Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: ASM is increased in AD and complete ASM gene deficiency exacerbates pathology of APP/PS1 mice. (A and B) ASM was estimated in the blood plasma (A; control, n = 30; AD, n = 40; and PD, n = 20) and fibroblast (B; control, n = 24; PS1-FAD, n = 24; ApoE4, n = 24; and PD, n = 12) with AD, PD, or normal controls. (C) ASM activity did not show passage differences between AD and normal fibroblasts ( n = 8 per passage group). (D) Detection of sphingomyelin, ceramide, and AC in plasma (control, n = 20–22; and AD, n = 33–35) and fibroblast (control, n = 12; PS1-FAD, n = 18; and ApoE4, n = 18). (E) Crossing scheme to generate WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice. PCR-based genotyping to detect WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice. (F) Survival curves of WT ( n = 26), APP/PS1 ( n = 30), ASM −/− ( n = 30), and APP/PS1/ ASM −/− ( n = 25) mice. (G) Body weights of WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice were determined at the indicated ages ( n = 6–7 per group). (H–J) Brain sections from 7-mo-old mice were immunostained with anti–active caspase3 (H; n = 4 per group; bars, 50 µm), anti-GFAP (I; n = 4 per group; bars, 100 µm), and anti–Iba-1 (J; n = 4 per group; bars, 100 µm). Data are representative of three independent experiments. A–D and G, Student’s t test. H–J, one-way ANOVA, Tukey’s post hoc test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.

Article Snippet: Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.

Techniques: Clinical Proteomics, Control, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: Partial genetic inhibition of ASM leads to decreased AD pathology in the APP/PS1 mice. (A) Generation of the APP/PS1/ ASM +/− mice. (B) Body weights of WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice were determined at 9 mo of age ( n = 14 per group). (C) ASM activity in blood plasma ( n = 14–15 per group), brain ( n = 13–14 per group), and fibroblast ( n = 8 per group) derived from WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice. (D) ASM activity was assessed in neuron and microglia isolated from mouse brain (WT, n = 8; APP/PS1, n = 6; and APP/PS1/ ASM +/− , n = 6). (E) Detection of sphingomyelin, ceramide, and AC in plasma ( n = 8–10 per group), brain ( n = 7–9 per group), and tail ( n = 5–6 per group) fibroblast. (F) Mice brain sections were stained with thioflavin S in APP/PS1 and APP/PS1/ ASM +/− mice. The relative area occupied by Aβ plaques were determined ( n = 6–7 per group; bars, 100 µm). (G–I) Analysis of Aβ40 and Aβ42 depositions from the mice brain samples using immunofluorescence staining (G and H; n = 6–7 per group; bars, 200 µm) and ELISA kits (I; n = 8 per group). (J and K) Confocal laser microscope images and quantification of cerebral amyloid angiopathy (J; n = 6 per group; bars, 50 µm) and tau hyperphosphorylation (K; n = 6 per group; bars, 20 µm) in APP/PS1 and APP/PS1/ ASM +/− mice. Data are representative of two (D and K), three (B, C, and E), or four (F–J) independent experiments. B–E, one-way ANOVA, Tukey’s post hoc test. F–K, Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005. All error bars indicate SEM.

Article Snippet: Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.

Techniques: Inhibition, Activity Assay, Clinical Proteomics, Derivative Assay, Isolation, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: Partial genetic inhibition of ASM reverses defective autophagy in APP/PS1 mice. (A) Western blot analysis of LC-3 and beclin-1 levels in controls, PS1-FAD, and ApoE4 fibroblasts. (B) LC3-II and beclin-1 levels were quantified ( n = 4 per group). (C) Immunocytochemistry for LC3 in controls, PS1-FAD, and ApoE4 fibroblast ( n = 4–5 per group; bars, 20 µm). (D) Degradation of long-lived proteins was measured in controls, PS1-FAD, and ApoE4 fibroblasts ( n = 6 per group). (E) Representative images and quantification of LBPA in control, PS1-FAD, and ApoE4 fibroblast ( n = 4 per group; bars, 50 µm). (F) Western blot analyses for LC3, beclin-1, p62, and cathepsin D in tail fibroblast derived from WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice. (G) Densitometric analysis of LC-3-II, beclin-1, p62, and cathepsin D ( n = 7–8 per group). (H) Cathepsin D activity in mice tail fibroblast ( n = 4 per group). (I) Rates of proteolysis of long-lived proteins in fibroblasts ( n = 6 per group). (J) Representative images and quantification data of SA-β-gal staining in the mice tail fibroblasts ( n = 5 per group; bars, 50 µm). (K) Western blot analyses for LC3, beclin-1, p62, and cathepsin D in the brains of 9-mo-old WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice. (L) Densitometric quantification of LC-3-II, beclin-1, p62, and cathepsin D ( n = 6–8 per group). (M) Cathepsin D activity in brain extracts of WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice ( n = 4 per group). (N) EM images and quantification data of cortical region. Higher magnification of boxed area shows detail of AVs (arrow; n = 5 per group; bars: [low magnification] 2 µm, [high magnification] 1 µm). (O) Western blot analysis of Rab5 and Rab7 levels in the brain lysates ( n = 5 per group). Data are representative of two (A–E and N) or three (F–M and O) independent experiments. B–O, one-way ANOVA, Tukey’s post hoc test. *, P < 0.05. All error bars indicate SEM.

Article Snippet: Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.

Techniques: Inhibition, Western Blot, Immunocytochemistry, Control, Derivative Assay, Activity Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: Autophagic processes are affected by lysosomal ASM. (A and C) Western blot analysis of LC3, Beclin-1, and p62 in human fibroblast (A; n = 7 per group) and human neuron (C; n = 6 per group). (B) Immunocytochemistry and quantification for LC3 after ASM treatment ( n = 7–8 per group; bars, 20 µm). (D) ASM activity was assessed in the ASM-treated fibroblast with or without M6P ( n = 6 per group). (E) Confocal microscopic analysis of Lamp1- and ASM-positive vesicles (bars, 20 µm). (F and G) The effect of lysosomal ASM on LC3-II expression. (F) 10 µM ASM was added to fibroblast for 24 h with or without 10 mM M6P. LC3-II expression was determined by Western blot analysis ( n = 5–6 per group). (G) LC3-II levels were examined in 10 µM ASM-treated fibroblast with or without M6P receptor suppression using siRNA ( n = 5–6 per group). (H) The effect of 10 µM ASM on cell viability was estimated by MTT assay ( n = 5–6 per group). (I and J) Representative images and quantification data of LC3 (I; bars, 20 µm) and SA-β-gal staining (J; bars, 100 µm) in P5, P10, and P20 human fibroblasts. NH 4 Cl and H 2 O 2 were used for positive control ( n = 5 per group). Data are representative of three (B, E, I, and J) or four (A, C, D, and F–H) independent experiments. A, C, F, G, I, and J, one-way ANOVA, Tukey’s post hoc test. B, D, and H, Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005. All error bars indicate SEM.

Article Snippet: Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.

Techniques: Western Blot, Immunocytochemistry, Activity Assay, Expressing, MTT Assay, Staining, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: ASM causes abnormal autophagic protein degradation by altering ALP. (A) Autophagic flux assay. Human fibroblasts were cultured in: (1) complete medium with or without 10 µM ASM in the presence or absence of NH 4 Cl (left), (2) complete medium or starvation condition in the presence or absence of NH 4 Cl (middle), or (3) complete medium or starvation condition with or without 10 µM ASM (right). The LC3-II levels were examined by Western blotting ( n = 6–7 per group). (B) The accumulation of p62 was assessed in the human fibroblast cultured with 10 µM ASM, 20 mM NH 4 Cl, or starvation condition ( n = 4 per group). (C) Western blot analysis of LC3-II levels in controls, PS1-FAD, and ApoE4 fibroblasts in the presence or absence of NH 4 Cl ( n = 6 per group). (D) Western blot analysis for LC3-II levels in fibroblasts derived from WT, APP/PS1, and APP/PS1/ ASM +/− mice in the presence or absence of NH 4 Cl ( n = 6 per group). (E) Effect of ASM on lysosomal pH. FACS and histological analysis of fibroblasts stained with LysoTracker red ( n = 5 per group; bars, 20 µm). H 2 O 2 - and NH 4 Cl-treated cells were used as positive and negative controls, respectively. (F and G) Western blot analyses for TFEB and Lamp1 in human fibroblasts (F; n = 6 per group) and neurons (G; n = 6 per group) after treatment with ASM. (H) Immunocytochemistry of Lamp1 in control and ASM-treated fibroblast ( n = 5 per group; bars, 20 µm). (I) Western blot analysis for nuclear localization of TFEB in ASM-treated cells ( n = 5 per group). (J) Quantitative real-time PCR analysis of TFEB-target gene expression in normal ( n = 6) and ASM-treated ( n = 10) fibroblasts. (K) ASM activity was estimated in the fibroblast with or without NH4Cl ( n = 5 per group). Data are representative of two (E, H, and I) or three (A–D, F, G, J, and K) independent experiments. A, B, and E–G, one-way ANOVA, Tukey’s post hoc test. C, D, and H–K, Student’s t test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.

Article Snippet: Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.

Techniques: Flux Assay, Cell Culture, Western Blot, Derivative Assay, Staining, Immunocytochemistry, Control, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: Restoration of ASM to the normal level reverses impaired autophagy in the AD patient-specific cells. (A) SMPD1 gene suppression by ASM-siRNA in human fibroblasts. ASM activity was assessed after ASM siRNA treatment in the control and AD fibroblast ( n = 6 per group). (B) LC3-II and p62 levels were examined in human AD fibroblast with or without ASM inhibition. siRNA-mediated suppression of ASM reduced LC3-II and p62 levels in PS1-FAD (left; n = 7 per group) and ApoE4 fibroblast (right; n = 6 per group). (C) Protein expression of TFEB and Lamp1 in the PS1-FAD and ApoE4 fibroblast after ASM inhibition ( n = 5–6 per group). (D–G) Generation of PS1 iPSC lines from patient fibroblast. (D) Established iPSCs showed embryonic stem cell–like morphology (Phase; bar, 1 mm), AP activity (bar, 200 µm), and expressed pluripotent stem cell markers SSEA4 (bar 100 µm), TRA1-60 (bar 100 µm), and TRA1-81 (bar 100 µm). (E) Normal karyotype of PS1 iPSC. (F) Quantitative real-time PCR analysis of hESC marker gene of PS1 iPSC ( n = 3 per group). (G) Gross morphology and hematoxylin-eosin staining of representative teratomas generated from PS1-4 iPSCs (bars, 50 µm). (H) Estimation of neural differentiation from control and PS1-4 iPSCs. Representative images of immunocytochemical staining the β-III tubulin after neural differentiation (bars, 50 µm). (I) The amount of Aβ42 secreted from control iPSC-derived neuron and PS1 iPSC-derived neuron ( n = 5 per group). (J) Characterization of ASM activity in the control and PS1 iPSC and iPSC-derived neurons ( n = 6 per group). (K) Western blot analyses for LC3, beclin-1, p62, TFEB, and Lamp1 in the control and PS1-4 iPSC–derived neuron after ASM siRNA treatment ( n = 5–6 per group). (L) EM images and quantification data of control and PS1 iPSC-derived neurons. Higher magnification of boxed area shows detail of AVs (arrow; n = 4 per group; bars: [low magnification] 1 µm, [high magnification] 500 nm). (M) Quantitative real-time PCR analysis of TFEB-target gene expression in iPSC-derived neurons after ASM siRNA treatment ( n = 5–6 per group). Data are representative of two (A, D–G, I, and L), or three (B, C, H, J, K, and M) independent experiments. A, C, F, I, J, and M, Student’s t test. B, K, and L, one-way ANOVA, Tukey’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. A–K, error bars indicate SEM. L and M, Error bars indicate SD.

Article Snippet: Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.

Techniques: Activity Assay, Control, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Marker, Staining, Generated, Derivative Assay, Western Blot, Targeted Gene Expression