Abstract
Paired helical filaments (PHFs) are the major structural elements of Alzheimer's disease neurofibrillary lesions, and these filaments are formed from hyperphosphorylated brain tau known as PHF-tau. Recent studies showed that many previously identified phosphorylated residues in PHF-tau also are phosphate acceptor sites in fetal and rapidly processed adult brain tau. However, Ser262 has been suggested to be uniquely phosphorylated in PHF-tau and a key regulator of the binding of tau to microtubules. For these reasons, we generated a monoclonal antibody (12E8) specific for phosphorylated Ser262 and showed that 12E8 binds to PHF-tau, rat and human fetal brain tau, as well as to rapidly processed adult rat and biopsy-derived human brain tau. Further, phosphorylation Ser262 was developmentally regulated, and endogenous brain phosphatases rapidly dephosphorylated Ser262 in biopsy-derived brain tau isolates. Finally, the phosphorylation of Ser262 did not eliminate the binding of tau to microtubules. Thus, we speculate that the binding of tau to microtubules is regulated by phosphorylation at multiple sites and that the generation of PHF-tau in Alzheimer's disease results from the reduced efficiency of phosphatases leading to the incremental accumulation of hyperphosphorylated tau.
Highlights
Paired helical filaments (PHFs) are the major structural elements ofAlzheimer's disease neurofibrillary lesions, and these filaments are formed from hyperphosphorylated brain tau known as PHF-tau
Since the latter study emphasized the critical importance of Ser262 in regulating the binding of tau to MTs and the pivotal role that Ser262 may play in the pathogenesis ofPHF-tau in Alzheimer's disease (AD) [36], we explored this issue further using a novel monoclonal antibody (MAb) raised to a synthetic phosphopeptide spanning amino acid residues 257-270 in human tau with a phosphoserine at position 262
Since 12E8 recognized PHF-tau, fetal brain tau, and biopsy-derived adult brain tau, we probed nitro-5-thiocyanobenzoic acid (NTCB) cleavage products of tau and demonstrate that only Ser262 is phosphorylated in tau from normal and AD brain
Summary
Materials-NJ mice were purchased from Jackson Labs, and Sprague-Dawley rats were purchased from Charles River. Preparation of Monoclonal Antibody 12EB-The immunogen was a synthetic phosphopeptide corresponding to amino acid residues 257-270 in tau (according to the numbering of the longest human tau isoform) [10], with a phosphoserine at position Ser, as well as with two additional Gly residues and a Cys residue added at the carboxyl terminus for ease of coupling. This peptide (KSKIGS(PO,)TENLKHQPGGC) was coupled to sheep anti-mouse IgG using maleimidohexanoyl-N-hydroxysuccinimide, and 100 IJ-g of the immunogen was emulsified in Freund's complete adjuvant and injected subcutaneously into NJ mice.
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