APP intracellular domain-WAVE1 pathway reduces amyloid-β production

Abstract

An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aβ production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aβ. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aβ levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Aβ production.

Figure 1

Downregulation of WAVE1 expression by overexpression of APP or AICD. (a) Immunohistochemistry of WAVE1 and APPswe in 12 month-old WT and 3xTg male mice. High magnification images (bottom) of the rectangular regions (yellow) in top images. CA1 (red), and CA2, CA3 and dentate gyrus (green). Scale bars, 500 μm. (b, c) Immunoblotting of WAVE1 and actin in the hippocampus from 12 month-old WT (n = 4) and 3xTg (n = 8) (b) or 8 month-old WT (n = 10) and Tg/APPswe (n = 10) (c) male mice. The quantified protein level of WAVE1 was normalized to the level of actin. (d) N2a cells were transiently transfected as indicated. Representative immunoblotting images (left), and quantification (right, n = 5). (e) WAVE1 protein (left, n = 6) and mRNA (right, n = 6) levels in normal N2a and N2a/APPwt cells. (f) Effect of the β-secretase (BACE1-IV) or γ-secretase (DAPT) inhibitors on WAVE1 protein level in N2a/APPwt cells (Cont and BACE1-IV, n = 6; DAPT, n = 8). (g) WAVE1 protein (left, n = 4) and mRNA (right, n = 6) levels in N2a cells transiently transfected with AICD. (h) ChIP analysis of N2a cells transiently transfected with 3xFlag-tagged AICD. Immunoprecipitation (IP) was performed with preimmune (Cont) IgG, anti-RNA polymerase antibody (anti-RNA pol) as a positive control, or anti-Flag antibody. A fragment of the Wasf1 gene promoter in the immune complex was amplified by PCR and quantified (n = 9). (i) N2a cells were transiently co-transfected as indicated. Luciferase activity was measured (n = 6). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, two-tailed t-test.

Figure 2

Lowering WAVE1 expression reduces Aβ. (a–g) N2a/APPswe.PS1ΔE9 cells were transfected with control siRNA plus control plasmid (Control), Wasf1 siRNA plus control plasmid (Wasf1 siRNA), or Wasf1 siRNA plus siRNA-resistant plasmid for WAVE1 (WAVE1 add-back). WAVE1 (a, n = 9), Aβ40 (b, n = 6), Aβ42 (c, n = 6), total APP (f, n = 4) and actin were measured in cell lysates. Surface APP was measured by a biotinylation assay (d, n = 6). Soluble ectodomain of APP (sAPPβ) produced by β-secretase (e, n = 4), and sAPPα, a product of α-secretase (g, n = 4), were measured in the medium. Data represent mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed t-test. (h, i) The levels of Aβ40 (h) and Aβ42 (i) were measured in mouse brains of 4–5 month-old 2xTg AD male mice (tg/+) harboring Wasf1^+/+ (n = 13), Wasf1^+/– (n = 13) or Wasf1^–/– (n = 6). Data represent means ± SEM. *P < 0.05 and **P < 0.01, one-way analysis of variance (ANOVA), Dunnett's post hoc test.

Figure 3

WAVE1 facilitates budding of APP-containing vesicles from the Golgi apparatus. (a–c) Immunocytochemistry of WAVE1 and APP in N2a/APPswe.PS1ΔE9 cells (a, low magnification; c, high magnification; a and c are different cells) and in N2a/APPwt cells (b). Cells were infected with a viral vector expressing a fluorescent protein fused-Golgi targeting sequence (GFP-Golgi). DAPI counterstaining was used to show the nucleus. Line scan (white line in merge 2 of c) shows coinciding fluorescence signal (cyan arrows) for WAVE1, APP and GFP-Golgi (right panel of c). Scale bars, 10 μm (a, b) and 2 μm (c). Arrows (a, b) and arrow heads (c) indicate the co-localization of WAVE1 and APP in the Golgi apparatus. (d) A detergent soluble membrane fraction from N2a/APPwt cells was used for immunoprecipitation with preimmune IgG or anti-WAVE1 antibody. WAVE1 co-precipitated with APP. (e, f) Golgi membrane (from N2a/APPwt cells) and cytosol (from N2a cells) were prepared from cells transfected with control siRNA or Wasf1 siRNA for in vitro budding assay. Reconstituted Golgi membrane and cytosol were incubated at 37 °C for the indicated times, and released vesicles were separated from Golgi membrane by centrifugation. Representative images showing time course of APP-containing vesicle formation (e) and time-dependent fold increase of APP-containing vesicle formation from six independent experiments (f, n = 6). Data represent mean ± SEM. ****P < 0.0001, Control versus Wasf1 siRNA, two-way ANOVA.

Figure 4

Behavioral consequence and clinical relevance of WAVE1 downregulation, and a model for negative feedback regulation of Aβ production. (a–c) WT (non-Tg) (Wasf1^+/+, n = 17; Wasf1^+/–, n = 15) and 2xTg (tg/+) (Wasf1^+/+, n = 15; Wasf1^+/–, n = 10) AD mice (7 month-old, males) were analyzed in the Morris water maze. The number of platform crossings (a) and time spent swimming in the target quadrant (b) were measured (mean ± SEM). ***P < 0.001, *P < 0.05, two-tailed t-test. Representative swim paths recorded during the probe trial (c). (d–g) WASF1 exon expression profiles (d, e) and the mean value of the exon intensities (f, g) from the amygdala (d, f) or the parietal-occipital neocortex (e, g) in 19 AD cases and 10 controls. Each red triangle or blue square represents least squares mean expression of an exon in AD or control tissues, respectively. The upper part in d shows the structure of the four WASF1 isoforms retrieved from the UCSC browser. Mean ± SEM. ****P < 0.00001, one-way ANOVA. (h) Differences in WASF1 transcript expression between 173 AD cases and 187 controls in an independent publicly available dataset. Mean ± SEM. ***P < 0.001, one-way ANOVA. (i)Proposed model of negative feedback circuit. The processing of APP in the amyloidogenic pathway increases Aβ and AICD. AICD negatively regulates WASF1 gene promoter activity. Downregulation of WAVE1 inhibits the trafficking of APP from the Golgi apparatus to the cell surface and endosomal membranes, resulting in reduced production of Aβ. Colored backgrounds indicate different subcellular compartments.