Role of phosphorylation of Alzheimer's amyloid precursor protein during neuronal differentiation

Abstract

Alzheimer's amyloid precursor protein (APP), the precursor of beta-amyloid (Abeta), is an integral membrane protein with a receptor-like structure. We recently demonstrated that the mature APP (mAPP; N- and O-glycosylated form) is phosphorylated at Thr668 (numbering for APP695 isoform), specifically in neurons. Phosphorylation of mAPP appears to occur during, and after, neuronal differentiation. Here we report that the phosphorylation of mAPP begins 48-72 hr after treatment of PC12 cells with NGF and that this correlates with the timing of neurite outgrowth. The phosphorylated form of APP is distributed in neurites and mostly in the growth cones of differentiating PC12 cells. PC12 cells stably expressing APP with Thr668Glu substitution showed remarkably reduced neurite extension after treatment with NGF. These observations suggest that the phosphorylated form of APP may play an important role in neurite outgrowth of differentiating neurons.

Fig. 1.

Phosphorylation of APP at Thr668 during neuronal differentiation of PC12 cells. PC12 cells (∼1 × 10⁶ cells) were cultured in the presence of NGF for the indicated times (0–120 hr). a, APP was immunoprecipitated from cell lysates (1.5 mg of protein) using UT-421, and samples were analyzed by SDS-PAGE [6% (w/v) polyacrylamide] and Western blotting using either UT-421 (APP,top) or UT-33 (PiAPP,bottom). b, APP and PiAPP were quantified using a Fuji BAS 2000 Imaging Analyzer, and the level of PiAPP was normalized to that of APP. The results shown are the average of duplicate assays, and error bars are indicated. mAPP, Mature APP isoforms; imAPP, immature APP isoforms. The size of the molecular weight standards (in kilodaltons) is indicated.

Fig. 2.

Localization of APP and phosphorylated APP in differentiating PC12 cells. PC12 cells ∼2–3 × 10⁴) were cultured for 72 hr with NGF. Cells were double-stained with UT-421 (a) and TU-01 (b) antibodies, or double-stained with UT-33 (c) and TU-01 (d). Scale bar, 25 μm. Arrowhead indicates a growth cone.

Fig. 3.

Domain organization of wild-type and mutant fAPP695 molecules and analysis of their expression and phosphorylation in PC12 cells. a, The domain organization of fAPP695 is illustrated. The positions of the FLAG sequence (FLAG), the transmembrane domain (TM), the β-amyloid (Aβ) domain, and the phosphorylation site, T668 (668), are indicated. The mutation of Thr668 is also indicated: in fAPP695T668A, threonine is replaced by alanine; in fAPP695T668E, threonine is replaced by glutamate. b, APP was immunoprecipitated from cell lysates (1.5 mg of protein) from PC12 cells stably expressing fAPP695wt (wt), fAPP695T668A (T668A) or fAPP695T668E (T668E) using M2 (FLAG) antibody, and the samples were subjected to SDS-PAGE [6% (w/v) polyacrylamide]. Samples were analyzed by Western blot using UT-421 (APP) and UT-33 (PiAPP) antibodies. Immunocomplexes were detected with ¹²⁵I-protein A, and APP and PiAPP were quantified using a Fuji BAS 2000 Imaging Analyzer.mAPP, Mature fAPP695; imAPP, immature fAPP695.

Fig. 4.

Localization of fAPP695 in PC12 cells after treatment with NGF. PC12 cells stably expressing fAPP695wt (a, d), fAPP695T668A (b, e), and fAPP695T668E (c, f) were treated with NGF for 72 hr and double-stained with M2 (FLAG) (a–c) and T-3526 (tubulin) (d–f) antibodies. Scale bar, 25 μm.

Fig. 5.

Neurite extension of PC12 cells expressing fAPP695 after treatment with NGF. PC12 cells stably expressing fAPP695wt (WT), fAPP695T668A (T668A), or fAPP695T668E (T668E), and nontransfected cells (PC12) were cultured in the presence of NGF for the indicated times. a, The numbers of neurites was divided by the cell number examined (n = 100).b, The length (in micrometers) of the neurites was measured, and the average length of neurites from 100 cells is indicated. Experiments were performed using five independent clones of cell lines expressing the various constructs, and the averages and SD are shown (n = 5). Asterisksindicate statistical significance by standard t test relative to fAPP695wt (*p < 0.01; **p < 0.005; ***p < 0.001).

Fig. 6.

Proposed model for the role of APP, phosphorylated at Thr668, in neurite outgrowth. Endogenous wild-type APP is phosphorylated at Thr668 after incubation with NGF, and PiAPP present in growth cones influences neurite extension via a putative protein factor (gray box). Exogenous wild-type APP is also phosphorylated in response to NGF treatment and contributes to normal neurite extension. Exogenous fAPP695T668A(T668A) cannot bind the putative protein, but neurite extension progresses normally by virtue of the interaction of endogenous phosphorylated APP with protein factor. In contrast, exogenous fAPP695T668E (T668E) binds the putative protein factor, but this association either initially blocks neurite outgrowth or interferes with normal function of the phospho-APP/protein factor complex by a “dominant negative” effect.