UNIQUE ALZHEIMERS-DISEASE PAIRED HELICAL FILAMENT SPECIFIC EPITOPES INVOLVE DOUBLE PHOSPHORYLATION AT SPECIFIC SITES

Alzheimer's disease (AD) paired helical filaments (PHFs), building blo cks of neurofibrillary tangles (NFTs) are composed of hyperphosphoryla ted forms of the microtubule-associated protein tau (i.e., PHF-tau). C urrently, much effort is devoted to the development of diagnostic anti bodies specific for PHF-tau since elevated tau levels are found in the cerebral spinal fluid of AD patients. To this end, we have mapped the epitopes of a large panel of monoclonal antibodies (mAbs) that recogn ized only phosphorylation dependent epitopes on PHF-tau. These mAbs in clude the PHF-tau specific mAb AT10 and 12 newly developed anti-PHF mA bs that recognize PHF-tau but not autopsy-derived normal adult tau on Western-blot and enzyme-linked immunosorbent assay (ELISA). Epitope an alysis, together with data on known binding sites of previously publis hed mAbs, revealed that Ser214, Thr231, and Ser396 are immunodominant phosphorylated amino acids in PHF-tau. Six of the 12 new mAbs recogniz ed one of these three phosphorylated sites. With the exception of AT10 and PHF-27, all the mAbs also labeled fetal tau and biopsy-derived ta u. Since mAbs AT10 and PHF-27 had little or no affinity for fetal tau and biopsy tau, they can be considered as the first ''true'' PHF-speci fic antibodies capable of distinguishing tau isoforms from normal vers us AD subjects, suggesting a possible utility of these mAbs as diagnos tic markers. Remarkably, the true PHF-specific antibodies recognized p eptide sequences phosphorylated on more than one amino acid residue. T he peptide recognition of mAb AT10 required the simultaneous phosphory lation of Thr212 and Ser214, and the peptide recognition of mAb PHF-27 was markedly increased when both the primary site Thr231 and the subs ite Ser235 were phosphorylated. Since AT10 and PHF-27 are the only mAb s currently available that bind specifically to PHF-tau, these data su ggest that double phosphorylation at Thr212/Ser214 and Thr231/Ser235 m ay be unique to PHF-tau. These data may facilitate the development of mAbs that can be used as specific diagnostic reagents for the detectio n of altered a in cerebrospinal fluid of AD patients.