Figure 5.

Cytoplasmic FMRP granules sequester HDAC6 and enhance TDP-43 aggregation. A, FMRP–GFP expressing cells were double-labeled to show co-localization between FMRP granules (green) and endogenous HDAC6 (red). Cyan arrowheads highlight FMRP granules that recruited HDAC6. Scale bar, 20 μm. B, QBI-293 cells co-expressing cytoplasmic TDP-43 (TDP-43–ΔNLS) in the presence or absence of FMRP–GFP were treated with the HDAC6-specific inhibitor tubastatin A (TBST) (5 μm for 12 h). Cell lysates were sequentially extracted to generate soluble (RIPA-extracted) and insoluble (UREA-extracted) fractions followed by immunoblotting with TDP-43 and GAPDH antibodies. Myc-tagged TDP-43 was distinguished from endogenous TDP-43 (end. TDP-43) based on their distinct molecular weight differences. C, protein band intensities were quantified using ImageQuant TL software (version 8.1), and the values were represented as relative intensities normalized to the GAPDH loading control. D and E, cells co-expressing TDP-43–ΔNLS–2KQ and FMRP–GFP were treated with TBST, where indicated, and triple-labeled to detect FMRP (green), total Myc-tagged TDP-43 (red), and P-409/410 (blue). The number of inclusions and average inclusion size per cell were measured using ImageJ software and plotted in E. F, QBI-293 cells co-expressing aggregate-prone TDP-43–ΔNLS–2KQ and FMRP–GFP along with either control vector (pcDNA3.1, lanes 1–3), WT HDAC6 (lanes 4–6), HDAC6-ΔBUZ lacking residues 1045–1215 (lanes 7–9), or HDAC6 1–840 lacking residues 841–1215 (lanes 10–12) were extracted, analyzed by immunoblotting, and quantified by densitometry in G. All HDAC6 constructs were functional, as determined by deacetylation of tubulin. Error bars indicate S.E., and p values were calculated by Student's t test. ***, p < 0.001; **, p < 0.01; *, p < 0.05. Ectopic expression of FMRP–GFP or inhibition of HDAC6 increased TDP-43 aggregation while restoring HDAC6 expression suppressed TDP-43 aggregation. ns, not significant.