Tangle evolution linked to differential 3- and 4-repeat tau isoform deposition: a double immunofluorolabeling study using two monoclonal antibodies.

Double immunofluorolabeling for 3-repeat (3R) and 4-repeat (4R) tau was performed with two monoclonal antibodies, RD3 and RD4, after an additional pretreatment with potassium permanganate and oxalic acid to eliminate nonspecific 3R tau cytoplasmic staining. This method involves hyperdilution of one of the primary monoclonal antibodies (≥100-fold), making it undetectable by usual secondary antibodies. The hyperdiluted primary antibody can then only be detected after tyramide amplification. Subsequent application of the other monoclonal antibody at its usual concentration allows double immunofluorolabeling without cross-reaction. This novel method revealed that tau immunoreactivity (IR) in the hippocampal pyramidal neurons of Alzheimer's disease (AD) brains is heterogeneous in that pretangle neurons exhibit 4R-selective (3R-/4R+) IR, ghost tangles exhibit 3R-selective (3R+/4R-) IR, and neurofibrillary tangles exhibit both 3R and 4R (3R+/4R+) IR. Some nigral neurons exhibited RD3 IR in both AD and corticobasal degeneration/progressive supranuclear palsy (CBD/PSP) brains. However, in CBD/PSP cases, 3R IR was always superimposed on 4R IR, while 3R-selective neurons were present in AD cases. These differential isoform profiles may provide a pivotal molecular reference, closely related to the morphological evolution of tau-positive neurons, which may be variable according to disease (CBD/PSP vs. AD), lesion site (cerebral cortex and substantia nigra), or the stage of evolution (from pretangles to ghost tangles). These findings should provide a more comprehensive understanding of the histological differentiation of various tau deposits in human neurodegenerative disease.