Bidirectional regulation of Aβ levels by Presenilin 1

Abstract

Alzheimer's disease (AD) is characterized by accumulation of the β-amyloid peptide (Aβ), which is generated through sequential proteolysis of the amyloid precursor protein (APP), first by the action of β-secretase, generating the β-C-terminal fragment (βCTF), and then by the Presenilin 1 (PS1) enzyme in the γ-secretase complex, generating Aβ. γ-Secretase is an intramembranous protein complex composed of Aph1, Pen2, Nicastrin, and Presenilin 1. Although it has a central role in the pathogenesis of AD, knowledge of the mechanisms that regulate PS1 function is limited. Here, we show that phosphorylation of PS1 at Ser367 does not affect γ-secretase activity, but has a dramatic effect on Aβ levels in vivo. We identified CK1γ2 as the endogenous kinase responsible for the phosphorylation of PS1 at Ser367. Inhibition of CK1γ leads to a decrease in PS1 Ser367 phosphorylation and an increase in Aβ levels in cultured cells. Transgenic mice in which Ser367 of PS1 was mutated to Ala, show dramatic increases in Aβ peptide and in βCTF levels in vivo. Finally, we show that this mutation impairs the autophagic degradation of βCTF, resulting in its accumulation and increased levels of Aβ peptide and plaque load in the brain. Our results demonstrate 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 also decreases Aβ levels by increasing βCTF degradation through autophagy. Elucidation of the mechanism by which PS1 regulates βCTF degradation may aid in the development of potential therapies for Alzheimer's disease.

Keywords: Alzheimer’s disease; Aβ; Presenilin 1; autophagy; phosphorylation.

Fig. 1.

PS1 is phosphorylated at Ser367 by CK1γ2. (A) Analysis of the level of knockdown of the CK1 isoforms by quantitative PCR. n = 3. (B) Knockdown of CK1γ2 isoform decreases phosphorylation of PS1 at Ser367 in N2A cells. (C) Densitometric analysis of pS367/total PS1. n = 3. (D and E) Inhibition of CK1γ decreases PS1-Ser367 phosphorylation. N2A cells were treated for 24 h with 5 μM HPB, a CK1γ-specific inhibitor, resulting in a 57% decrease in the phosphorylation of PS1 at Ser367. n = 6. (F and G) Mouse fibroblasts transfected with CK1γ2 showed a 49% increase in the phosphorylation of PS1 at Ser367. n = 3. (H and I) N2A cells were treated for 12 h with 5 μM HPB, resulting in an increase in intracellular (H) and released (I) Aβ. n = 4. (J and K) Overexpression of CK1γ2 induces a 33% decrease in Aβ40 in N2A695 cells. n = 3. Data represent means ± SEM [*P < 0.05, **P < 0.01, ***P < 0.001 compared with control (Ctrl), t test].

Fig. 2.

Phosphorylation of PS1 at Ser367 reduces Aβ levels and plaque burden in brains of Alzheimer’s disease model mice. (A–D) J20-S367A mice were generated by crossing the J20 Alzheimer model mice to PS1-S367A knock-in (KI) mice. The levels of soluble Aβ40 (A), soluble Aβ42 (B), insoluble Aβ40 (C), and insoluble Aβ42 (D), in 9-mo-old mouse brains were analyzed by ELISA. KI/WT refers to mice with the heterozygous mutation, and KI/KI refers to mice with the homozygous mutation. Data represent means ± SEM (J20: n = 6; J20-S367AKI/WT: n = 6; J20-S367AKI/KI: n = 6). (E) Amyloid plaques in sections from 9-mo-old J20, J20-S367AKI/WT, and J20-S367AKI/KI mouse brains. (F) Quantification of plaque burden. Amyloid plaques were revealed by 6E10 immunostaining. Data represent means ± SEM (J20: n = 5; J20-S367AKI/WT: n = 7; J20-S367AKI/KI: n = 6; *P < 0.05, ***P < 0.001, ANOVA test).

Fig. 3.

Phosphorylation of PS1 at Ser367 reduces Aβ levels in brains expressing endogenous levels of APP. Endogenous Aβ40 (A) and Aβ42 (B) are increased in the brains of knock-in PS1-S367A mice compared with wild-type mice. n = 7; ***P < 0.001, ANOVA test.

Fig. S1.

Mutation of PS1 at Ser367 to aspartic acid does not mimic phosphorylation. (A–D) J20-S367A and J20-S367D mice were generated by crossing the J20 Alzheimer model mice to S367A-PS1 or S367D-PS1 knock-in mice. The levels of insoluble Aβ42 (A), insoluble Aβ40 (B), soluble Aβ42 (C), and soluble Aβ40 (D) in 9-mo-old mouse brains were analyzed by ELISA. Data represent means ± SEM (J20: n = 6; J20-S367A: n = 6; J20-S367D: n = 7; ***P < 0.001 ANOVA test). (E–G) Amyloid plaques in sections from J20 (E); J20-S367A (F); J20-S367D (G) mouse brains. (H) Quantification of plaque burden. Amyloid plaques were revealed by 6E10 immunostaining. Data represent means ± SEM (J20: n = 5; J20-S367A: n = 7; J20-S367D: n = 6; ***P < 0.001, ANOVA test).

Fig. 4.

βCTF degradation is increased by the phosphorylation of PS1 at Ser367. (A) In vitro γ-secretase activity assay. Indicated concentrations of βCTF-Flag were added to CHAPSO-solubilized brain membranes derived from WT or S367A knock-in mice. After 90 min, aliquots were assayed for Aβ40 by ELISA. Aβ40 levels are expressed as arbitrary units. n = 3. (B) Solubilized brain membranes used in A are phosphorylated at PS1-Ser367. (C) Analysis of active γ-secretase levels. Membranes isolated from brains of WT or S367A mutant mice were incubated with JC-8 (a photoactivatable γ-secretase probe), photolysed, pulled down with streptavidin beads, and subject to immunoblot analysis using PS1-NTF antibody. Addition of l-685,458, a γ-secretase inhibitor, blocks the binding of JC-8 to γ-secretase, indicating binding specificity. n = 3. (D–H) Levels of βCTF were increased in the brains of J20-S367A knock-in mice compared with J20 mice (D and E), but no changes were observed in the levels of αCTF (D and F), sAPPβ (D and G), or full-length APP (D and H). (I and J) Fibroblasts derived from WT or S367A knock-in mice were transfected with APPswe and subject to pulse–chase analysis for the indicated time points. n = 3. Data represent means ± SEM (**P < 0.01, ***P < 0.001; t test for E and ANOVA test for J).

Fig. S2.

Characterization of the specificity of pS367-PS1 antibody. (A) GST-PS1 third intracellular loop was incubated with or without CK1γ2, and phosphorylation was analyzed by Western blots using pS367-PS1 antibody. (B) The specificity of the phospho-antibody was characterized by dot blot with a panel of peptides: 1, ESRAAVQELSSS₃₆₇ILAGEDP; 2, ESRAAVQELSS(pS₃₆₇)ILAGEDP; 3, ESRAAVQELS(pS₃₆₆)S₃₆₇ILAGEDP; 4, ESRAAVQEL(pS₃₆₅)SS₃₆₇ILAGEDP; 5, ESRAAVQEL(pS₃₆₅)S(pS₃₆₇)ILAGEDP. Five hundred nanograms of the indicated peptide was spotted on a nitrocellulose membrane and incubated with pS367-PS1 antibody.

Fig. S3.

Levels of N-terminal fragment of PS1 in wild-type and PS1-S367A levels measured by Western blot.

Fig. 5.

Autophagy is regulated by the phosphorylation of PS1 at Ser367. (A) MEFs derived from WT or PS1-S367A were transfected with APPswe. In Upper, autophagy was inhibited by incubating with 10 nM Bafilomycin a1 for 3 h. In Lower, the proteasome was inhibited by incubating with 10 μM MG132 for 8 h. Cell lysates were immunoblotted with the 6E10 antibody to detect βCTF. (B–D) LC3-II and SQSMT1/p62 levels were elevated in the brains of S367A knock-in mice compared with WT mice as measured by analysis of immunoblots. In B, results from three different WT and S367A mutant mice are shown. (E) Immunoblot against LC3 and p62 from lysates prepared from WT or PS1-S367A cultured neurons. (F) Immunofluorescence against LC3 (green) and DAPI (blue) in WT and PS1-S367A neurons in culture. (G) Tandem GFP-RFP-LC3 in MEFs derived from WT or PS1-S367A mice. (H) Pearson’s correlation coefficient was used as a measure of colocalization of RFP and GFP signals. n = 20. Data represent means ± SEM (**P < 0.01, ***P < 0.001).