Journal: bioRxiv

Article Title: Ketone body β-hydroxybutyrate restores neuronal Tau proteostasis via ketolysis-independent mechanism

doi: 10.64898/2026.01.30.702936

Figure Lengend Snippet: (A) Normalized protein abundances of Tau and Tubulin interactor OPTN from the interactome study. * p < 0.05 by linear regression model. (B) Representative Western blot of input lysate and co-IP with HT7 (hTau) in HEK293T cells overexpressing OPTN-eGFP and either mScarlet-Tau WT or mScarlet-Tau V337M , treated with βHB. IP, immunoprecipitation; IB, immunoblot; Tau5, total Tau. (C) Representative Western blot of input lysate and IP with HT7 (hTau) in HEK293T cells overexpressing mScarlet-Tau V337M , treated with βHB for 1 hr. IP, immunoprecipitation; IB, immunoblot; DAKO, total Tau; Ub, ubiquitin. (D) Schematic of Ub-dependent recognition of Tau at the autophagosome membrane by the LC3B-adapter protein OPTN. (E) Normalized protein abundances of Tau and Tubulin interactor LC3B-II. # p < 0.05, ## p < 0.01, #### p < 0.0001 by pairwise limma test. (F) Graphic illustrating mCherry-GFP-LC3B construct. mCherry was pseudocolored to magenta. APG, autophagosome; AL, autolysosome. (G-H) Representative immunofluorescent images (G) and quantification of autophagic vesicles (H) in primary neurons transfected with lenti-mCherry-GFP-LC3B and treated with βHB for 1 hr. Magenta-only vesicles were counted as autolysosomes, and double-positive bright green and magenta vesicles (white) were counted as autophagosomes. Each point represents an individual Map2+ neuron, with cells in the same well stacked into one column. Thick, color-coded bars represent the well mean (n = 8-15 cells/well), and black bars represent the overall group mean ± SD (n = 3 wells/group, from separate batches). Scale bar: 10 μm. (I) Quantification of percent of Tau secreted into the conditioned media (CM) in primary neurons infected with either lenti-shScramble or lenti-shOPTN for >5 days and treated with βHB for 1 hr. Value calculated by Tau levels in the CM divided by the sum of the CM Tau and intracellular (lysate) Tau, measured by ELISA. Each point represents one independent well, normalized to the control (shScramble NaCl) wells from its respective plate. (J-K) Representative immunofluorescent images (J) and quantification of Halo-Tau P301L (pink) co-localized with lysosomes (LysoTracker, green) in Map2+ neurons (K) co-transfected with lenti-Halo-Tau P301L and either lenti-shScramble or lenti-shOPTN for 5 days then labeled and co-treated with βHB and LysoTracker for 1 hr. Values represent the area of Halo-Tau co-localized to lysosomes over the total Halo-Tau area within a single neuron. Each point represents an individual Map2+ neuron, with cells in the same well stacked into one column. Thick, color-coded bars represent the well mean (n = 10-20 cells/well), and black bars represent the overall group mean ± SD (n = 3 wells/group, from separate batches). Scale bar: 10 μm. Data for A, E, and I are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant by linear mixed-effects model with Tukey post-hoc test (H, K) or by Šídák’s multiple comparisons test (I).

Article Snippet: Primary neurons were plated at a density of 600K per mL onto PDL-coated 12-well plates or coverslips and were infected on DIV3 with the homemade lentivirus for 5 days. shOPTN efficacy was validated via Western blot with anti-OPTN antibody (Proteintech, #10837-1-AP).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Construct, Transfection, Infection, Enzyme-linked Immunosorbent Assay, Control, Labeling