Alzheimer proteopathic tau seeds are biochemically a forme fruste of mature paired helical filaments

Abstract

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils.

Keywords: PTM; Tau protein; human Alzheimer disease; mass spectrometry; neurodegeneration; proteomics.

Figure 1

Preparation and characterization of HMW, LMW and PHF tau. (A) Schematic of workflow used in this study. Human Alzheimer’s disease (AD) and control (CT) subjects brain tissue homogenate was used for (i) size exclusion chromatography (SEC); and (ii) for the sarkosyl enrichment method. SEC high molecular weight (HMW) and low molecular weight (LMW) fractions from Alzheimer’s disease and control subjects were tested for seeding ability using biosensor cells. HMW and LMW fractions were further processed and analysed by mass spectrometry (MS) to identify the post-translational modification sites and to determine the extent of modification, respectively. Created using BioRender software. (B) Brain extracts from seven Alzheimer’s disease and three control participants in our study were separated using a SEC column. HMW and LMW fractions were run on a semi-denaturing detergent agarose gel electrophoresis and revealed by a total tau antibody (Dako). (C) Post-translational modification (PTM) map of Alzheimer’s disease and control HMW tau is shown. Position of the PTM is mapped on 2N4R tau isoform. (D) PTM of Alzheimer’s disease and control LMW tau is shown. Position of the PTM is mapped on 2N4R tau isoform. (E) Using biosensor cell assay, seeding analysis for Alzheimer’s disease and controls, HMW and LMW tau was performed.

Figure 2

HMW seed competent tau PTMs correlate with seeding competency. (A) Absolute quantification of high molecular weight (HMW) seed competent tau concentration by the FLEXITau mass spectrometry (MS) assay. n = 7 individual human participants. (B) Correlation between HMW seed competent tau absolute concentrations and tau seeding. (C and D) Associations between tau seeding and the number of phosphorylation sites, ubiquitination sites (E and F) or acetylation sites (G and H). Correlations were performed using a two-tailed Spearman’s rank non-parametric test, and P- and r-values are indicated on each plot. FRET = fluorescence resonance energy transfer; PTM = post-translational modification.

Figure 3

Proteopathic seed competent tau is ubiquitinated. (A–C) Tau extracted from the brain homogenate of seven different Alzheimer’s disease (AD) cases as well as immunoprecipitated (IP) fractions using an anti-ubiquitin antibody were analysed by western blot using the following primary antibodies: N-terminal domain of tau (A), C-terminal domain of tau (B) and ubiquitin (C). (D) Tau was immunoprecipitated using an anti-ubiquitin antibody and the immunodepleted fractions were run on a seeding biosensor assay showing a high and reproducible reduction of tau seeding. Samples were normalized for the amount of tau present in the input. HMW = high molecular weight.

Figure 4

Similarities between HMW and PHF tau isolated from Alzheimer’s disease patients. (A) Cumulative post-translational modifications (PTMs) map of high molecular weight (HMW) and sarkosyl insoluble (paired helical filaments, PHF) tau extracted from Alzheimer’s disease (AD) brain tissue. The modification sites are schematically mapped on 2N4R tau from the N-terminal domain (amino acid 1) to the C-terminal domain (amino acid 441). Phosphorylation sites are indicated in black, ubiquitination sites in blue and acetylation sites in pink. (B–D) Examples of PTM maps of HMW and sarkosyl insoluble (PHF) tau extracted from Alzheimer’s disease patients with low, medium and high tau load, respectively. (E) FLEXITau quantification heat map of HMW and sarkosyl insoluble (PHF) tau across all the subjects. The number e.g. [6–23], on the y-axis represents the position of the amino acids of the detected 2N4R tau peptides. The heat map shows the extent of modification of each peptide, red means highly modified and blue means less or no modification. (F and G) Shows the cosign similarity between HMW and sarkosyl insoluble (PHF) tau extracted from Alzheimer’s disease brain tissue, based on PTM and FLEXITau quantification data, respectively.