Preparation of stable tau oligomers for cellular and biochemical studies

Abstract

Increasing evidence suggests that small oligomers are the principal neurotoxic species of tau in Alzheimer's disease and other tauopathies. However, mechanisms of tau oligomer-mediated neurodegeneration are poorly understood. The transience of oligomers due to aggregation can compromise the stability of oligomers prepared in vitro. Consequently, we sought to develop an efficient method which maintains the stability and globular conformation of preformed oligomers. This study demonstrates that labeling a single-cysteine form of the pro-aggregant tau four-repeat region (K18) with either Alexa Fluor 488-C5-maleimide or N-ethylmaleimide in reducing conditions stabilizes oligomers by impeding their further aggregation. Furthermore, the use of this approach to study the propagation of labeled extracellular tau K18 oligomers into human neuroblastoma cells and human stem cell-derived neurons is described. This method is potentially applicable for preparing stabilized oligomers of tau for diagnostic and biomarker tests, as well as for in vitro structure-activity relationship assays.

Keywords: Alzheimer's disease; Maleimide; Oligomer; Tau; Tauopathies; iPSC-derived neurons.

Fig. 1

Schematic illustration of maleimide conjugation to the tau K18 protein. (A) Molecular structure of AF-maleimide, as provided by the vendor [42]. (B) Molecular structure of NEM. (C) Illustration of the reaction of the maleimide functional group of AF-maleimide with the sulfhydryl group of tau K18 to produce a stable thioether conjugate. (D) Illustration of the specific binding of NEM with the sulfhydryl group of tau K18 to produce a tau K18-NEM conjugate.

Fig. 2

Representative non-denaturing SDS-PAGE images demonstrating the outcome of AF-maleimide labeling of tau K18 in reducing and non-reducing conditions. Migration of tau K18 labeled with AF-maleimide with (A) or without (B) prior TCEP treatment. The figures show representative (n = 3) non-denaturing SDS-PAGE analysis of tau aggregate patterns in the two labeling methods.

Fig. 3

Structural characterization of tau K18 oligomers stabilized by labeling with maleimide derivatives. Representative TEM micrographs of globular tau K18 oligomers prepared: (A) without labeling, (B) with AF-maleimide labeling, or (C) with NEM labeling. Scale bars = 50 nm for all images.

Fig. 4

Labeling with AF-maleimide stabilizes tau K18 oligomers. (A) Representative electron micrographs of AF-maleimide-labeled tau K18 structures identified after co-incubation with heparin at 37 °C for 48 h. Figures A(i) and A(ii) show globular oligomers with different degrees of negative staining. (B) Representative TEM micrographs of aggregates identified for the unlabeled control samples treated similarly to the test samples. Globular oligomers (i), protomers (ii), short and mature fibrils (iii and iv respectively) were observed, suggesting heparin-induced structural transition to form fibrils. n = 2, with images taken from at least 5 different areas of each TEM grid. Scale bars = 100 nm for all images.

Fig. 5

NEM labeling stabilizes tau K18 oligomers in their globular conformation. NEM-labeled and unlabeled control tau K18 samples were each incubated with heparin at 37 °C for 48 h, and aliquots analyzed using negative-stain TEM. (A) Representative TEM micrographs of NEM-labeled tau K18 aggregates. The globular nature of these non-fibrillar aggregates in A(i) and A(ii) suggests oligomer stabilization. (B) Representative TEM micrographs of endpoint samples demonstrating the aggregation of unlabeled tau K18 into oligomers, protomers, short and mature fibrils. n = 2, with images taken from at least 5 different areas of each grid. Scale bars = 100 nm for all images.

Fig. 6

Immunological analysis of the conformation of AF-maleimide-labeled tau K18 oligomers using dot blotting. (A) Representative dot blot data for the reactivity of labeled and unlabeled tau K18 to two tau antibodies (A0024, top panel) and T22 (bottom panel). (B) Semi-quantitative analysis of dot blot intensity using Image J. UNL = unlabeled; MAL = AF-maleimide labeled. Mann Whitney test, * = p < 0.05, ns = not significant (n = 4). Data expressed as mean ± standard deviation.

Fig. 7

Dot blot analysis of the conformation of NEM-labeled tau K18 oligomers. (A) Representative dot blot data for the immuno-reactivity of labeled and unlabeled tau K18 to two tau antibodies (A0024, top panel) and T22 (bottom panel). (B) Semi-quantitative analysis of dot blot intensity using Image J. UNL = unlabeled; NEM = NEM labeled. Mann Whitney test, * = p < 0.05, ns = not significant (n = 4). Data expressed as mean ± standard deviation.

Fig. 8

Extracellularly-applied tau K18 oligomers are internalized by SH-SY5Y neuroblastoma cells and hiPSC-derived neurons. (A) Internalization of AF-maleimide-labeled tau K18 oligomers in SH-SY5Y neuroblastoma cells. (B) Internalization of extracellular oligomers was also studied in hiPSC-derived neurons, revealing their localization both in neurites and the cell soma. Scale bars = 10 μm for all images.

Fig. 9

Schematic illustration of the facile method of tau oligomer stabilization described in this study. The method involves first treating highly-purified monomers with the reducing agent TCEP to monomerize apparent oligomers. Monomers are then labeled overnight with either maleimide derivative (AF-maleimide or NEM), followed by removal of excess fluorophore by dialysis. Labeled oligomers can then be characterized using an array of biochemical and biophysical tools, including non-denaturing SDS-PAGE, dot/Western blotting and TEM. Oligomer stabilization can be studied by inducing aggregation with heparin and studying the structures and conformation of the aggregates formed by TEM and dot blotting respectively, or by similar techniques.

Fig. S1

AF-maleimide labeling of tau K18 at RT generates a mixture of globular oligomers and fibrillar aggregations. (A) Representative TEM micrographs of tau K18 labeled with AF-maleimide at RT showing presence of both globular oligomers and fibrils (shown by arrows). (B) Unlabeled control tau K18 samples aggregated into a range of pre-fibrillar and fibrillar structures. Scale bars = 100 nm.