α-Synuclein Oligomers Induce a Unique Toxic Tau Strain

Abstract

Background: The coexistence of α-synuclein and tau aggregates in several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, raises the possibility that a seeding mechanism is involved in disease progression.

Methods: To further investigate the role of α-synuclein in the tau aggregation pathway, we performed a set of experiments using both recombinant and brain-derived tau and α-synuclein oligomers to seed monomeric tau aggregation in vitro and in vivo. Brain-derived tau oligomers were isolated from well-characterized cases of progressive supranuclear palsy (n = 4) and complexes of brain-derived α-synuclein/tau oligomers isolated from patients with Parkinson's disease (n = 4). The isolated structures were purified and characterized by standard biochemical methods, then injected into Htau mice (n = 24) to assess their toxicity and role in tau aggregation.

Results: We found that α-synuclein induced a distinct toxic tau oligomeric strain that avoids fibril formation. In vivo, Parkinson's disease brain-derived α-synuclein/tau oligomers administered into Htau mouse brains accelerated endogenous tau oligomer formation concurrent with increasing cell loss.

Conclusions: Our findings provide evidence, for the first time, that α-synuclein enhances the harmful effects of tau, thus contributing to disease progression.

Keywords: Alpha-synuclein; Oligomeric complexes; Seeding; Strain; Tau oligomers; Toxicity.

Figure 1.

Seeds of α-synuclein enhance tau oligomer (tauO) toxicity in cells in culture. Tau strains were generated by adding seeds of preformed tauO or α-synuclein oligomers (α-synO) to 8 μM tau monomer in 1 × phosphate-buffered saline at a ratio of 1:140 (weight/weight). Atomic force microscopy images of tau seeded with (A) preformed tau oligomers (Tau/tauO) or (B) preformed α-synO (Tau/α-synO). Scale bar = 100 nm. (A) Seeded tau showed some oligomers arranged in a chain, suggesting the formation of tau protofibrils (Tau/tauO; dotted area). (C–E) Live cell imaging and (G–O) confocal images of CV-1 cells transfected with human tau linked to yellow fish (YFP-tau) plasmid treated with vehicle (phosphate-buffered saline) (C, G, J, M), or 1 μM tauO obtained by seeding (Tau/tauO) (D, H,K,N) or cross-seeding (Tau/α-synO) (E, I, L, O). Seeds of α-synuclein induced tau assembly into a distinct toxic oligomeric strain, as demonstrated by the reduced number of viable cells after treatment. The graph (F) shows the relative luminescence units (RLUs) of CellTiter Glo to cellular adenosine triphosphate. Bars represent the mean and SEM (one-way analysis of variance, Tukey multiple comparisons test; F ratio = 17.5, n = 4 independent experiments; tau/α-synO, **p < .006, F₆ = 12; Tau/tauO, *p < .03, F₆ = 12). (G–O) Representative confocal images showing the merge (yellow), between tubulin (green), and T22 (red) antibodies. Cross-seeded tau (Tau/α-synO) induces abnormal cell morphology. Scale bar = 12 μm. Representative images of cortical primary neurons derived from Htau mice treated with (P, S, V) vehicle, (Q, T, W) 1 μM tauO seeded with preformed tau oligomers (Tau/tauO), or (R, U, X) preformed Tau/α-synO for 6 hours. (P–X) Immunofluorescence staining using postsynaptic density protein 95 antibody to visualize synaptic spines and Mapt2 showed that cells exposed to tau cross-seeded with α-synO have a dramatic dendritic spine reduction after treatment (white arrows) compared with cells treated with (P) vehicle and (Q) seeded tau (Tau/tauO). (Y) Quantification of postsynaptic density protein 95 puncta revealed a decrease in dendrites after treatment with Tau/α-synO (**p < .001; Tau/tauO, *p < .02, F₁₁ = 12.5; error bars, SEM, n = 5 independent experiments, one-way analysis of variance, Tukey multiple comparisons test). Scale bar = 5 μm.

Figure 2.

Seeds of α-synuclein shift the tau aggregation pathway, extending lifespan of tau toxic conformation. (A) Kinetic analyses of recombinant tau protein (8 μM) incubated in phosphate-buffered saline (pH 7.2) with either preformed oligomeric tau (tauO; green line) or α-synuclein (α-synO; blue line) at a ratio of 1:140 (weight/weight) or 10 μM of heparin (red) at 22°C for 360 hours. A tau strain induced by α-synO evades fibril formation (blue line), whereas tau readily fibrillizes in the presence of inducers, such as heparin (red line) or seeds of preformed tau oligomers (green line). Bars represent the mean and SEM (****p < .0001, ***p < .001; n = 3 independent experiments; two-way analysis ofvariance, Bonferroni post hoc multiple comparisons test). (B) Dot blot analyses oftauO at different time points using T22 and Tau13antibodies. Both seeds of preformed tauO (Tau/tauO) and heparin induced tau fibril formation, while cross-seeded tau (Tau/α-synO) remains oligomeric. Atomic force microscopy images of tau aggregates induced by (C) preformed tauO (Tau/tauO), (D) α-synO (Tau/α-synO), and (E) heparin. (F–H) Graphs represent the length distribution of tau aggregates. Scale bar = 400 nm.

Figure 3.

Parkinson’s disease (PD) brain-derived α-synuclein oligomers (α-synO) propagate endogenous tau misfolding and neuronal loss in Htau mice. Brain-derived tau oligomers (tauO) and oligomeric complexes of α-synO/tauO were immunoprecipitated from patients with progressive supranuclear palsy (PSP) and PD using T22 and F8H7 antibodies. One-month-old Htau mice were intracerebroventricularly injected bilaterally with (B–E) 1 × phosphate-buffered saline (PBS) (vehicle, n = 8), (F–I) 0.5 μg of PSP brain–derived tauO (n = 8), or (J–M) PD brain–derived α-synO/tauO (n = 8) into the hippocampi. Fourteen months postinjection, mouse brains were immunostained using the neuronal marker NeuN (B, C, F, G, J, K), T22 antibody (D, H, L), and Thr231(E, I, M). Images show increased neuronal loss in mice receiving PD α-synO/tauO at the injection site compared with mice injected with PSP tauO or PBS (C, G, K, N). Stereological quantification of (N) NeuN⁺ cells (*p < .03, F₁₄ = 4.7, n = 6), (O) T22⁺ cells (*p < .03, F₁₇ = 4.3, n = 8), and (P) Thr231⁺ cells (*p < .04, F₉ = 3.02, n = 4). One-way analysis of variance Bonferroni post hoc comparisons test. (Q–S) Western blot analyses of brain homogenate from mice injected with PBS, PSP tauO, and PD α-synO/tauO probed with T22 and reprobed with caspase-3. (R–S) Graphs represent the protein band intensity relative to actin (T22, **p < .003, *p < .02, F₆ = 15.7, n = 3 independent experiments; caspase-3, **p < .006, F₆ = 14.6, n = 3 independent experiments). One-way analysis of variance Tukey multiple comparisons test. Immunohistochemistry analyses of the hypothalamic brain region from mice injected with (T) PBS (vehicle), (U) PSP tauO, and (V) PD α-synO/tauO using LB509 antibody. Mice injected with PD α-synO/tauO developed PD-like Lewy body deposits (arrow). (W) Stereological quantification of LB509⁺ cells (Lewy body-like deposits) (*p < .01, F₁₂ = 5.9, n = 5; one-way analysis of variance, Tukey multiple comparisons test). Scale bar = 50 μm.

Figure 4.

Parkinson’s disease (PD) brain-derived α-synuclein/tau oligomers (α-synO/tauO) delayed neurofibrillary tangle formation. Immunostaining of cortical brain sections from mice injected with (A) phosphate-buffered saline, (B) progressive supranuclear palsy (PSP) tauO, or (C) PD α-synO/tauO probed with AT8 antibody. (C) Mice injected with PD α-synO/tauO showed fewer neurofibrillary tangles (brown). PSP tauO showed an increased immuno-reaction to AT8 compared with mice injected with PD α-synO/tauO or phosphate-buffered saline. (D) Stereological quantification of AT8⁺ cells. (E–G) Immunofluorescence staining with AT8. Scale bar = 25 μm. (H–J) Gallyas silver staining for the detection of neurofibrillary tangles (red arrow) and neuropil threads (arrowhead). Scale bar = 25 mm. *p < .03, n = 5, one-way analysis of variance, Bonferroni post hoc comparisons test.