3R and 4R tau isoforms in paired helical filaments in Alzheimer’s disease

presence of 4r tau in AD pathologies may be underestimated when rD4 is used.However, anti-4r (available from Cosmo Bio Co., Ltd.) raised against rD4 peptide with N279D substitution stained both wild-type and deamidated 4r tau, and strongly stained rD3+/rD4-tangles and smearing tau fragments in sarkosyl-insoluble fraction of AD brain [2].It was reported that rD3 stained abundant ghost tangles in entorhinal cortex and tangles in CA1, but failed to stain fine processes of tangles and threads [3], while rD4 failed to detect ghost tangles in entorhinal cortex [3].To understand these findings, we examined the influence of protease on immunoreactivity.paraffin sections of AD brains were treated with 10 μg/mL proteinase K (pro-K) for 30 min after autoclaving (Ac) and formic acid (FA) treatment.rD3 staining was strongly enhanced (Fig. 1a,b).Conversely, rD4 immunoreactivity almost completely disappeared after pro-K treatment (Fig. 1c,d).Not only ghost tangles but also rD3-/rD4+ tangles and their processes became rD3-positive after pro-K treatment (Fig. 1a,b), strongly suggesting that the rD3 epitope was buried in tau filaments of intracellular tangles and threads, and was exposed by pro-K treatment.Contrary to expectation, anti-4r staining was also enhanced by pro-K treatment (Fig. 1e,f).It is possible that the recognition site of anti-4r is distinct from that of rD4 and is exposed by pro-K treatment of sections.Anti-4r antibody may recognize the carboxyl-half of the antigen peptide, while rD4 recognizes the amino-terminal half around N279. pro-K treatment was also effective in immunostaining of free-floating AD sections with a lower concentration.To confirm these findings biochemically, sarkosylinsoluble fractions from two AD brains were treated with trypsin or pro-K, then immunoblotted with rD3, rD4, anti-4r and anti-ps396 (Fig. 1g-j).rD3 strongly stained many bands and smears, as seen with ps396 (Fig. 1g,j),