Abstract
The intracellular deposition of fibrils composed of the microtubule-associated protein Tau is a characteristic feature of Alzheimer's disease (AD) and other fatal neurodegenerative disorders collectively known as tauopathies. Short Tau fibrils spread intracerebrally through transfer between interconnected neurons. Once taken up by a recipient cell, Tau fibrils recruit Tau monomers onto their ends. Based on the number of microtubule-binding repeats, there are two distinct groups of Tau isoforms: three-repeat (3R) Tau and four-repeat (4R) Tau. In AD, all Tau isoforms are deposited, whereas in other tauopathies, only 3R or 4R Tau isoforms are deposited. The molecular basis for these isoform-specific depositions is poorly understood, although conformation-based cross-seeding barriers are key. Here, we used sedimentation assays, EPR spectroscopy, and other structural readouts to better understand the cross-seeding barriers of 4R Tau fibrils. We observed that fibrils formed from truncated Tau (K18), but not full-length Tau (htau40), exhibit a barrier that inhibits 3R Tau recruitment. Investigating an array of differently sized fragments, we found that the Tau C terminus modulates the cross-seeding barrier and that the N terminus plays a synergistic role. Two disease-associated Tau variants, P301S and P301L, also established strong cross-seeding barriers. EPR analysis indicated that fibrils seeded with truncated and mutated Tau, but not htau40, are structurally disordered in the second half of repeat four and onward. These findings suggest that the disorder in this region diminishes the ability of 4R Tau fibrils to recruit 3R Tau monomers, revealing a new mechanism for Tau cross-seeding barriers.
Highlights
The intracellular deposition of fibrils composed of the microtubule-associated protein Tau is a characteristic feature of Alzheimer’s disease (AD) and other fatal neurodegenerative disorders collectively known as tauopathies
These findings suggest that the disorder in this region diminishes the ability of 4R Tau fibrils to recruit 3R Tau monomers, revealing a new mechanism for Tau crossseeding barriers
The present study focused on 4R Tau fibrils
Summary
In a first set of experiments, we sought to determine the seeding properties of htau (largest isoform, 4R Tau) [18]. The EPR spectra of the paramagnetically labeled proteins revealed three sharp lines (Fig. S3B), in agreement with the high mobility and intrinsic disorder of monomeric Tau [47, 48] These proteins were mixed with the preformed seeds of variant Tau fibrils (htau, P301S, P301L, and K18). When grown onto htau seeds, all paramagnetically labeled mutants produced single-line EPR spectra with reduced amplitudes (Fig. 8B, left column), characteristic for parallel, in-register arrangement of -strands, with identical residues in neighboring Tau molecules stacking on top of each other [25]. To obtain spectra free of these distortions, we repeated the seeding experiments using a mixture of paramagnetically and nonparamagnetically labeled mutants (0.2:0.8 molar ratio) In this case, all spectra of htau40seeded fibrils exhibited three broad lines with a 68-G separation between outer peaks (Fig. S4A). The clouds of htau fibrils are distinct from those of P301S and P301L fibrils
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