Figure 8.

Pharmacological and genetic inactivation of HDAC6 impairs GR chaperoning and blunts hormone- and stress-induced GR nuclear translocation. a, Confocal scan of the DR (40×) shows dual immunostaining for GR (red) and the 5-HTergic marker tryptophan hydroxylase (TPH, green). GR is expressed by most TPH⁺ cells but also by some TPH⁻ cells. b, c, Effect of HDAC6 on GR responses to hormonal stimulation in the RN46A immortalized raphe precursor cell line. b, In the absence of HDAC inhibitor pretreatment (VEH), DEX (1 μm) produces a shift from predominantly cytoplasmic GR immunostaining to a predominantly nuclear signal. Pretreatment with TSA (5 μm) and tubacin (Tub, 10 μm), two inhibitors with nanomolar affinity for HDAC6 prevented DEX-induced nuclear localization of GR, while the selective class I HDAC inhibitor NaBu (1 mm) was devoid of effect. c, Corresponding quantitative measures of DEX-induced changes in GR signal normalized to respective vehicle conditions (*p < 0.05, **p < 0.01, compared with VEH; n = 22–69 cells per condition). d, Thirty minutes following social defeat, the amount of nuclear GR increases significantly in the DR of WT mice, while pan-neuronal HDAC6 KO mice show no increase in nuclear GR in the DR at this time point. Representative bands are shown for each group (*p < 0.05, compared with WT control; n = 4–7 samples/group). e, Lysates from HDAC6 KO tissues were immunoprecipitated for total Hsp90 and blotted for acetylated Hsp90 (AcHsp90, K294), GR, or total Hsp90. While total levels of Hsp90 were not different between WT and KO mice, Hsp90 acetylation was increased upon neuronal depletion of HDAC6. Under these conditions, the association between Hsp90 and GR was dramatically diminished. Error bars indicate SEM.