Visualization of newly deposited tau in neurofibrillary tangles and neuropil threads

Neurofibrillary tangles (NFTs) and neuropil threads (NTs), the major hallmark of Alzheimer disease (AD), are composed of the microtubule-associated protein tau that has undergone posttranslational modifications, including deamidation and isomerization on asparaginyl or aspartyl residues. Because such modifications represent protein aging, we generated 2 antibodies, TM4, specific for Asp-387 of tau, and iD387, specific for isoAsp-387 of tau, to investigate the evolution of NFTs and NTs. On Western blots of Sarkosyl-insoluble fractions, TM4 strongly labeled paired helical filament-tau (PHF-tau), whereas iD387 preferentially labeled PHF smear. Thus, it is reasonable to postulate that TM4-labeled tau (unmodified tau species) represents more recent deposition, and iD387-labeled tau (modified tau species) represents earlier deposition. Unexpectedly, TM4 immunostained even highly evolved NFTs, suggesting that deposition of newly produced tau continues until neuronal death. iD387 labeled the whole profile of NFTs up to distal dendritic branches, whereas TM4 staining was localized to particular portions of NFTs in proximal dendrites and neuronal perikarya. In NTs, TM4 preferentially labeled the outer portion, whereas iD387 intensely labeled the core portion. Based on TM4-positive NFT counts and total NFT counts, we speculate that NFTs in the human hippocampus are produced at a constant rate irrespective of the disease stage.