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) Schematic depicting enantiomer strategy for dissecting ketolysis-dependent and -independent features of βHB. AcAc, acetoacetate. (B) Climbing score for diet-fed WT and hTau+ flies at 7, 14, and 21 days old. Data represents the average scores of 4 vials per group from three trials. n = 15 flies/vial. AUC, area under the curve. (C) Survival curve for diet-fed WT and hTau+ flies. n = 20 flies/vial, 5 vials per group. (D) Schematic of organotypic brain slice culture (BSC) paradigm used in E-G. PFF, pre-formed fibrils. (E-G) Representative immunofluorescent images (E) and quantification (F, G) of BSC treated with AAV-Tau P301S , K18-Tau P301L PFF, and βHB or NaCl for 10 days, stained for MC1 (magenta) and DAPI (white). Each point represents an individual brain slice, with slices in the same well stacked into one column. Thick, color-coded bars represent the well mean (n = 4-6 slices/well), and black bars represent the overall group mean ± SD (n = 3 wells, from separate batches). Each batch was normalized to the average of its respective control (NaCl) well. Scale bars: 500 μm. (H) Schematic of primary neuron Tau secretion paradigm for 4I. (I) Quantification of percent of Tau secreted into the conditioned media (CM) in primary neurons treated with various metabolites, with or without glucose, for 1 hr. Value calculated by the amount of Tau in the CM divided by the sum of the CM Tau and intracellular (lysate) Tau, and each point represents one independent well, normalized to the control (NaCl + glucose) well/s from its respective plate. Tau levels were measured using ELISA. aCSF, artificial cerebrospinal fluid. (J) Quantification of relative intracellular Tau levels in primary neurons, corresponding to 4I. Each point represents one independent well, normalized to the control (NaCl + glucose) well/s from its respective plate. (K) Lactate dehydrogenase (LDH) assay as a proxy for cytotoxicity. 100% represents the amount of LDH within the media upon complete cell lysis/death. (L) Schematic of HaloTag-Tau P 301 L turnover assay in induced pluripotent stem cell-derived neurons (i 3 Ns). i 3 Ns do not express 4R-Tau normally, so an anti-4R-Tau antibody identifies total HaloTag-Tau. TMR, tetramethylrhodamine-conjugated Halo ligand. (M-N) Representative Western blot (M) and quantification (N) of Halo-Tau in-gel fluorescence. Each point represents one independent well, normalized to the average of the NaCl-treated values for its respective plate. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns not significant by AUC analysis (B, C), linear mixed-effects model with Tukey post-hoc test (F, G), and Dunnett’s multiple comparison test (J-K, N). For I, **** p < 0.0001, ns not significant compared to NaCl + glucose, #### p < 0.0001 compared to NaCl - glucose group by Šídák’s multiple comparisons test.

Article Snippet: Human TauRD P301S FRET biosensor-expressing HEK293T cells (ATCC CRL3275) were maintained and passed in complete DMEM media (DMEM with 10% FBS, 100 μg P/S).

Techniques: Slice Preparation, Staining, Control, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Lysis, Turnover Assay, Derivative Assay, Western Blot, Fluorescence, Comparison

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) Representative Western blot showing RAB-soluble cortical fractions from WT and hTau+ mice fed with CTL or KE diets. Arrows indicate p-hTau band quantified for AT8 and PHF1. hTau, human Tau; msTau, mouse Tau. (B-G) Quantifications of p-hTau (B-D), hTau (E-F), and total Tau (G) in RAB-soluble cortical fractions. (H) Representative Western blot for Tau species in PBS-soluble cortical lysates from WT and hTau+ mice fed with CTL or KE diets. Lysates were used for a Tau seeding assay in HEK293T biosensor cells . (I-K) Quantifications of p-hTau (I-J) and total hTau (K) from PBS-soluble cortical lysates. Data are represented as mean ± SD. Each point represents one mouse. Data were normalized to the hTau+ CTL group. * p < 0.05, ns not significant by Welch’s t-test.

Article Snippet: Human TauRD P301S FRET biosensor-expressing HEK293T cells (ATCC CRL3275) were maintained and passed in complete DMEM media (DMEM with 10% FBS, 100 μg P/S).

Techniques: Western Blot

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) Representative Western blot showing the RIPA-soluble cortical fractions from WT and hTau+ mice fed with CTL or KE diets. Arrows indicate p-hTau band quantified for AT8 and PHF1. hTau, human Tau; msTau, mouse Tau. (B-F) Quantification of hTau from RIPA-soluble cortical fractions for pTau (B-D) and total hTau (E-F). LMW, low molecular weight (∼37 kDa). (G) Representative Western blot showing the formic acid-insoluble cortical fractions from WT and hTau+ mice fed with CTL or KE diets. Arrow and bracket indicate p-hTau band quantified for PHF1. Input was normalized by volume proportional to the initial amount (mg) of starting tissue. (H-M) Quantification of hTau from insoluble cortical fractions for pTau (H-K) and total hTau (L-M). HMW, high molecular weight (120-180 kDa). (N-P) Representative immunofluorescent images (N) and quantifications (O-P) of TauRD biosensor HEK293T cells seeded with PBS-soluble cortical fractions. Quantification represents the average value of two wells per mouse, four images per well. Scale bars: 200 μm. Data are represented as mean ± SD. Each point represents one mouse. Data were normalized to the hTau+ CTL group. * p < 0.05, ** p < 0.01, ns not significant by Welch’s t-test.

Article Snippet: Human TauRD P301S FRET biosensor-expressing HEK293T cells (ATCC CRL3275) were maintained and passed in complete DMEM media (DMEM with 10% FBS, 100 μg P/S).

Techniques: Western Blot, Molecular Weight, High Molecular Weight

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: Human TauRD P301S FRET biosensor-expressing HEK293T cells (ATCC CRL3275) were maintained and passed in complete DMEM media (DMEM with 10% FBS, 100 μg P/S).

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