Phosphorylation of human APP695 at Thr668 seems to be specific to neuronal tissue and could affect A beta production. Metabolism of APP mutated at Thr-668 residue was analyzed in CHO cell line and primary cultures of rat cortical neurons. By site-directed mutagenesis, T668A or T668D substitutions were introduced in wild-type APP695. In CHO cells, wild-type APP695 was very slightly phosphorylated at Thr668 and produced similar levels of extracellular A beta 40 as compared to APPT668A. On the contrary, APPT668D was more efficiently cleaved by beta-secretase. However, accumulated beta CTF were less cleaved by gamma-secretase and less extracellular A beta 40 was produced. Decreased susceptibility to cleavage by gamma-secretase was confirmed upon expression of C99T668D. In neurons, part of APP695 was phosphorylated at Thr-668. Following neuronal expression of APPT668A, extracellular A beta 40 production was increased. In conclusion, phosphorylation of human APP695 at Thr668 increases APP P-cleavage but decreases its gamma-cleavage and extracellular A beta 40 production. (c) 2007 Elsevier Inc. All rights reserved.