Figure 1.
The IGF-1R β subunit is enriched at the distal-most third of the axon in adult RGC regenerating in culture.
A) Western blot of protein from total retina disaggregated cells (RC), a fraction enriched in RGC (RGCF-see methods) and the same fraction after 4 days in culture (RGCF 4DIV) detected using an anti-βgc antibody. Equal amounts of protein from the three different fractions were loaded onto each lane. Note the increased intensity of βgc staining in the RGC 4DIV fraction. Numbers below are the O.D. (arbitrary units) +/− s.e.m. n = 3 independent experiments. B) Western blot prepared as in Fig. 1 A developed using a polyclonal antibody to the IGF-1R β subunit (C20-Santa Cruz Biotechnology). Note the increased intensity of C20 staining in the RGC 4DIV fraction. C) Triple immunofluorescence micrographs of a rat adult RGC regenerating in culture after 3 DIV showing the distribution of the RGC marker NF200 (blue, top panel), the axonal marker Tau-1 (red, second panel) and the β subunit of the IGF-1R (βgc) (green, third panel) in adult RGC neurons regenerating in culture. The lower panel shows a merged image of the three stainings. Note the enrichment of βgc at the distal end of the axon (arrow). Calibration bar = 20 µm.
Figure 2.
Stimulation with IGF-1 triggers the polarized activation of the IGF-1R and PI3K in the axons of adult rat RGC regenerating in culture.
A) Triple immunofluorescence micrographs of a rat adult RGC regenerating in culture after 3 DIV showing the distribution of the RGC marker NF200 (blue), the axonal marker Tau-1 (red) and the phosphorylated (active) form of the IGF-1R (green, pIGF-1R). Cells were cultured for 3 days, deprived of insulin for 4 h and stimulated for 5 min in control medium or medium containing 10 nM IGF-1. Note the significant activation of the IGF-1R, especially at the distal third of the axon (arrow). B) Triple immunofluorescence micrographs of a rat adult RGC regenerating in culture after 3 DIV showing the distribution of the RGC marker NF200 (blue), the axonal marker Tau-1 (red) and the phosphorylated form of the PI3k regulatory subunit p85 (green, p-p85). Cells were treated as described above (Fig. 1A). Note the pronounced activation of PI3K, especially within the distal axon (arrow). Calibration bar = 20 µm.
Figure 3.
Treatment with the PI3K inhibitor LY 294002 or the IGF-1R blocking antibody αIR3 prevents axon formation in adult RGC in culture.
A) Double immunofluorescence showing the distribution of the RGC marker NF200 and the axonal marker Tau-1 in cultured adult RGC. Cells were cultured for 3 days in control conditions or in the presence of 20 nM LY 294002 (LY) or αIR3 (10 mg/ml). Calibration bar = 40 µm. B) Percentage (+/− s.e.m.) of adult RGC cultured for 3 days in the different conditions described above (Fig 3A), exhibiting or lacking axons. n = 3 independent experiments. At least 100 cells were scored for each condition. * Significantly different from LY and a IR3 p≤0,0005. C) Cell viability (determined by the criterion of propidium iodide exclusion [52]) after 1 or 4 DIV. n = 3 independent experiments. At least 100 neurons were scored for each condition.
Figure 4.
Silencing IGF-1R expression prevents axonal formation in adult RGC in culture.
A) Double immunofluorescence of adult RGC after 3 days in culture transfected with scrambled sequence RNA (ssRNA) or shRNA directed to the IGF-1R (shRNA) showing the distribution of the IGF-1R β subunit and the axonal marker Tau-1. GFP shows the efficiency of cell transfection. Calibration bar = 10 µm. B) Percentage (+/− s.e.m.) of adult RGC treated as described above (Fig. 4A) exhibiting or lacking axons. n = 3 independent experiments. At least 100 cells were scored for each condition. * Significantly different from shRNA p≤0,0005. C) Cell viability (determined by the criterion of propidium iodide exclusion [52]) after 4 DIV. n = 3 independent experiments. At least 50 neurons were scored for each condition.
Figure 5.
Co-transfection of IGF-1R-suppressed adult RGC with a constitutively active form of the PI3k catalytic subunit p-110 restores the ability of the neurons to form axons.
Double immunofluorescence of adult RGC showing the distribution of NF200 (displaying neuronal shape) and myc (marker of transfection with cDNA encoding for a constitutively active form of the PI3K regulatory subunit p110). GFP is a marker of transfection efficiency with the shRNA directed to IGF-1R. Cells were transfected and cultured for 4 days. Calibration bar = 10 µm.