; (E-H) =50 μm.