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: IR-AMES uncovers structure and composition of Alzheimer’s tau oligomers

doi: 10.64898/2026.03.12.711458

Figure Lengend Snippet: a, Schematic of oligomerization from recombinant human 2N4R human tau monomers (rTauM) to oligomers (rTauO) and imaging workflow. b, Amide-I band intensity histograms of rTauM (green) and rTauO (orange) particles with Gaussian fits ( n = 200 for each). c, Amide-I band intensity versus apparent oligomer order (rTauM, green; rTauO, orange). Order 1 corresponds to the rTauM population. Data points represent the mean of each peak in b . Error bars indicate the fitted Gaussian FWHM. d–e, Heatmaps of IR-AMES spectra from monomers ( d ) and oligomers ( e ), sorted by integrated intensity. Cartoons illustrate that monomers, although structurally dynamic, remain predominantly random coil, whereas oligomers exhibit more heterogeneous secondary structures. Detailed conformations predicted by AlphaFold3 are provided in the Supplementary Note 5 and Supplementary Fig. 13. f, Quantification of fitted spectral components obtained from Lorentzian deconvolution of the amide-I band (see Extended Data Fig. 4 for representative fitting examples). Monomers show a narrow distribution dominated by random-coil features, whereas oligomers exhibit a broader heterogeneity with increased β-sheet structures. All groups were expressed as mean ± s.d. g, Representative average spectra for monomers (green) and oligomers (orange). Solid lines: mean spectra, shaded regions: standard deviation. Ensemble averages show minimal spectral differences, highlighting that conformational diversity is primarily resolved in IR-AMES.

Article Snippet: Recombinant human tau protein was purchased from R&D Systems, Inc (SP-495).

Techniques: Recombinant, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: IR-AMES uncovers structure and composition of Alzheimer’s tau oligomers

doi: 10.64898/2026.03.12.711458

Figure Lengend Snippet: a, Recombinant human 2N4R tau monomers (rTauM). b, Recombinant human 2N4R tau oligomers (rTauO). Each spectrum was normalized to 0–1 and decomposed into five secondary-structure components within the amide-I region: parallel β-sheet, random coil, α-helix, β-turn, and antiparallel β-sheet. Colored areas represent the contribution of each component, and solid black lines represent the fitted total spectrum. The integrated areas of these components were used to generate the single-particle structural distributions shown in .

Article Snippet: Recombinant human tau protein was purchased from R&D Systems, Inc (SP-495).

Techniques: Recombinant, Single Particle

Journal: bioRxiv

Article Title: IR-AMES uncovers structure and composition of Alzheimer’s tau oligomers

doi: 10.64898/2026.03.12.711458

Figure Lengend Snippet: a, Atomic force microscopy images of Alzheimer’s disease patient derived tau oligomers (AD TauO) and fibrils (AD TauF). Scale bars: 250 nm. AD TauO appear as spherical or ellipsoidal particles wih heights of 5–8 nm and lateral dimensions of ∼40 nm. AD TauF appear as short fragmented rods with heights of ∼10 nm, lateral widths of ∼30–50 nm, and lengths ranging from 100 to 500 nm. Dimensions were measured along the white dashed lines, details are provided in Supplementary Fig. 14. b–c, Cytotoxicity of iPSC-derived neurons treated with human tau for 24 h, quantified by LDH release ( b ) and cleaved caspase-3–positive area relative to TUJ1 ( c ). n = 6. Data were expressed as mean ± s.d. Column means were compared using two-way ANOVA, with ****p < 0.0001. d, IR-AMES image of AD TauO and age-matched normal human derived tau oligomers (Ctrl TauO) at the amide-I band. Scale bars: 1 µm. e, Heatmaps of IR-AMES spectra from AD TauO and Ctrl TauO, n = 150. Spectra were normalized to 0–1. f–g, IR-AMES image of AD TauO and Ctrl TauO at the antiparallel β-sheet channel ( f ) and RNA channel ( g ). Scale bars: 1 µm. h–j, Heatmaps of IR-MAES spectra from AD TauO with endonuclease benzonase (AD TauO w/Benz) treatment ( h ), AD TauF ( i ) and normal human derived tau fibrils (Ctrl TauF) ( j ), n = 150. Spectra were normalized to 0–1. k, Quantification of antiparallel β-sheets and RNA content from human tau in e and h–j . Values were derived from Lorentzian deconvolution of the amide-I region (see Extended Data Fig. 6 for representative fits). All groups were expressed as mean ± s.d. Column means were compared using one-way ANOVA, with ****p < 0.0001, and ns for not significance. l, t-SNE visualization of all spectra from individual tau assemblies, revealing structure-dependent clustering patterns. Each dot indicates a single-particle spectrum.

Article Snippet: Recombinant human tau protein was purchased from R&D Systems, Inc (SP-495).

Techniques: Microscopy, Derivative Assay, Single Particle

Journal: bioRxiv

Article Title: IR-AMES uncovers structure and composition of Alzheimer’s tau oligomers

doi: 10.64898/2026.03.12.711458

Figure Lengend Snippet: a ,: Schematic illustrating the co-incubation of human-derived tau aggregates with lipid nanodiscs (NDs) to form tau–ND complexes for IR-AMES imaging. b, Representative IR-AMES images of NDs composed of phosphatidylcholine and phosphatidylserine (PC+PS) or PC only, shown for integrated amide-I and lipid signals. Scale bars, 1 µm. c, Heatmaps of IR-AMES spectra from ND (PC+PS) ( n = 119) and ND (PC) ( n = 165). Spectra were normalized to 0–1 and ordered by lipid intensity. d, Average spectra of NDs. Solid lines: mean spectra, shaded regions: standard deviation. e, Representative images of AD TauO and Ctrl TauO following NDs incubation. Scale bars, 1 µm. f, Heatmaps of spectra from AD TauO–ND (PC+PS) ( n = 225), AD TauO–ND (PC) ( n = 115), and Ctrl TauO–ND (PC+PS) ( n = 140), highlighting distinct protein secondary-structure and lipid-associated spectral features. Spectra for AD TauO–ND spectra were ordered by antiparallel β-sheet contribution. g, Average spectra of lipid-poor ( n = 25) and lipid-enriched ( n = 25) subsets derived from f , compared with tau aggregates alone ( n = 150 for each). Spectra were normalized to 0–1 and vertically offset for display in d and g . h, Quantification of antiparallel β-sheet and lipid contributions from IR-AMES spectra using Lorentzian fitting. All groups were expressed as mean ± s.d. Column means were compared using one-way ANOVA, with ****p < 0.0001, and ns for not significance. i, Single-particle correlation analysis of lipid content and antiparallel β-sheet contribution in AD TauO–ND (PC+PS), revealing a moderate negative correlation (Pearson’s r = –0.56). j, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectra of human tau ( n = 3, solid lines: mean spectra, shaded regions: standard deviation), indicating enhanced surface hydrophobicity of AD TauO relative to controls.

Article Snippet: Recombinant human tau protein was purchased from R&D Systems, Inc (SP-495).

Techniques: Incubation, Derivative Assay, Imaging, Standard Deviation, Single Particle, Fluorescence