dGAE(297-391) Tau Fragment Promotes Formation of Chronic Traumatic Encephalopathy-Like Tau Filaments

Abstract

The microtubule-associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at the atomic level. Here, we used the tau fragment dGAE, which aggregates spontaneously, to seed the formation of full-length tau filaments. The structures of dGAE and full-length tau filaments were investigated by magic-angle spinning (MAS) solid-state NMR, showing that dGAE allows propagation of a chronic traumatic encephalopathy (CTE)-like fold to the full-length tau. The obtained filaments efficiently seeded tau aggregation in HEK293T cells. This work demonstrates that in vitro preparation of disease-specific types of full-length tau filaments is feasible.

Keywords: NMR spectroscopy; dGAE fragment; filamentous aggregates; tau protein; tauopathies.

Figure 1

(a) Full‐length tau 2N4R domain architecture. N1 and N2 are exon‐coded inserts, P1 and P2 indicate the proline‐rich regions, and R1 to R’ indicate repeat domains. dGAE includes residues I297‐E391 from the repeat domains of the 2N4R full‐length tau. (b) ThT fluorescence curve of dGAE aggregation kinetics (average of three replicates with standard deviation values). (c) Negative‐stain EM micrograph of dGAE(297–391) tau filaments. (d) Comparison of secondary structures between dGAE filaments presented here, patient‐derived tau filaments,[ ⁴ , ⁶ ] and other in vitro dGAE studies.[ ¹⁵ , ¹⁹ , ²⁰ ]

Figure 2

(a) A selected region of a 2D ¹³C‐¹³C DARR spectrum (150 ms mixing time) showing V339Cβ‐I354Cδ1, V337Cβ‐I354Cγ2, and V363Cβ‐I328γ2 correlations. (b) A selected region of a ¹³C‐¹³C DARR spectrum (150 ms mixing time) showing V337Cβ‐G355Cα correlation. (c) Selected regions of a ¹³C‐¹³C DARR spectrum (150 ms mixing time) showing L344Cγ‐F346Cζ and V350γ1‐F346Cζ correlations. (d) Selected regions of a ¹³C‐¹³C DARR spectrum (150 ms mixing time) showing V363Cγ1‐L325Cδ, V363Cγ1‐I328Cγ1, V363Cγ1‐L325Cγ correlations. (e) A selected region of a ¹³C‐¹³C DARR spectrum (150 ms mixing time) showing V363Cγ1‐I328Cδ1 correlation. (f) A selected region of a ¹³C‐¹³C DARR spectrum (150 ms mixing time) showing V363Cγ2‐I328Cβ correlation. (g) A selected region of a ¹³C‐¹³C DARR spectrum (100 ms mixing time, valine, isoleucine, leucine, and lysine suppressed sample) showing P322Cδ‐N359Cβ correlation. (h) A selected region of a ¹³C‐¹³C DARR spectrum (500 ms mixing time, lysine suppressed sample) showing H330Cγ‐N359Cδ and H330Cδ2‐N359Cδ correlations. (i) Selected regions of a ¹³C‐¹³C DARR spectrum (500 ms mixing time) showing E338Cδ‐H329Cγ and E338Cδ‐H329Cδ2 correlations. (j) V337, V339, and I354 side‐chain packing in CTE filaments. (k) L344, F346, and V350 side‐chain packing in CTE filaments. (l) L325, I328, and V363 side‐chain packing in CTE filaments. (m) H330, P322, and N359 side‐chain packing in CTE filaments. (n) H329 and E338 locations in the CTE type II filament structure.

Figure 3

(a) SDS‐PAGE of the full‐length tau sample with dGAE seeds before and after aggregation in PBS pH 7.4, 5 mM DTT. Lane R is a reference sample at the beginning of aggregation, lane P corresponds to the pellet fraction after aggregation, lane S—the supernatant fraction after aggregation, lane W‐whole reaction mixture after aggregation, and lane Seed is the dGAE seed. Figure S14A contains uncropped/full‐size SDS‐PAGE image. (b)ThT fluorescence curves of dGAE‐seeded 2N4R aggregation kinetics (average of four replicates with standard deviation values). (c) Negative‐stain EM micrograph of the seeded full‐length tau filaments. (d) SDS‐PAGE of the trypsin and pronase E treated full‐length tau filaments. Lane R is a reference sample before digestion, lane P corresponds to the pellet fraction after digestion. Figures S14B and S14C contain uncropped/full‐size SDS‐PAGE images. (e) Overlay of 2D ¹³C‐¹³C DARR spectra of dGAE and seeded full‐length tau filaments. (f) Overlay of 2D NCA spectra of dGAE and seeded full‐length tau filaments. The missing residues are identified.

Figure 4

Tau aggregation in biosensor cell line induced by dGAE and 2N4R filaments. (a) Fluorescence microscopy of tau inclusions after transfection with filaments. dGAE monomers and sarkosyl insoluble AD tau filaments were used as negative and positive control, respectively. White bars represent 20 μm. (b) Quantification of intracellular tau inclusions in FRET‐positive cells based on averaged median fluorescence intensity in the FRET channel. Error bars represent standard deviation.