Specific Peptide from the Novel W-Tau Isoform Inhibits Tau and Amyloid β Peptide Aggregation In Vitro

W-Tau, a new tau human-specific splicing isoform generated by intron retention, has been recently described. This isoform contains an 18-residue unique sequence corresponding to the translation of the retained region of intron 12. In this work, we have described that such 18-amino-acid peptide from the retained intron 12 can inhibit tau and β amyloid peptides aggregation under in vitro conditions. This inhibitory function is also present in smaller fragments of the 18-residue peptide.


■ INTRODUCTION
Recently, a new tau isoform generated by the retention of intron 12 of the human MAPT gene has been described. 1 Soon after the start of intron 12 of human MAPT, a stop codon appears, followed by a canonical polyadenylation sequence, resulting in the truncation of the protein at this point. Thus, this isoform differs from other human tau isoforms by lacking exon 13 of the MAPT gene and including an 18-amino-acid sequence corresponding to the translation of the retained fragment of the intron 12 in its place, at its carboxyl-terminal region, right after exon 12. 1 The 18-residue sequence contains two tryptophan residues (W), an amino acid that cannot be found at any other point of the human tau sequence, and thus, the isoform has been named w-Tau. The 18-amino-acid peptide, termed in turn w-Tau peptide, may be involved in the decreased aggregation shown by w-Tau compared to that of other full-length and truncated tau isoforms. 1 Indeed, analyzing the primary structure of the w-Tau peptide, some similarities were found with the sequences of a peptide family that prevents both tau and amyloid aggregation. 1,2 In this short report, we have studied, in vitro, the inhibitory effect of the w-Tau peptide on the aggregation of full-length tau and on a self-aggregating tau fragment (residues 317−335 from full-length 4R2N Tau). 3,4 In parallel, the inhibition of the aggregation of β amyloid peptide by w-Tau peptide was also analyzed.

■ METHODS
In Vitro Polymerization Analyses: Tau Protein, β Amyloid Peptide. Tau protein and its peptides were dissolved at a concentration of 10 mg/mL in distilled water, aliquoted, and immediately used or frozen to be used only once, to avoid several  freezing/thawing cycles. Amyloid β peptide was stored in small aliquots as a solid at 4°C. Recombinant human Tau 3R or 1/2R peptide (10 μg) was incubated in 10 μL of Buffer A (0.1 MES (pH 6.4), 0.5 mM MgCl 2 , and 2 mM EGTA) and 0.5 M NaCl, in the absence or presence of different concentrations of heparin. The optimal Tau protein:heparin ratio to visualize fibrillar polymers was 1:4 (mass/mass). For the 1/2R tau peptide, the optimal peptide:heparin ratio was found to be 1:1 (mass/mass). To study the effect of the 18aa Tau peptide on the polymerization of Tau 3R, equimolar amounts of Tau and peptide were used. To analyze the effect of w-Tau (18aa and fragments) peptides on 1/2R tau peptide polymerization, w-Tau peptides were added at final concentrations of 1, 0.1, and 0.01 μg/μL. The reactions were allowed to proceed at room temperature for 7 days before analysis. In the presence of heparin, protein assembly is accelerated and reaction conditions could be set at 3 days at a lower temperature (4°C). 4 Tau polymers were partially quantified by observing filamentous polymers at electron micrographs of several fields. For a proper quantification of Tau proteins, the protein aggregates were pelleted by centrifugation at 28 psi for 30 min, using a table Beckman Airfuge ultracentrifuge, and the pelleted protein was analyzed by Western blot using tau antibody 7.51. 7 As for the corresponding amyloid β peptide polymerization assays, 10 μL of w-Tau peptide at a concentration of 3, 0.3, or 0.03 μg/μL in buffer A was added to 10 μg of lyophilized amyloid β peptide. After 10 min of incubation at room temperature, fibrillar polymers in the absence or presence of w-Tau peptides can be visualized by electron microscopy.
Amyloid β polymers quantifications were done by pelleting the polymers, as indicated for tau aggregates, and measuring protein amount by dot blot using Coomassie blue. 8 Electron Microscopy Analysis. Polymerization reaction samples were added to a Formvar (400 mesh) carbon-coated grid for 5 min. The grid was stained with 2% uranyl acetate for 40 s. The grids were examined with a JEM1010 (Jeol) transmission electron microscope. Images were taken with a TemCam F416 (TVIPS) camera at a magnification of 20,000×.
■ RESULTS w-Tau Peptide Inhibits the Polymerization of Tau Protein In Vitro. It was already described that highly purified tau protein can polymerize in vitro, 9 yielding filaments similar to those found in the brain of Alzheimer's disease patients, 10 but not identical. 11,12 Also, it is known that, in the presence of heparin, filament polymerization accelerates for ON3R tau isoform. 3,4,13 Thus, we have tested the action of w-Tau peptide on tau protein aggregation in the presence or absence of heparin. Figure 1 shows that, in both situations, w-Tau peptide (18aa) inhibits tau filament assembly. When 0N4R tau isoform was tested, inhibition of its polymerization by w-Tau peptide was found as well (Supporting Information 1).
Assembly of Tau Peptide (317−335aa) Is Inhibited by w-Tau Peptide. In a pioneer study, it was shown that the microtubule-binding region of tau protein, containing similar, but not identical, three or four repeated sequences was the region involved in tau−tau interaction. 14 Focusing on the third repeat (residues 306−335), it was found that it may be able to self-assemble into filamentous peptides. 3 Even small fragments of that 306−335 peptide, like peptides comprising 306−311 15 or that containing residues 317−335 (1/2R peptide) 3,4 are able to self-polymerize. Figure 2 shows the dose-dependent inhibition of the tau 1/2R peptide (317−335) polymerization mediated by the presence of w-Tau peptides.
Fragments of w-Tau Peptide Could Inhibit Tau Polymerization. Since w-Tau peptide contains 18aa (KKVKGVGWVGCCPWVYGH), we have tested if fragments of this peptide like a 10aa peptide (VGWVGCCPWV) or a 6aa peptide (GWVYGH) can also inhibit the self-assembly of the 1/2R tau fragment (317−355). Figure 2B,C indicates that also those peptides could inhibit in a dose-dependent manner this fragment's assembly. As a negative control, Figure 2D indicates the lack of inhibition of tau assembly in the presence of the peptide DHGAEIV, a peptide containing the residues 387− 393 of the 2N4R tau molecule.
w-Tau Peptide Inhibits Amyloid β Peptide Assembly. The sequence of w-Tau peptide is similar to that of the LYIWVQ family peptides that prevent the assembly of tau and amyloid peptide. 2 Thus, we have tested if w-Tau peptides might also prevent amyloid peptide assembly (Figure 3). To do that, w-Tau peptides at different concentrations in buffer A, or buffer A alone as a control, were added to a known amount of lyophilized (solid) β amyloid peptide and properly mixed. The polymerized protein in an aliquot of the resulting preparation was visualized by electron microscopy. The rest of the preparation was subjected to centrifugation, and the pelleted (polymerized/aggregated) protein was measured by electrophoresis. Figure 3A shows that 18-aa w-Tau peptide inhibits amyloid polymerization in a dose-dependent manner. In addition, 10-aa ( Figure 3B) fragments but not 6-aa fragments ( Figure 3C) of 18-aa w-Tau peptide also slightly inhibit the formation of amyloid polymers.

■ DISCUSSION
In this work, we have shown that w-Tau peptide, the sequence of intron 12 retained in the new w-Tau isoforms, 1 is able to inhibit tau protein assembly. This feature could explain the decreased capacity of w-Tau isoform for self-assembly 1 and suggests its action as a potential inhibitor for the assembly of the other tau isoforms. The inhibitory role of w-Tau peptide not only for tau protein assembly but also for the inhibition of amyloid β peptide polymerization may suggest a potential use as a tool to prevent tauopathies that are mainly characterized by the presence of aberrant tau protein aggregates. 16,17 The inhibition of amyloid aggregation could be also of interest for the most relevant tauopathy, Alzheimer's disease.