To elucidate the role cAMP-dependent protein kinase (PKA) phosphorylations on tau play in Alzheimer's disease, we have generated highly specific monoclonal antibodies, CP-3 and PG-5, which recognize the PKA-dependent phosphorylations of ser214 and ser409 in tau respectively. The present study demonstrates by immunohistochemical analysis, CP-3 and PG-5 immunoreactivity with neurofibrillary pathology in both early and advanced Alzheimer's disease, but not in normal brain tissue and demonstrates that cAMP-dependent protein kinase phosphorylations on tau precede or are coincident with the initial appearance of filamentous aggregates of tau. Studies using heat-stable preparations demonstrate that neither site appears to be phosphorylated to any appreciable extent in normal rodent or human brain. Further analysis demonstrates that the ␤ catalytic subunit of PKA (C␤), the ␤ II regulatory subunit of PKA (RII␤), and the 79 kDa A-kinase-anchoringprotein (AKAP79), are tightly associated with the neurofibrillary pathology, positioning cAMP-dependent protein kinase to participate directly in the pathological hyperphosphorylation of tau seen in Alzheimer's disease.

Hyperphosphorylated tau (PHF‐tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation‐dependent epitopes on tau, and (c) those recognizing conformation‐dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation‐dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF‐tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation‐dependent phosphoepitopes on tau, typified by TG‐3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site‐directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG‐3. Additional studies demonstrate that TG‐3 reacts with neonatal mouse tau and PHF‐tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG‐3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.