Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99)

Abstract

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer's disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down ayndrome (trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP) and is specifically mediated by the β cleaved carboxy terminal fragment of APP (APP-βCTF, C99). In primary fibroblasts from individuals with DS, lysosomal degradation of autophagic and endocytic substrates is selectively impaired, causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) as a basis for slowed LC3 turnover and the inactivation of cathepsin D and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, whereas RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one Bace1 allele from adult Ts2 mice had similar rescue effects in vivo The modest elevation of endogenous APP-βCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-βCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD.SIGNIFICANCE STATEMENT Down syndrome (trisomy 21) (DS) is a neurodevelopmental disorder invariably leading to early-onset Alzheimer's disease (AD). We showed in cells from DS individuals and neurons of DS models that one extra copy of a normal amyloid precursor protein (APP) gene impairs lysosomal acidification, thereby depressing lysosomal hydrolytic activities and turnover of autophagic and endocytic substrates, processes vital to neuronal survival. These deficits, which were reversible by correcting lysosomal pH, are mediated by elevated levels of endogenous β-cleaved carboxy-terminal fragment of APP (APP-βCTF). Notably, similar endosomal-lysosomal pathobiology emerges early in sporadic AD, where neuronal APP-βCTF is also elevated, underscoring its importance as a therapeutic target and underscoring the functional and pathogenic interrelationships between the endosomal-lysosomal pathway and genes causing AD.

Keywords: AD; APP-βCTF; Down syndrome; cathepsin D; lysosomal pH; lysosome.

Figure 1.

Decreased lysosomal turnover of autophagic protein substrates in DS fibroblasts. A–D, Fibroblasts from 3 different DS patients and age-matched control were labeled for 2 d with [³H] leucine. After extensive washing, cells were plated in complete medium (A, B) or in serum-depleted media (C, D); and where indicated, (B, D) 20 mm NH₄Cl/100 μm leupeptin (NL) was added into the incubation medium. The rate of total protein degradation at the indicated times was calculated as the percentage of total radiolabeled protein transformed in soluble amino acids and small peptides at each time. Values are the mean ± SEM (n = 10 replicates). E, Western blotting of autophagy-related proteins in 2N and DS fibroblasts treated with vehicle or AZD (0.5 μm for 2 h) and with or without leupeptin and pepstatin A (LP, 10 μm for 24 h), with actin as an internal control, and (F) quantification of LC3-II protein levels normalized with actin in 2N and DS fibroblasts (n = 6 replicates). G, Representative confocal microscopic images and (H) the size quantification of lysosome identified by dextran-AlexaFluor-546 uptake (4 h pulse and 20 h chase) in 2N and DS fibroblasts with or without AZD treatment shown as scatter plot (n ≥ 52 lysosomes). Red lines indicate mean values. I, Quantifications of size and number of endosomes and lysosomes in 2N and DS fibroblasts (n ≥ 30 cells). J, Representative EM images of HRP uptake in 2N and DS fibroblasts and (K) the quantification of HRP-positive lysosomes at 1 and 24 h uptake time point (n ≥ 9 field). Arrows indicate HRP-positive vesicles. + sign indicates mean in Bar and Whiskers plots. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005. Scale bar, 5 μm.

Figure 2.

Lysosomal acidification deficiency in DS fibroblasts. A, Representative confocal microscopic images of 2N and DS fibroblasts transfected with mRFP-eGFP-LC3 for 48 h and dextran-AlexaFluor-647 uptake as lysosome marker and (B) less acidic ALs as a percentage of total ALs (n ≥ 31 cells). Arrows indicate a less acidic AL. + sign indicates mean in Bar and Whiskers plot. ***p ≤ 0.005. Scale bar, 5 μm.

Figure 3.

Altered lysosomal enzymatic activity in DS fibroblasts. A, Representative confocal microscopic images and (B) the quantification of BODIPY FL-Pepstatin A (BP) labeling in 2N and DS fibroblasts (n ≥ 42 cells). C, Representative Western blotting of CTSD (n = 9 replicates) and CTSB (n = 4 replicates) from total extracts of 2N and DS fibroblasts, and their quantification (D). E, Representative images of BODIPY FL-Pepstatin A staining and (F) the quantification of fluorescence intensity in 2N and DS fibroblasts treated with nanoparticles for 24 h (n ≥ 74 cells). G, Specific activity of CTSD, ACP2, HEXA, and CTSB measured by their corresponding in vitro activity assay (n ≥ 6 replicates). H, Representative confocal microscopic images and (I) the quantification of Magic Red CatB (MRB) labeling in 2N and DS fibroblasts (n ≥ 131 cells). J, mRNA expression level of APP, and lysosomal enzymes from RNA-Seq analysis of 2N and DS fibroblasts (n ≥ 10 replicates). + sign indicates mean in Bar and Whiskers plots. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005. Scale bar, 5 μm.

Figure 4.

Regulation of CTSD activity in 2N and DS fibroblasts by downregulation of APP. A, Representative Western blot of 2N and DS fibroblast after treatment with siRNA for either negative control (siNC) or for APP (siAPP) for 72 h (n = 3 replicates). B, Lysosome pH of 2N and DS fibroblasts treated with siNC or siAPP for 72 h (n ≥ 9 replicates) determined by LysoSensor Yellow/Blue dextran. C, Quantitation of BODIPY FL-Pepstatin A fluorescence intensity in 2N and DS fibroblasts treated with siNC or siAPP (n ≥ 200 cells). D, Lysosomal CTSD specific activity of 2N and DS cells treated with siNC or siAPP for 72 h. E, Quantitation of CTSD protein levels after siNC and siAPP treatment, and GAPDH as internal control. F, mRNA level of APP, and lysosomal enzymes and (G) TFE3 and TFEB in 2N and DS fibroblasts treated with siNC or siAPP for 72 h (n = 6 replicates). + sign indicates mean in Bar and Whiskers plots. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.

Figure 5.

Accumulation of βCTF in lysosome of DS fibroblast and βCTF dependence of lysosomal CTSD activity in 2N and DS fibroblasts. A, Representative images of colabeling of CTSD (red) and APP-βCTF (green) antibodies in 2N and DS fibroblasts treated with either vehicle (DMSO) or γ-secretase inhibitor, L-685,458 (10 μm for 24 h), and (B) the Pearson correlation coefficient between the CTSD and βCTF (n ≥ 15 cells). C, Representative Western blot of APP-βCTF in total lysate and lysosome after lysosomal isolation with dextran-conjugated magnetite beads (n = 3 replicates), and (D) its quantitation in total cellular (load 15 μg protein/lane) and lysosomal fractions (load 5 μg protein/lane). E, Lysosome pH measured by LysoSensor Yellow/Blue dextran in 2N and DS fibroblast after 24 h L685,458 or BACE inhibitor IV treatment (n ≥ 6 replicates). F, Representative images of BODIPY FL-Pepstatin A staining and (G) its quantitative graph in 2N and DS fibroblasts treated with DMSO or L685,458 (n ≥ 150 cells). H, Representative images of BODIPY FL-Pepstatin A staining, with arrow indicating cell with positive mCherry-C99 transfection, and (I) BP quantitative graph in 2N fibroblasts either transfected with or without mCherry-C99 (n ≥ 62 cells). J, Representative confocal colocalization image of mCherry-C99 and dextran-AlexaFluor-488 uptake as lysosomal marker. + sign indicates mean in Bar and Whiskers plots. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005. Scale bar, 5 μm.

Figure 6.

Endosomal and lysosomal defects in Ts2 mouse cortical neuronal culture. A, Representative confocal images of EEA1 staining of 2N and Ts2 neurons, and (B) quantification of EEA1-positive (n ≥ 58 neurons) and (C) Rab7-positive endosomes (n ≥ 59 neurons). D, Lysosome pH of 2N and Ts2 neurons measured by LysoSensor Yellow/Blue dextran (n = 8 replicates). Representative images of (E) BODIPY FL-Pepstatin A and (F) Magic Red CatB (MRB) labeling and (G) their intensity quantification in 2N and Ts2 neurons (n ≥ 33 neurons). H, Scatter plot with mean designated by bars showing specific activity of CTSD and CTSB measured by in vitro enzymatic activity assay (n = 3 replicates). I, Representative Western blot and (J) scatter plot with mean designated by bars showing quantitation of APP, βCTF, CTSD, and CTSB in 2N and Ts2 neurons, GAPDH as internal control (n ≥ 4 replicates). + sign indicates mean in Bar and Whiskers plots. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005. Scale bar, 5 μm.

Figure 7.

Restoration of endosomal and lysosomal defects in Ts2 mice. A, Representative Western blot (n = 4 replicates) and (B) representative confocal image of APP labeling and (C) quantitation of APP in 2N and Ts2 neurons after treatment with siRNA for either negative controls (siNC) or for mouse-specific APP (siAPP) for 72 h (n = 16 neurons). D, Representative confocal images of EEA1 staining and (E) quantitation of EEA1-positive endosomes of 2N and Ts2 neurons 72 h after siRNA transfection (n ≥ 30 neurons). F, Representative images of BODIPY FL-Pepstatin A and (G) quantitation of 2N and Ts2 neurons 72 h after siRNA transfection (n = 36 neurons). H, Specific in vitro activity of CTSD in 2N, Ts2, and Ts2BACE1^+/− mouse brain homogenate (n ≥ 3 mice). + sign indicates mean in Bar and Whiskers plots. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005. Scale bar, 5 μm.