Figure 7. De-acetylation of p150^Glued increases its interaction with dynein.

A. Inhibition of dynein blocks p75NTR retrograde apoptotic signaling. Sympathetic neurons were cultured in microfluidic chambers and cell bodies (CB) and distal axons (DAx) were treated with 12.5 mM KCl (K) or 200 ng/ml BDNF, as indicated. During the last 8 hrs, 25 μm ciliobrevin A together with BDNF was applied to the distal axons (B+C). The neurons were then fixed and stained for TUJ1 and DAPI to quantify pyknotic nuclei. The bars depict the means ± SEM for n=3; **, p < 0.01, 2-way ANOVA with Tukey’s multiple comparisons test.

B. Inhibition of HDAC1 reduces the interaction of p150^Glued with dynein. (Left) HEK293 cells were treated with MS275 or left untreated (control), then fractionated and the cytosolic lysates immunoprecipitated with anti-p150^Glued, then western blotted for dynein intermediate chain (DIC). Also shown are the lysates blotted for p150^Glued and DIC. (Right) The average ratio of DIC pulled down to total DIC in the lysates ± SEM for n=3; *, p < 0.05, student’s t-test.

C. Activation of p75NTR enhances the interaction of p150^Glued with dynein. (Left) Neurons were treated with 200 ng/ml BDNF or 20 ng/ml NGF + 5 μM MS275 for 48 hrs. The neurons were then lysed, and immunoprecipitated with DIC and western blotted for p150^Glued or DIC. (Right) The average ratio of p150^Glued pulled down with DIC to total DIC in the lysates ± SEM for n=3; *, p < 0.05, student’s t-test.

D. Mutation of K230 to R in p150^Glued increases its interaction with dynein. (Left) HEK293 cells were transfected with Myc-p150^Glued wild type, K230R or K230Q mutant. The cells were then fractionated and the cytosolic lysates immunoprecipitated with anti-Myc and western blotted for DIC or p150^Glued, as indicated. Also shown are the lysates blotted for DIC. (Right) The average ratio of DIC pulled down to total DIC in the lysates + SEM for n=6; *, p < 0.05, student’s t-test.

E. Summary model. Axonal HDAC1 is required for retrograde apoptotic signaling by activation of p75NTR or trophic factor deprivation. HDAC1 enhances p75ICD transport in axons by deacetylating p150^Glued, which facilitates its binding to the dynein intermediate chain.