p301s mutation ps19  (Jackson Laboratory)

Journal: bioRxiv

Article Title: Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation

doi: 10.1101/2024.07.22.604618

Figure Lengend Snippet: ( A, B ) Integrated FRET quantification from cells transfected with TBS-based brain lysates from 2-9 months old P301S (0N4R) ( A ) and P301S (1N4R) ( B ) mouse models. Each dot represents the average of three technical replicates per lysate. ( C , D ) representative integrated FRET signals ( C ) and fluorescent images ( D ) from HEK tau-biosensor cell lines used from tau seeding activity qualifications. Experiments were performed with an n=3 using technical triplicates for each n. Values are represented as the mean ± s.e.m.; n=3. Scale bar: 100 µm.

Article Snippet: The PS19 mouse model , which overexpresses human 1N4R tau with the P301S mutation, was directly purchased from Jackson Laboratories (stock number 008169).

Techniques: Transfection, Activity Assay

Journal: bioRxiv

Article Title: Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation

doi: 10.1101/2024.07.22.604618

Figure Lengend Snippet: ( A, B ) Characterization of different tau species (HT7 for total human tau, AT8 for pTau-Ser202/Thr205, AT100 for pTau-Thr212/Ser214, PHF6 for pTau-Thr231, anti-pTau-T212, anti-pTau-Sser214, anti-pTau-Thr217, anti-pTau-Ser262, PHF1 for pTau-Ser396/S404, and MC1 that recognizes misfolded tau) in full brain TBS-lysates from 2-9 months P301S (0N4R) ( A ) and P301S (1N4R) ( B ) mouse models. ( C, D ) Correlation between percentage of FRET signal in the P301S (0N4R) ( C ) and P301S (1N4R) ( D ) mouse models with phosphorylated tau species. Dots indicate the average of three animals, each one quantified in technical triplicates. Red values indicate significance for positive correlations. Values are represented as the mean ± s.e.m.; n=3.

Article Snippet: The PS19 mouse model , which overexpresses human 1N4R tau with the P301S mutation, was directly purchased from Jackson Laboratories (stock number 008169).

Techniques:

Journal: bioRxiv

Article Title: Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation

doi: 10.1101/2024.07.22.604618

Figure Lengend Snippet: Sagittal hippocampal brain sections from both ( A ) Tau P301S (0N4R) and ( B ) Tau P301S (1N4R) tauopathy models were stained using specific antibodies against pTau-Ser214 and AT8 at 3, 6, and 9-month-old to characterize the histopathological deposition of these phospho-tau species. No positive immunostaining was observed in wild-type mice (right panel). Staining was performed with an n=3.

Article Snippet: The PS19 mouse model , which overexpresses human 1N4R tau with the P301S mutation, was directly purchased from Jackson Laboratories (stock number 008169).

Techniques: Staining, Immunostaining

Journal: bioRxiv

Article Title: Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation

doi: 10.1101/2024.07.22.604618

Figure Lengend Snippet: ( A ) Immunohistochemical staining of pTau-Ser214 and AT8 at 9 months old in both the P301S (0N4R, upper panel) and P301S (1N4R, lower panel) mouse models. Scale bar: 250 µm (low magnification hippocampus), and 100 µm (region-specific hippocampal insets); n=3.

Article Snippet: The PS19 mouse model , which overexpresses human 1N4R tau with the P301S mutation, was directly purchased from Jackson Laboratories (stock number 008169).

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation

doi: 10.1101/2024.07.22.604618

Figure Lengend Snippet: ( A, B ) Double immunofluorescence using specific antibodies against pTau-Ser214 (red) and AT8 (green) was performed in brain sections of 9-month-old brains in the ( A ) Tau P301S (0N4R) and ( B ) Tau P301S (1N4R) tauopathy models to analyze their spatial distribution in the hippocampus; n=3.

Article Snippet: The PS19 mouse model , which overexpresses human 1N4R tau with the P301S mutation, was directly purchased from Jackson Laboratories (stock number 008169).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation

doi: 10.1101/2024.07.22.604618

Figure Lengend Snippet: (A) Tau seeding activity in cytosolic and synaptic TBS-soluble hippocampal fractions from 6 months old Tau P301S (1N4R) mouse model. Significance was determined by ordinary one-way ANOVA; n=6. ( B-E ) Quantifications of pTau-Ser214 (B) , AT8 (C), and HT7 (D) levels of a western blot ( E ) using cytosolic ( C ) and synaptic ( S ) fractions. PSD95 antibody was used to confirm synaptic compartment enrichment. Values are represented as the mean ± s.e.m, and significance was determined by two-way ANOVA; n=3.

Article Snippet: The PS19 mouse model , which overexpresses human 1N4R tau with the P301S mutation, was directly purchased from Jackson Laboratories (stock number 008169).

Techniques: Activity Assay, Western Blot

Journal: Nature Communications

Article Title: Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy

doi: 10.1038/s41467-023-37840-y

Figure Lengend Snippet: A – C Soluble aggregated tau internalization triggers endogenous tau phosphorylation in primary human brain microvascular endothelial cells (HBEC). A Soluble tau aggregates accumulate in brain microvasculature of P301S(PS19) mice. Representative images of cortical brain sections from 8-month-old male and female P301S(PS19) and WT controls showing soluble tau aggregates (T22, green) and total tau (Tau5, cyan) immunoreactivity in lectin-stained microvasculature (red). Nine independent samples (three brain sections from each of three mice) were examined over three independent experiments. B Representative images of primary HBEC exposed to soluble aggregates of V5-tagged recombinant human tau-441 or vehicle (control) in the presence or absence of heparin, immunostained with antibodies for V5 (green) and α/β-tubulin (red), and counterstained with DAPI (blue). Scale bar is 200 µm. Nine independent samples were examined over 3 independent experiments. C Representative electropherograms from capillary electrophoresis immunoassays for phosphorylated tau (T231) and β-actin in lysates from HBEC treated with recombinant human cytokeratin-8 (KRT8), monomeric tau protein (M. Tau), unlabeled soluble tau aggregates (O.Tau), or vehicle (control). D Quantitative analyses of data in C (F(3,22) = 3.115, ANOVA, p = 0.047. Control vs. O. Tau, *, P < 0.05. Control, n = 12; KRT8, n = 4; M. Tau, n = 4, O. Tau, n = 6 biologically independent samples examined over 6 independent experiments. Data are means ± SEM. E , F Atomic force microscopy profiling of tau. E Representative AFM images of field fragments with soluble tau aggregates, tagged (V5) and untagged. F Size distribution of tau particles as measured by AFM show overlapping distributions of soluble tau aggregates (>12 nm in length) in non-tagged and V5-tagged soluble tau aggregate preparations (Soluble tau aggregates V5, n = 405 particles; soluble tau aggregates, n = 403 particles [i.e., biologically independent samples (particles measured) examined over 1 independent experiment]. Data are frequency profiles. For post-hoc analyses, lack of a specific P value in the legend reflects the information reported by GraphPad Prism Version 9.4.0.

Article Snippet: Male and female P301S(PS19) mice expressing human tau carrying the P301S mutation in the C57BL/6JxC3H were generated in our laboratory from original breeders obtained from The Jackson Laboratory (Stock #008169).

Techniques: Phospho-proteomics, Staining, Recombinant, Control, Electrophoresis, Microscopy

Journal: Nature Communications

Article Title: Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy

doi: 10.1038/s41467-023-37840-y

Figure Lengend Snippet: A Impaired endothelium-dependent cerebral blood flow (CBF) responses in male and female P301S(PS19) mice in response to topical acetylcholine (ACh) stimulation as compared to WT animals worsen with age (F(3,125) = 53.04, ANOVA, p < 0.0001. * indicates Tukey’s q(126) = 3.80, ** indicates Tukey’s q(126) = 4.66, p < 0.007; **** indicates Tukey’s q(126) = 6.16, p < 0.0001). The bracket highlights significant worsening of endothelial dysfunction with age in P301S(PS19) mice, ** Tukey’s q(126) = 4.61, WT 4 months, n = 6; PS19 4 months, n = 6; WT 8 months, n = 7; PS19 8 months, n = 6 mice. Four-month-old animals were males. Eight-month-old animals were males and females. Data are means ± SEM, plotted against ACh-induced vasodilation in C57BL/6 J mice, that serve as reference and were not included in the analysis. B , C Accumulation of tau in brain microvasculature isolated from 8-month-old P301S(PS19) mice. B Representative electropherograms from capillary electrophoresis immunoassays showing increased tau in brain vasculature isolated from 8-month-old, male and female P301S(PS19) mice. C Quantitative analyses of data in ( C ) (Two-sided Student’s t-test t(5.064) = 3.116, *, p = 0.026. Control, n = 6; PS19, n = 6 mice). Data are representative images and means ± SEM. For post-hoc analyses, lack of a specific P value in the legend reflects the information reported by GraphPad Prism Version 9.4.0.

Article Snippet: Male and female P301S(PS19) mice expressing human tau carrying the P301S mutation in the C57BL/6JxC3H were generated in our laboratory from original breeders obtained from The Jackson Laboratory (Stock #008169).

Techniques: Isolation, Electrophoresis, Control

Journal: Nature Communications

Article Title: Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy

doi: 10.1038/s41467-023-37840-y

Figure Lengend Snippet: A , B Decreased levels of acetylated tubulin in microvasculature of P301S(PS19) mice. A Representative immunofluorescent images of cortical brain sections from WT control and P301S(PS19) mice stained with DAPI (blue) and lectin (red) and immunostained for acetyl-α-tubulin (green); B Quantitation of acetyl-α-tubulin immunoreactive signal co-localized with lectin-reactive brain microvasculature (Unpaired, two-sided Student’s t-test, t(21) = 2.582, *, p = 0.018. Control, n = 12; PS19, n = 11 mice). C , D Decreased eNOS activation in microvasculature isolated from P301S(PS19) mice. C Representative electropherograms from capillary electrophoresis immunoassays of phospho-eNOS (S1176) and total eNOS in microvasculature purified from brains of WT and P301S(PS19) mice. D Quantitative analyses of phospho-eNOS (S1176) normalized to total eNOS levels in isolated brain vasculature of P301S(PS19) mice (Unpaired, two-sided Student’s t-test, t(6.248)=2.662, * p = 0.036. n = 6 mice per group). E , F Increased inhibition of eNOS in microvasculature isolated from P301S(PS19) mice. E Representative electropherograms from capillary electrophoresis immunoassays of phospho-eNOS (T495) and β-actin in microvasculature isolated from brains of WT control and P301S(PS19) mice. F Quantitative analyses of data in E (Student’s t test with Welch’s correction, t(5.966) = 3.370, *, p = 0.0152, n = 6 mice per group). G , H Increased markers of senescence in microvasculature isolated from P301S(PS19) mice. G mRNA abundance for cell cycle arrest mediators cyclin dependent kinase inhibitor 2 A (Cdkn2a, p16, unpaired, two-sided Student’s t test with Welch’s correction, t(19) = 0.77, p = 0.45. WT control, n = 9; PS19, n = 12), cyclin dependent kinase inhibitor 1 A (Cdkn1a, p21, unpaired, two-sided Student’s t test with Welch’s correction (t(13.3) = 2.794, *, p = 0.015. WT control, n = 8; PS19, n = 13) and tumor protein 53 (Tp53, p53, unpaired, two-sided Student’s t test (t(19) = 0.5973, p = 0.56. WT control, n = 10; PS19, n = 11 mice); H Senescence-associated secretory phenotype components interleukin 6 ( IL-6 , unpaired two-sided Student’s t test, t(17)=1.023, p = 0.32. WT control, n = 9; PS19, n = 10 mice), interleukin 1β (IL-1β, unpaired, two-sided Student’s t test with Welch’s correction, t(12.87) = 2.497, * p = 0.027. WT control, n = 7; PS19, n = 12 mice), tumor necrosis factor α (TNFα, unpaired, two-sided Student’s t test with Welch’s correction, t(10.46) = 2.309, *, p = 0.043. WT control, n = 8; PS19, n = 10 mice), monocyte chemoattractant protein-1 (MCP-1, unpaired, two-sided Student’s t test with Welch’s correction, t(11.07) = 2.794, *, p = 0.018. WT control, n = 7; PS19, n = 12 mice), and plasminogen-activator inhibitor-1 (PAI-1, unpaired, two-sided Student’s t test with Welch’s correction, t(15.53) = 1.701, p = 0.11. WT control, n = 10; PS19, n = 12 mice), measured with quantitative real-time PCR (qRT-PCR) in isolated brain vasculature of WT and P301S(PS19) mice. Data are representative images and electropherograms, and means ± SEM. For post-hoc analyses, the lack of a specific P value in the legend reflects the information reported by GraphPad Prism Version 9.4.0.

Article Snippet: Male and female P301S(PS19) mice expressing human tau carrying the P301S mutation in the C57BL/6JxC3H were generated in our laboratory from original breeders obtained from The Jackson Laboratory (Stock #008169).

Techniques: Control, Staining, Quantitation Assay, Activation Assay, Isolation, Electrophoresis, Purification, Inhibition, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy

doi: 10.1038/s41467-023-37840-y

Figure Lengend Snippet: A, B Treatment with TOMA ameliorates profound deficits in endothelium-dependent vascular responses in somatosensory cortex of P301S(PS19) mice, restoring vascular responses to levels comparable to those of 4-month-old animals. A ACh-stimulated vascular responses; B Quantitative analyses of data at 25 min in A. Interaction of time and treatment group by 2WRM ANOVA, F(10,50) = 4.91, p < 0.0001; difference from WT by Holm-Sidak’s p ost hoc indicated as *, t(60) = 2.86, p = 0.02; **, t(60)=3.33, p = 0.003; ***, t(60) = 4.04, p = 0.0005; ****, t(60) = 5.31, p < 0.0001; # indicates difference from PS19 + TOMA, for all #s, t(60)>2.34, p < 0.046 by Holm-Sidak’s post hoc. WT, n = 4; PS19, n = 4; PS19 + TOMA, n = 5 mice. Data from 4-month-old WT and P301S(PS19) groups (Fig. ) serve as reference and were not included in the analysis. C , D Treatment with TOMA reduces levels of soluble tau aggregates (tau oligomers) in brains of P301S(PS19) mice. C Representative immunoblots of cortical lysates of P301S(PS19) mice treated with isotype-matched IgG (PS19) or TOMA (PS19 + TOMA); D, Quantitative analyses of data in ( C ) (F (2, 19) = 6.807, ANOVA, p = 0.0081. WT vs. PS19, **, p < 0.01; PS19 vs PS19 + TOMA, *, p < 0.05 by Tukey’s post hoc test. WT, n = 8; PS19, n = 7; PS19 + TOMA, n = 7 mice). E , F TOMA treatment reduces levels of vascular tau in brains of P301S(PS19) mice. E Representative images (100X) of tau immunoreactivity (green) associated with lectin-reactive cortical brain vasculature (red), counterstained with DAPI (blue) staining. Scale bar is 50 µm. F Quantitative analyses of data in ( E ) (H = 6.839, Kruskal–Wallis test, *, p = 0.026. WT vs. PS19, * p < 0.05 from Dunn’s. n.s., no significant difference between experimental means for WT vs PS19 + TOMA. WT, n = 5; PS19, n = 7; PS9 + TOMA, n = 8 mice per group). G , H Cortical brain vascular density in P301S(PS19) mice is unchanged by tauopathy or by TOMA immunotherapy. G Representative images (40X) of lectin-reactive cortical brain vasculature (red) of P301S(PS19) animals treated with TOMA (PS19 + TOMA) or isotype-matched IgG (PS19). Scale bar is 250 µm. H Quantitative analyses of data in ( G ) (H = 1.787, Kruskal–Wallis, p = 0.426. WT, n = 5; PS19, n = 8; PS19, n = 7 mice). Data are representative images and means ± SEM. For post-hoc analyses, a lack of a specific P value in the legend reflects the information reported by GraphPad Prism Version 9.4.0.

Article Snippet: Male and female P301S(PS19) mice expressing human tau carrying the P301S mutation in the C57BL/6JxC3H were generated in our laboratory from original breeders obtained from The Jackson Laboratory (Stock #008169).

Techniques: Western Blot, Staining