Fig. 2. HDAC6 co-aggregates with tau and accumulates in pathological tau lesions.

a Primary cortical neurons from mouse embryos (E16) of tau knockout (KO) mice were cultured for 10 days in vitro (10 DIV) and transfected with P301L Tau-GFP (green) and HDAC6-FLAG (FL) (red) expression constructs followed by imaging. Arrows indicate regions of extensive co-localization along processes. Scale bar = 50 μm. b Primary cortical neurons (E16) from WT (top) and PS19 tau transgenic (bottom) mice were analyzed by immunofluorescence (IF), detecting co-localized focal swellings of tau (green), endogenous HDAC6 (red), and the nuclei marked by DAPI (blue). The white rectangle highlights a magnified insert of a neuronal process harboring tau-positive foci. Scale bar = 50 μm. c Biochemical fractionation of soluble high-salt fractions and insoluble urea fractions were extracted from frontal cortex of control and AD brains followed by immunoblotting to detect HDAC6, tau, Hsc70, and acetylated tubulin. The arrows highlight acetylated tubulin fragments ~37 kDa and higher molecular weight species ~150 kDa. d, e Immunoblotting results were quantified by densitometry. f AD cortical sections were analyzed by double-labeling IF using either conformational tau (MC1) or total tau (TAU-5) antibodies (blue), combined with Thioflavin S (ThS) (green), and HDAC6 (red) to evaluate localization in proximity to AD neuritic plaques. White arrows highlight regions of tau and HDAC6 immunoreactivity that are associated with ThS positive plaques. Scale bar = 50 μm. Statistical tests: p value determined by two-sided unpaired t-test with Welch’s correction from n = 5 biologically independent samples (d, e). Representative neurons are shown from n = 3 biologically independent experiments (a, b) and n = 3 independent AD brains (f). Error bars represent means ± SEM and are plotted relative to control (d) or relative to AD (e). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.