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

Abstract

BackgroundTau aggregation and propagation are hallmark features of Alzheimer's disease and related tauopathies. The molecular identity and post-translational modifications that contribute to tau seeding activity remain incompletely understood.ObjectiveTo characterize the temporal dynamics of tau seeding activity and identify specific phosphorylated tau species associated with tau propagation in vivo.MethodsWe profiled tau seeding activity using a FRET-based biosensor cell line and correlated it with the abundance of phospho- and conformational tau species in two transgenic mouse models of tauopathy (P301S-1N4R and P301S-0N4R). Immunohistochemistry and subcellular fractionation were used to examine the spatial distribution of tau species. Functional relevance of serine 214 phosphorylation was assessed via phospho-dead mutants in Drosophila and primary neuronal cultures using a microfluidic platform.ResultsTau seeding activity was detected as early as 2 months and increased with age in both mouse models, preceding AT8-positive neurofibrillary tangles. Phosphorylation at serine 214 (pTau-Ser214) positively correlated with tau seeding in both models and was observed earlier and in distinct histological compartments compared to AT8. pTau-Ser214, but not AT8, localized to the synaptic compartment. Mutation of serine 214 to alanine reduced tau propagation in primary neurons and seeding activity in Drosophila, without affecting total tau levels.ConclusionspTau-Ser214 marks an early, synaptically enriched tau species that correlates with seeding activity and promotes transsynaptic propagation. These findings highlight the functional diversity of tau species and support pTau-Ser214 as a potential biomarker and therapeutic target for early-stage tauopathy.

Keywords: Alzheimer's disease; propagation; tau; tau phosphorylation.

Figure 1.

Tau seeding activity is an early pathological manifestation and increases with age in two models of tauopathy. (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. Each dot represents the average of 3 technical replicates for one individual, and the experiment was performed using 3 different animals (n = 3). Values are represented as the mean ± s.e.m.; n = 3. Scale bar: 100 µm.

Figure 2.

Tau phosphorylation at serine 214 correlates with seeding activity in disease progression in two mouse models of tauopathy. (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-Thr212, anti-pTau-Ser214, 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.

Figure 3.

Tau phosphorylated at serine 214 is observed at an early stage of the disease and develops before the formation of AT8-positive tau inclusions. 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.

Figure 4.

The staining pattern of tau phosphorylated at serine 214 differs from AT8-positive tau species in two different tauopathies mouse models. (A) Immunohistochemical staining using antibodies against 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.

Figure 5.

Tau phosphorylated at serine 214 is differentially distributed compared to AT8-positive staining. (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.

Figure 6.

Tau phosphorylation at serine 214 is deposited in the synaptic compartment. (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.

Figure 7.

Phosphorylation of tau^P301L at serine 214 is involved in spreading pathological tau. (A) Schematic of the Gal4-UAS crosses utilized to overexpress human tau^P301L or tau^P301L/S214 under the GMR driver (eye promoter). (B) Integrated FRET density (seeding activity) in fly head lysates from WT controls, and flies overexpressing human tau^P301L or tau^P301L/S214A mutations. Significance was determined by one-way ANOVA. Each dot represents a pool of 20 heads (each pool of 20 heads is represented as a single dot in the quantification; equal number of males and females). (C) Total tau levels were quantified by western blot using the HT7 antibody to specifically detect human tau. Significance was determined by one-way ANOVA. Each dot represents a pool of 20 heads. (D) Immunofluorescence against hTau (HT7, Red) in primary mouse neonatal cortical neurons plated in both microfluidic chambers. Neurons were transduced in the first chamber at 1 DIV with an AAV9 harboring plasmids coding for either tau^P301L or tau^P301L/S214A and tagged with V5. The second chamber shows the spreading of tau from the first chamber. Scale bar: 100 µm. (E) Quantification of Tau spreading in Chamber 2 and normalized by the number of nuclei in (F); Triangle for N1, Square for N2, and Circle for N3. Values are represented as the mean ± s.e.m from 3 independent experiments (n = 3, each n represent a different mouse brain for neuronal culture), and significance was determined by t-test.