Fig. 8
TFEB acetylation at K310 is required for HDAC7 deletion-mediated TFEB nuclear translocation, lysosomal biogenesis and tau clearance in astrocytes. A Identification of TFEB acetylation atK310 by mass spectrometry analysis. B Quantification of TFEB transcriptional activity in HEK293T cells with different point mutations (K to Q). n = 4 per group. C Protein sequence alignment of TFEB homologues in different species. The red mark represents the identified K310 residue. D, E Immunoprecipitation assay showing the acetylation level of exogeneous WT/K310R Flag-TFEB in control and HDAC7 knockout astrocytes. The acetylation level of Flag-TFEB was quantified. n = 3 per group. F Representative immunostaining images of WT/K310R Flag-TFEB in control and HDAC7 knockout astrocytes. Scale bar: 10 μm. G Quantification of Flag-TFEB nuclear/cytoplasmic ratio in F. n = 30 (HDAC7+/++WT-TFEB), 34 (HDAC7-/-+WT-TFEB), 30 (HDAC7+/++K310R-TFEB), 31 (HDAC7-/-+K310R-TFEB). H Analysis of Lamp1, Ctsb, Ctsd and Uvrag mRNA levels by qPCR in control and HDAC7 knockout astrocytes treated as indicated. n = 4 per group. I Representative images showing colocalization of tau-pff and Lysotracker Red in primary astrocytes overexpressed with WT/K310Q/K310R-TFEB or vector. Scale bar = 10 μm. Cells were treated with tau-pff for 4 h and then co-staining with Lysotracker Red. J Manders’ coefficient analysis of tau-pff and lysotracker colocalization in I. n = 10 per group. K Analysis of intracellular tau by ELISA in astrocytes overexpressed with WT/K310Q/K310R-TFEB or vector and then treated with tau-pff for indicated time period. n = 3 per group. L After incubation with tau-pff for 4 h, tau-pff was removed and cells are continuously cultured for 30–180 min. At various time points, intracellular tau was quantified by ELISA. n = 3 per group. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis. Data are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, n. s, not significant