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. 2008 Jul 2;28(27):6926–6937. doi: 10.1523/JNEUROSCI.0800-08.2008

Figure 6.

Figure 6.

Vinblastine effects on AV accumulation and autophagy induction. A, Western blot analyses of p-p70 and total p70 after exposure of neurons to vinblastine (10 μm) for 1–24 h. B, Densitometric analyses of immunoblots in A (n = 6): ratios of p-p70 relative to total p70 are expressed as percentages of the control value from each set of treatments (*p < 0.05; **p < 0.01; ***p < 0.001). Error bars indicate SEM. C, Representative immunoblots of LC3-I and LC3-II in neurons exposed to vinblastine (10 μm) for 1, 6, and 24 h. D, Densitometry of LC3-I, LC3-II, and tubulin levels after neurons exposed to vinblastine (10 μm) for 1, 6, and 24 h (n = 6; *p < 0.05, **p < 0.01). E–H, Immunostaining of primary neurons stained with TUJ-1 antibody for neuron-specific β-III-tubulin. Tubulin remains intact when autophagy is induced (F) or lysosomal proteolysis is inhibited by leupeptin (20 μm) (G), but is disrupted after 24 h treatment with vinblastine (10 μm) (H). Scale bars, 5 μm. I, J, Live image of BODIPY-pepstatin-FL in DsRed-LC3 transfected neurons after treatment with vinblastine (10 μm). Autophagosomes that have not fused with lysosomes are the predominant AVs accumulating after 1 h of vinblastine (I, right panel). J, After 6 h of vinblastine, LC3 mostly colocalizes with BODIPY-pepstatin vesicles indicating that fusion between autophagosomes and lysosomes occurs despite impairment in microtubule-dependent lysosome transport. K, AV accumulation seen in vinblastine (10 μm; 1 h)-treated neurons immunostained with LC3 antibody. L, Quantification of vesicular profiles in DsRed-LC3 transfected cells loaded with BODIPY-pepstatin. The average number of each vesicle type is shown as a percentage of the total number: red, autophagosome (AP); orange, autolysosome (AL); yellow, lysosomes with minimal traces of DsRed-LC3; green, lysosomes. In untreated neurons transiently expressing DsRed-LC3 loaded with BODIPY-pepstatin, most vesicles (∼80%) are lysosomes. Autophagosomes are efficiently fused with lysosomes after rapamycin because the proportion of autophagosomes remains very low, and only autolysosomes are increased. Between 1 and 6 h of vinblastine treatment, autophagosome maturation also occurs as indicated by the increased proportion of autolysosomes.