Phosphorylated Presenilin 1 decreases β-amyloid by facilitating autophagosome-lysosome fusion

Abstract

Presenilin 1 (PS1), the catalytic subunit of the γ-secretase complex, cleaves βCTF to produce Aβ. We have shown that PS1 regulates Aβ levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 decreases Aβ levels by increasing βCTF degradation through autophagy. Here, we report the molecular mechanism by which PS1 modulates βCTF degradation. We show that PS1 phosphorylated at Ser367, but not nonphosphorylated PS1, interacts with Annexin A2, which, in turn, interacts with the lysosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vamp8. Annexin A2 facilitates the binding of Vamp8 to the autophagosomal SNARE Syntaxin 17 to modulate the fusion of autophagosomes with lysosomes. Thus, PS1 phosphorylated at Ser367 has an antiamyloidogenic function, promoting autophagosome-lysosome fusion and increasing βCTF degradation. Drugs designed to increase the level of PS1 phosphorylated at Ser367 should be useful in the treatment of Alzheimer's disease.

Keywords: Annexin A2; Presenilin 1; autophagosome–lysosome fusion; autophagy; phosphorylation.

Fig. 1.

Incompletely fused autophagosomes–lysosomes are increased in brains of PS1-S367A mice. (A) Representative electron micrographs of autolysosomes in a pyramidal neuron from the CA1 area of the hippocampus from S367A knock-in mice. (Scale bar, 500 nm.) (B) Higher magnification of an unfused autolysosome. Arrows point to the double membrane. (Scale bar, 100 nm.) (C) Autolysosomes in the CA1 area of the hippocampus in PS1-S367A mice contain Cathepsin D, as revealed by immunoelectron microscopy. (Scale bar, 500 nm.) (D) Quantification of the percentage of neurons bearing autolysosomes, as seen by electron microscopy. Sixty fields from three different WT and PS1-S367A mice were used for quantification. N, nucleus. Data represent mean ± SEM. ***P < 0.001, t test.

Fig. S1.

Autolysosomes with incompletely fused membranes accumulate in the brains of PS1-S367A mice. Electron micrographs of representative autolysosomes that accumulate in the brains of PS1-S367A mice. (Scale bars, 500 nm.)

Fig. 2.

PS1 phosphorylated at Ser367 binds Annexin A2. (A) Immunoprecipitates (IP) from whole-mouse brain lysate using anti-Annexin A2 or anti-PS1 antibody were immunoblotted with an antibody against PS1 or Annexin A2. WB, Western blot. (B) Pull-down, showing that increasing the concentration of calcium increases the binding between recombinant Annexin A2 and the PS1-pSer367 biotinylated peptide. Numbers indicate calcium concentration (millimolar). P, phosphorylated PS1 peptide; S, phosphorylated scrambled control peptide. (C) Recombinant Annexin A2 binds to both PS1-pSer367 biotinylated peptide and a double-phosphorylated PS1-pSer365-pSer367 biotinylated peptide. It does not bind to S367D or S367A biotinylated peptide or to an S or WT sequence biotinylated peptide. (D) Recombinant Annexin A2 binds to PS1-pSer367 biotinylated peptide and to a mutant S366A-pS367 and L368A-pS367 biotinylated peptide. It does not bind to a S367D or S367A biotinylated peptide or to an S or WT sequence biotinylated peptide. (E) Biolayer interferometry between a PS1-pSer367 biotinylated peptide and an Annexin A2 (AnxA2) or AnxA2-p11 fusion protein at several Ca²⁺ concentrations. The y axis represents binding (nanometers). (F, Upper) Diagram showing the constructs used in this experiment. (F, Lower) Deletion mutant of Annexin A2 lacking the N terminus (ΔN) does not bind a pPS1 peptide, whereas a deletion mutant of Annexin A2 lacking the third and fourth calcium-binding domains (Δ3–4) is still able to bind a pPS1 peptide. (G) In situ PLA between Annexin A2 and PS1 in MEFs derived from WT or PS1-S367A mice. Note the loss of Annexin A2-PS1 binding in the absence of PS1 phosphorylation at Ser367. (Scale bars, 2 μm.)

Fig. 3.

Annexin A2 regulates Aβ metabolism. (A) Knock-down of Annexin A2 and/or p11 increases levels of Aβ40 in N2A cells overexpressing APP. Ctrl, control. (B) Knock-down of Annexin A2 and/or p11 increases βCTF levels in N2A cells. (C) Knock-down of Annexin A2 and p11 increases LC3-II and p62 protein levels in N2A cells. (D) CK1γ inhibitor compound 2-((4-(2-hydroxypropan-2-yl)phenyl)amino)-1H-benzo[d]imidazole-6-carbonitrile (HPB) increases Aβ levels in cells transfected with a control siRNA, but it fails to increase Aβ levels in cells transfected with siRNA against Annexin A2. Data represent mean ± SEM. *P < 0.05, ***P < 0.001; t test. Ctrl, control.

Fig. 4.

PS1 phosphorylated at Ser367 modulates binding of Annexin A2 to Vamp8. (A) Antibody against recombinant Vamp8 pulls down recombinant Annexin A2 in vitro. (B) Immunoprecipitates from whole-mouse brain lysate using anti-Annexin A2 antibody were immunoblotted with an antibody against Vamp8. (C, Left) Annexin A2 binds Vamp8 in cells, as measured by PLA. (C, Right) PLA control with fibroblasts derived from AnxA2-KO mice. (Scale bars, 5 μm.) (D) In situ PLA between Annexin A2 and Vamp8 in MEFs derived from WT (Left) or PS1-S367A mice (Right). Note the decrease of Annexin A2-Vamp8 binding in the absence of PS1 phosphorylation at Ser367. (Scale bars, 5 μm.) (E) Quantification of PLA from D (n = 15). Data represent mean ± SEM. ***P < 0.001, t test. Ctrl, control; WB, Western blot.

Fig. 5.

Annexin A2 facilitates binding of Vamp8 to Stx17. (A) Confocal images showing colocalization of Stx17 (green) with LC3⁺ (red) vesicles when autophagy is induced by starvation. Arrows indicate the site of colocalization. (Scale bar, 5 μm.) (B) Immunoprecipitates from whole-mouse brain lysate using anti-Vamp8 antibody were immunoblotted with an antibody against Stx17. Ctrl, control; IP, immunoprecipitation; WB, Western blot. (C) In situ PLA between Stx17 and Vamp8 in MEFs derived from WT, AnxA2-KO, or PS1-S367A mice in complete media (Upper) or starvation media (Lower). (Scale bar, 5 μm.) (D) Quantification of number of PLA reactions from C. Note the decrease of Stx17-Vamp8 binding in the absence of either Annexin A2 or PS1 phosphorylated at Ser367 (n = 20). Data represent mean ± SEM. **P < 0.01, ***P < 0.001; t test.

Fig. 6.

Proposed model by which PS1 increases autophagic flux and decreases Aβ levels. PS1, upon phosphorylation on Ser367 by CK1γ, interacts with Annexin A2 (AnxA2). In turn, Annexin A2 interacts with Vamp8, which facilitates the binding of Vamp8 to Stx17. This interaction induces the lysosome-autophagosome fusion, triggering the degradation of βCTF, leading to a decrease in Aβ levels. P, phosphorylation.