Rapid Dephosphorylation of τ in Heat‐Shocked Fetal Rat Cerebral Explants: Prevention and Hyperphosphorylation by Inhibitors of Protein Phosphatases PP1 and PP2A

: We heat shocked 21‐ and 35‐day‐old fetal rat cerebral explants at 45°C for 18 min and performed immunocytochemistry and immunoblot analysis of sodium dodecyl sulfate extracts using the monoclonal anti‐τ antibodies Tau‐1, Tau‐5, Tau‐46, and PHF‐1 and the peroxidase‐antiperoxidase technique or 125I‐labeled protein A. Tau‐1 and PHF‐1 recognize nonphosphorylated and phosphorylated epitopes, respectively, and both Tau‐5 and Tau‐46 recognize phosphate‐independent epitopes. τ immunoreactivity was confined to neurons and increased in heat‐shocked perikarya but not axons. At 0 h after heat shocking, there was dephosphorylation of τ exemplified by (1) faster migration of τ isoforms with resultant loss or attenuation of the 60‐ and 52‐kDa τ isoforms recognized by all four anti‐τ antibodies and concomitant accentuation of the fastest moving 50‐kDa τ isoform recognized by Tau‐1, Tau‐5, and Tau‐46; and (2) significant increase in the nonphosphorylated Tau‐1 epitope with resultant decreases in the ratio of total (phosphorylated plus nonphosphorylated) τ to nonphosphorylated τ and the difference of total τ minus nonphosphorylated τ. τ was phosphorylated back to the control level by 12 h and remained so at 24 and 48 h after heat shocking. Treatment of explants with cycloheximide, a protein synthesis inhibitor, did not prevent the heat shocking‐induced dephosphorylation of τ. Treatment of explants with the inhibitors of protein phosphatases PP1 and PP2A, okadaic acid or calyculin A, produced hyperphosphorylated τ polypeptides, prevented the heat shocking‐induced dephosphorylation of τ, and intensified the immunoreactivity of the neurofilament subunit H with the only antiphosphoneurofilament antibody that reacts with intraneuronal neurofibrillary tangles. In 35‐day‐old explants, in addition to the three 50‐, 52‐, and 60‐kDa τ isoforms seen in 21‐day‐old explants, a 66‐kDa τ polypeptide was also present.