Tau Oligomers in Alzheimer's Disease: Modulation Effect of Osmolytes on Amplified Brain-Derived Tau Oligomers

Abstract

Tau is bound to microtubules and plays a key role in their assembly and spatial organization. Under pathological conditions, tau detaches from the microtubules and develops a propensity to self-aggregate into soluble tau oligomers (TauO), paired helical filaments, and neurofibrillary tangles. Recent studies have revealed that TauO is the toxic species responsible for seeding, propagation, and development of tauopathies. Strategies that can modulate the process of TauO formation can help reduce the toxicity of TauO and stop the progression of the disease. Osmolytes are naturally occurring small-molecular-weight organic compounds, which crucially assist in the proper protein folding and thus impact the stability and solubility of proteins. Therefore, osmolytes can serve as good candidates for modulating TauO. Osmolytes cross the blood-brain barrier and act as chaperons to prevent the proteins from misfolding and aggregation. Here, we investigated the effect of different brain osmolytes against the amplified brain-derived tau oligomer (aBDTO). Our investigations have revealed that the brain osmolytes modulate the aBDTO differentially. The osmolytes sorbitol and glycerophosphocholine (GPC) displayed the potential to reduce the formation and accumulation of large aggregates by disaggregating the precursor tau oligomers into smaller assemblies with varying conformations. This may result from these osmolytes modulating the conformation of aggregated tau, which could lead to reduction in its seeding potential. However, trimethyl amine oxide (TMAO) has been found to prevent and clear out the formation of aBDTO significantly. Citrulline is less effective than TMAO and possibly affects more dimeric species. These osmolytes can become an indispensable tool for the management of Alzheimer's disease and part of hybrid therapeutic mechanisms, in addition to providing better understanding of tau oligomerization and seeding ability.

Keywords: Alzheimer’s disease; aBDTO; circular dichroism; osmolytes; proteinase K digestion, mass spectrometry.

1

Biochemical characterization of aBDTO alone and treated with different osmolytes done employing immunoblotting with (A) Tau 5, (C) Tau 13, (E) T22, and (G) T18 antibodies. It is clear from the protein bands that TMAO-aBDTO and most of the citrulline-aBDTO are confined to the region below 70 kDa. The bands above 70 kDa have disappeared. This suggests the TMAO and citrulline are effective in reducing the aBDTO. Other osmolytes (i.e., myo-inositol, taurine, betaine, and sorbitol) display no effect on aBDTO levels, as shown in the densitometric quantitation bands >70 kDa for all four antibodies (B) Tau 5, (D) Tau 13, (F) T22, and (H) T18.

2

Biochemical characterization of aBDTO alone and treated with different osmolytes employing dot blot analysis (n = 3) and immunodetection with (A) Tau13, (B) T22, and (C) T18. (D) Densitometric analysis of membrane indicating that TMAO strongly modulates aBDTO conformation which is similar to the monomer as is evident from the low value of the ratio of T22/Tau 13. Other osmolytes also modulate the conformation of the aBDTO which is different from the positive control conformation as is evident from the densitometric quantitation.

3

(A) FTA analysis (n = 2) of aBDTO alone and treated with osmolytes, probed with Tau13, T18, and T22. Antimouse (AM) and antirabbit (AR) antibodies were used to exclude nonspecific antibody-binding to the membrane, HRP-conjugated IgG antirabbit secondary antibody was used to detect T22 and T18, and antimouse secondary antibody was used to detect Tau13. Densitometric quantitation of aBDTO in the presence of different osmolytes and probed by Tau13 (B), T18 (C), and T-22 (D).

4

WB analysis of aBDTO treated with and without osmolyte at 37 °C and PK digested by three different concentrations: (A) 0.0 μg/mL, (B) 0.5 μg/mL, and (C) 1.0 μg/mL. Sequence-specific Tau5 antibody was used as the primary antibody to probe differential PK-digested fragments in the presence and absence of osmolytes (D,E). Significant peptides on the N-terminal (Tau 2–87). (F) Proline region (tau 164–221) and (G) repeat region (tau 243–280) after three tryptic digestions (n = 2); peptide codes designated with letters P1–P13; sequences and regions are as listed in Table S2; differential accessibility of peptide by trypsin was tested by multiple sample ANOVA test with permutation-based FDR < 0.01%. The intensity of each tau peptide was normalized with the intensity of the tau protein in each sample.

5

Biophysical characterization of aBDTO by (A) far-UV CD at pH 7.4 and 25 °C in the presence and absence of osmolytes. (B) Percentage secondary structure content of individual spectrum was estimated by Jasco spectra manger software based on Yang’s reference . Fluorescence intensity of an individual sample was compared with Tau+PBS after measuring the fluorescence spectra of aBDTO and aBDTO-osmolyte mixture in the presence of (C) bis-ANS and (D) ThT. Fluorescence spectra of fluorescence dye+osmolyte were measured for baseline to correct false positive readings that could be due to the intrinsic fluorescent properties of the osmolytes. Bars and error bars represent the mean and standard deviation (±SD) for n = 3.

6

Morphological characterization of aBDTO by AFM treatment with and without osmolytes. AFM image (A–H) (scale bars = 100 nm, left bottom corner of each image) and mean height distribution histograms (right side of each image). AFM image and mean height for each sample are as follows: (A) aBDTO, (B) myo-inositol-aBDTO, (C) taurine-aBDTO, (D) TMAO-aBDTO, (E) betaine-aBDTO, (F) sorbitol-aBDTO, (G) GPC-aBDTO, and (H) citrulline-aBDTO.