Figure 3.

Caspase-3 is required for axon degeneration following NGF withdrawal. A, E12.5 DRG explants from wild-type (WT) and Caspase-3 knockout (KO) mice were cultured for 2 days and subsequently deprived of NGF for 24 h using a function blocking antibody. WT explants had completely degenerated while we observed no apparent degeneration in the Caspase-3 KO. B, Identical findings were seen in Campenot chambers. Representative images are shown of three rows of axons per genotype. WT degeneration is shown after 36 h in anti-NGF, while the Caspase-3 KO cultures are shown following 60 h in anti-NGF to illustrate the magnitude of the protection observed. C, Quantification of phenotype from B. Axons are significantly protected from NGF deprivation at both 36 and 60 h; n = 6 Campenot chambers per genotype per time; *p < 0.001 at each time point, Student's t test. D, We observed protection against anti-NGF in Campenot chamber cultures treated with 30 μm z-DEVD-Fmk when the axons were from Caspase-3 heterozygous (Het) mice, whereas no protective effect was observed in WT axons. Representative images from the 36 h time point. E, Quantification of D. Significant protection is observed at the 36 h time point (p < 0.001, Student's t test, n = 5 per condition) but not at 60 h. Axons are visualized by TuJ1 immunoreactivity (A, B, D). F, Cleavage of Caspase-6 is visualized by immunostaining in WT explants deprived of NGF for 12 h. This cleavage is eliminated in Caspase-3 knockout explants. G, No apparent protection from injury-induced Wallerian degeneration of axons was observed in Caspase-3 knockout explants following physical severing of proximal axons in vitro. Caspase-6 knockout explants were also not protected (data not shown). H, Similarly, loss of Caspase-3 does not protect axons in Campenot chambers from degeneration elicited by the chemotherapeutic agent vincristine.