Figure 4. Tsc2 deficiency and hyperactive Rheb inhibit ephrin-A-induced growth cone collapse.

Purified RGCs from Tsc2^+/− and wild-type littermates at P8 were stimulated with LPA (1µM) or pre-clustered ephrin-A1-Fc or Fc (control) proteins (5 µg/ml). Growth cone collapse was determined by phalloidin staining. (a) Representative micrographs showing staining with rhodamine-phalloidin (red) and anti-Tuj1 (green) of RGCs with intact versus collapsed growth cone. Scale bar, 10 µm. (b) Quantification of the percentage of RGCs with collapsed growth cone. Data represent mean ± s.e.m.. Ephrin-A1-Fc stimulated collapse was dramatically reduced in Tsc2^+/− compared to wild-type RGCs (***: P < 0.001 by t-test). There is no significant difference in LPA-treated growth cones from the two genotypes. (c) The lentiviral construct was used to over-express either wild-type Rheb (wtRHEB) or a constitutively active mutant (caRHEB, S16H), or GFP-alone (GFP) as the control in Tsc2^+/+ cells. Western analyses verified higher levels of Rheb, phospho-S6K1 (Thr389) and phospho-S6 (Ser235/6) in wtRHEB and caRHEB infected hippocampal neurons than those in GFP or non-infected (N.I) neurons, in either 2% B27 medium or B27-lacking medium for 16 hours. Full-length gels are presented in Supplementary Figure S13. (d) Quantification of the percentage of growth cone collapse in purified RGC cultures infected with wtRHEB, caRHEB or GFP lentivirus. Results represent mean ± s.e.m. from 3 independent experiments. RGCs with over-expression of wtRHEB or caRHEB have significantly less collapse than GFP expressing RGCs (by t-test **P < 0.01 comparing GFP infected cells in Fc versus ephrin-A1 stimulation; *P < 0.05 comparing wtRHEB infected cells in Fc versus ephrin-A1 stimulation for; *P < 0.02 comparing ephrin-A1 stimulations between GFP and wtRHEB, or GFP and caRHEB infected neurons; and ns = not significant).