Journal: bioRxiv

Article Title: N -Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission

doi: 10.64898/2025.12.01.690311

Figure Lengend Snippet: (A) Primary amino acid sequence of 2N4R Tau with predicted (N167) and confirmed (N359 and N410) sites of N -linked glycosylation shown in blue. The red segment denotes the K18 Tau fragment. (B) Three classes of N -linked glycans attached at asparagine residues. N Acetylglucosamine (G) and chitobiose (C) serve as minimal model glycans that introduce limited structural perturbations to Tau, whereas the high-mannose heptasaccharide (M) with defined branching corresponds to the most abundant N -linked glycan characterized on PHFs from AD brain by Liu et al. 9 (C) Summary of K18 and 2N4R Tau glycoproteins used in this study. Residue numbering refers to asparagine positions in full-length 2N4R Tau as defined in panel A.

Article Snippet: HEK293T bioreporter cells stably expressing CFP/YFP K18 were obtained from ATCC (#CRL-3275) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with GlutaMAXTM and 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2.

Techniques: Sequencing, Glycoproteomics, Introduce, Residue

Journal: bioRxiv

Article Title: N -Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission

doi: 10.64898/2025.12.01.690311

Figure Lengend Snippet: (A) ThT-monitored aggregation of K18 and K18M monomers seeded with pre-formed K18 or 2N4R Tau fibrils, quantified as relative fluorescence over time. (B) Maximum ThT fluorescence signal for each condition, quantified at the individual reaction maxima. (C) Cross-seeding efficacy expressed as an acceleration factor relative to unseeded reactions, where the acceleration factor corresponds to the ratio of t 50 of unseeded versus seeded controls (t 50 ,unseeded / t 50 ,seeded). (D) FRET flow cytometry detection of seeding after transfection of K18 or 2N4R Tau proteoforms into HEK293T K18 CFP/YFP biosensor cells. (E) Representative confocal microscopy images of HEK293T CFP/YFP K18 biosensor cells transfected with vehicle, K18, or Tau proteoforms (20× objective; scale bar, 100 µm). (F) After seeding with vehicle, K18, or Tau proteoforms (100 nM, 24 h), integrated FRET density was quantified by flow cytometry. Vehicle denotes the lipofectamine-only negative control. Increases in FRET signal for glycosylated K18 and Tau proteoforms are shown relative to their respective WT constructs. n = 3 independent experiments for each proteoform; statistical significance was assessed by ordinary one-way ANOVA. (G) Representative flow cytometry scatter plots for each proteoform depicting FRET-positive cells (10,000 events analyzed per condition).

Article Snippet: HEK293T bioreporter cells stably expressing CFP/YFP K18 were obtained from ATCC (#CRL-3275) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with GlutaMAXTM and 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2.

Techniques: Fluorescence, Flow Cytometry, Transfection, Confocal Microscopy, Negative Control, Construct

Journal: bioRxiv

Article Title: N -Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission

doi: 10.64898/2025.12.01.690311

Figure Lengend Snippet: (A) Heat map of equilibrium dissociation constants (K D ) obtained by BLI for K18 and Tau binding to heparin and RNA (A40, C40, U40). K D values (nM) were derived from global fits across the full concentration series (20 µM–1.75 µM). (B) BLI binding curves for K18 proteoforms interacting with heparin and RNA. Experiments were performed in triplicate, and the shaded regions represent SD. After ligand immobilization, sensors were baselined for 30 s, followed by 120 s association and 120 s dissociation at each K18 concentration (20 µM–1.75 µM). (C) BLI binding curves for Tau proteoforms interacting with heparin and RNA ligands, measured under identical conditions as for K18. Kinetic parameters and K D values reflect global fitting across all concentrations.

Article Snippet: HEK293T bioreporter cells stably expressing CFP/YFP K18 were obtained from ATCC (#CRL-3275) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with GlutaMAXTM and 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2.

Techniques: Binding Assay, Derivative Assay, Concentration Assay

Journal: bioRxiv

Article Title: N -Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission

doi: 10.64898/2025.12.01.690311

Figure Lengend Snippet: (A) Schematic of the experimental workflow used to quantify cellular uptake. (B) Representative images of monomeric and aggregated pHrodo-labeled K18 uptake at 0 and 24 h. Left: brightfield; middle: TRITC; right: merge. (C) Representative images of K18 uptake at 24 h for different glycoforms. (D) Kinetic summary of K18 uptake at 50 nM (mean ± SD). Dextran is a pHrodo-labeled dextran control. (E) Comparison of monomeric versus aggregated K18 uptake at 24 h (mean ± SD; unpaired Welch’s t-test; n = 3). (F) Quantified uptake of Tau proteoforms (mean ± SD; two-way ANOVA with Dunnett’s multiple comparisons between glycoforms; n = 3).

Article Snippet: HEK293T bioreporter cells stably expressing CFP/YFP K18 were obtained from ATCC (#CRL-3275) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with GlutaMAXTM and 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2.

Techniques: Labeling, Control, Comparison