Phosphorylation of APP695 at Thr668 decreases gamma-cleavage and extracellular Abeta.

Phosphorylation of human APP695 at Thr668 seems to be specific to neuronal tissue and could affect Abeta production. Metabolism of APP mutated at Thr668 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 Abeta40 as compared to APPT668A. On the contrary, APPT668D was more efficiently cleaved by beta-secretase. However, accumulated betaCTF were less cleaved by gamma-secretase and less extracellular Abeta40 was produced. Decreased susceptibility to cleavage by gamma-secretase was confirmed upon expression of C99T668D. In neurons, part of APP695 was phosphorylated at Thr668. Following neuronal expression of APPT668A, extracellular Abeta40 production was increased. In conclusion, phosphorylation of human APP695 at Thr668 increases APP beta-cleavage but decreases its gamma-cleavage and extracellular Abeta40 production.

Calcium-mediated transient phosphorylation of tau and amyloid precursor protein followed by intraneuronal amyloid-beta accumulation.

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Lactacystin decreases amyloid-beta peptide production by inhibiting beta-secretase activity.

The human amyloid precursor protein (APP) is processed by the nonamyloidogenic and the amyloidogenic catabolic pathways. The sequential cleavage of APP by the beta- and gamma-secretase activities, known as the amyloidogenic processing of APP, leads to the formation of the amyloid-beta peptide (Abeta). Abeta is the main constituent of the amyloid core of senile plaques, a typical hallmark of Alzheimer's disease. In addition to secretases, other cellular proteolytic activities, like the proteasome, might participate in the metabolism of APP. We investigated the consequence of proteasome inhibition on the amyloidogenic processing of human APP. CHO cells and primary cultures of rat cortical neurons expressing human APP or a protein corresponding to its beta-cleaved C-terminal fragment (C99) were treated with lactacystin, an irreversible inhibitor of the chymotrypsin-like activity of the proteasome. Lactacystin significantly decreased the level of Abeta produced from APP in both cellular models, whereas the production of Abeta from C99 was not affected. Lactacystin did not inhibit gamma-secretase activity but was found to inhibit the beta-cleavage of APP, leading to a proportional decrease in Abeta production. Although lactacystin did not inhibit the catalytic activity of recombinant BACE1, a decrease in neuronal beta-secretase activity was measured after treatment with lactacystin.

Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3.

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.