The intracellular threonine of amyloid precursor protein that is essential for docking of Pin1 is dispensable for developmental function

Abstract

Background: Processing of Aβ-precursor protein (APP) plays an important role in Alzheimer's Disease (AD) pathogenesis. Thr residue at amino acid 668 of the APP intracellular domain (AID) is highly conserved. When phosphorylated, this residue generates a binding site for Pin1. The interaction of APP with Pin1 has been involved in AD pathogenesis.

Methodology/principal findings: To dissect the functions of this sequence in vivo, we created an APP knock-in allele, in which Thr(668) is replaced by an Ala (T(668)A). Doubly deficient APP/APP-like protein 2 (APLP2) mice present postnatal lethality and neuromuscular synapse defects. Previous work has shown that the APP intracellular domain is necessary for preventing early lethality and neuromuscular junctions (NMJ) defects. Crossing the T(668)A allele into the APLP2 knockout background showed that mutation of Thr(668) does not cause a defective phenotype. Notably, the T(668)A mutant APP is able to bind Mint1.

Conclusions/significance: Our results argue against an important role of the Thr(668) residue in the essential function of APP in developmental regulation. Furthermore, they indicate that phosphorylation at this residue is not functionally involved in those APP-mediated functions that prevent (NMJ) defects and early lethality in APLP2 null mice.

Figure 1. Survival analysis of APP^TA ki mice on APLP2 null background.

Analysis of genotypes of 128 offspring collected at P1 derived from crosses of APP^TA/−APLP2^+/− male and female mice. All genotypes were recovered at close to a Mendelian ratio (df = 8, p>0.95). Analysis of genotypes of these same offspring at P28 showed that the number of APP^−/−APLP2^−/− animals observed was much lower than expected (highlighted in bold, df = 8, p<0.001). On the contrary, the number of APP^TA/TAAPLP2^−/−, APP^TA/−APLP2^−/− was still close to a Mendelian ratio (B, df = 8, p>0.20).

Figure 2. No obvious neuromuscular synapse defects were observed in APP^TA/TA/APLP2^−/− mice.

A. Whole-mount staining of littermate APP^+/+/APLP2^−/−control (Ctrl) and APP^TA/TA/APLP2^−/− (TA) P0 diaphragm muscles with antibodies against synaptophysin (Syn). Anti-BTX was used to mark the AchRs. B. Quantification of the average band width of endplates marked by anti-BTX (band width in control 260.0±25.17 µm vs. TA 282.9±18.63 µm. p>0.05, student t-test. Mean ± SEM of 3 animals/genotype). C. Higher magnification images showing endplates closely apposed by Syn and no axonal Syn staining in the TA mutant. D. Quantification of the area percentage of AchR endplates covered by Syn (control 0.626±0.024 vs. TA 0.588±0.017. p>0.05, student t-test. Mean±SEM of 20 endplates/genotype). Scale bar: A, 100 µm; C, 20 µm.

Figure 3. APP^TA interacts with Mint1.

GST-Mint1 pull-down experiments show that Mint1 interacts with both wild-type APP (WT) and APP^TA (TA) mutant. The interaction is specific since GST alone does not interact with APP and GST-Mint1 does not pull down a protein of size similar to APP and cross-reacting with the anti-APP antibody 22C11 from brain lysates of APP KO (KO) mice. The faint doublet recognized by the 22C11 in the total lysates (TL) from APP KO mice probably represents low levels of cross-reactivity of the antibody with APLP1 and/or APLP2.