Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Bulk-RNA and single-nucleus RNA (sn-RNA) sequencing analyses reveales an association between ABCA1 and cellular senescence in APOE4 and AD. ( A ) Bulk-RNA sequencing analysis showing cellular senescence ssGSEA scores across no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD. ( B ) Bulk-RNA sequencing analysis of cellular senescence ssGSEA scores by Braak stage groups (0–3; 4; 5–6). ( C ) Bulk-RNA sequencing analysis of cellular senescence ssGSEA scores in the groups with and without neuritic plaques. ( D ) Bulk-RNA sequencing analysis of cellular senescence ssGSEA scores across different APOE genotypes and clinical groups. ( E ) Bulk-RNA sequencing analysis of ABCA1 expression levels across clinical groups. ( F ) Association between ABCA1 expression and cellular senescence ssGSEA scores in the bulk-RNA sequencing, analyzed using a linear model. ( G ) Single-nucleus RNA sequencing analysis of the association of cellular senescence ssGSEA scores in different brain cells with factors, including AD pathology, sex, APOE genotype, LSX/RXR co-expression genes and other AD risk genes. The heatmap shows t value (coefficient 𝛃 divided by standard error of 𝛃), with red indicating significant positive associations, blue indicates significant negative associations, gray indicating non-significant associations, and cutoff p = 0.1). The gray bar plot represents ABCA1 expression levels in different cell types. ( H ) total cholesterol, ( I ) 7-hydroxycholesterol levels of the postmortem human cortex tissues from participants with or without AD and carrying different APOE genotypes. ( J ) Correlation between 7-hydroxycholesterol levels and cellular senescence ssGSEA scores. Data were analyzed using an unpaired t-test ( C ) or one-way ANOVA ( A , B , D , E ) or a linear model ( F ). ** p < 0.01, *** p < 0.01

Article Snippet: An ABCA1 knockdown astrocyte cell line was created by transfection with ABCA1 CRISPR Gene Knockout Kit (ABCA1-sgRNAs and Cas9 recombinant protein, Synthego).

Techniques: RNA Sequencing, Expressing

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Lysosomal ABCA1 and STG are increased in AD compared to NCI in APOE3/4 carriers. All postmortem human brain tissues and slides were obtained from mid-frontal lobe tissue sections from the ROS. ( A ) Representative images showing DAPI, GFAP and STG, staining in human brain slides from AD and non-AD patients carrying APOE3/4 genotype. Arrows indicate STG signals quantified from GFAP-positive astrocytes. ( n ≥ 126 astrocytes from 5 individuals in NCI (blue) and 6 individuals in AD (red)). ( B ) ABCA1 protein levels in total membrane extracts from human brain tissue, obtained using TBSX buffer, were detected by western blotting (WB). Quantification of total membrane ABCA1 protein levels normalized to Na,K-ATPase (a plasma membrane marker) (NCI APOE3/3, n = 33; NCI APOE3/4, n = 19; AD APOE3/3, n = 44; AD APOE3/4, n = 42). ( C ) WB blot analysis of ABCA1 and LAMP2, with LAMP2 immunoprecipitated (IP) from homogenates of human brain tissues (ABCA1 IP / LAMP2; n = 10 per group). ( D ) WB analysis of phosphorylated mTOR and ABCA1 protein levels in human brain tissue homogenates, with association between phosphorylated mTOR and ABCA1 protein levels analyzed using simple linear regression ( n = 40). Data are represented as mean ± SD and analyzed using two-tailed t-test ( A , C ), one-way ANOVA followed by Tukey’s test ( B ). * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: An ABCA1 knockdown astrocyte cell line was created by transfection with ABCA1 CRISPR Gene Knockout Kit (ABCA1-sgRNAs and Cas9 recombinant protein, Synthego).

Techniques: Staining, Membrane, Western Blot, Clinical Proteomics, Marker, Immunoprecipitation, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Caveolin-1 regulates ABCA1 trafficking in cells and its expression is increased in APOE4 and AD biospecimens. ( A ) Schematic overview of the workflow for purification of the ABCA1 complex. ( B ) Co-immunoprecipitation of ABCA1 in ABCA1-GFP expressing and wild-type HeLa cells. ( C ) Co-immunoprecipitation in lysates of immortalized astrocytes using an ABCA1 antibody or species-matched IgG. ABCA1, Caveolin-1 and AP2B1 were detected by immunoprecipitation. ( D ) Co-staining for ABCA1 and caveolin-1 in immortalized astrocytes. Arrow indicates the cytoplasm, and arrowhead indicates the plasma membrane. ( E ) Co-staining for ABCA1 and AP2B1 in immortalized astrocytes. ( F ) Mouse primary astrocytes were transfected with non-target (NT), caveolin-1, and AP2B1 siRNA for 48 h. Plasma membrane protein was enriched by biotin agarose beads and the ABCA1 protein levels were detected by WB. Quantification was performed from two–three independent experiments. ( G ) Immortalized astrocytes were transfected with non-target or caveolin-1 siRNA for 24 h, followed by labeling with 3 H-cholesterol for 18 h. Cholesterol efflux was measured after treatment with recombinant ApoA1 or ApoE for 4 h ( n = 3 biological replicates). ( H ) Filipin staining of immortalized astrocytes loaded with or without low-density lipoprotein (LDL) (10 µg/mL) for 24 h. ( n = 7–12 random areas from two cultured wells). ( I ) Caveolin-1 protein levels in immortalized astrocytes loaded with or without LDL (10 µg/mL) for 24 h were detected by WB ( n = 3 biological replicates). ( J ) Protein levels of caveolin-1 and ABCA1 in the cortex of 8-months-old and 18-months-old APOE3 and APOE4-TR mice ( n = 5–6 mice for each genotype, mixed gender). ( K ) For 22-months-old APOE3 and APOE4-TR mice, the cortex was sequentially homogenate with TBS, TBSX and GnHCl. ABCA1 and caveolin-1 protein levels in TBSX-soluble (membrane enriched) and GnHCl-soluble (aggregated protein enriched) fractions were measured by WB. ( n = 7 mice for each genotype, both sexes). (L) Protein levels of caveolin-1 and ABCA1 in the cortex of 6-months-old APP/PS1/APOE3 and APP/PS1/APOE4 mice were detected by WB ( n = 6 mice of each genotype, both sexes). ( M ) Total membrane caveolin-1 protein levels in human postmortem mid-frontal lobe tissues from reactive oxygen species (ROS) were detected by WB. Quantification of the relative intensity of caveolin-1 normalized to the plasma membrane marker Na, K-ATPase (NCI APOE3/3, n = 29; AD APOE3/3, n = 44; NCI APOE3/4, n = 19; AD APOE3/4, n = 42). Data are represented as mean ± SD and analyzed using two-tailed t-test ( F , G , H , I , J , K and L ) or one-way ANOVA followed by Tukey’s test ( M ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: An ABCA1 knockdown astrocyte cell line was created by transfection with ABCA1 CRISPR Gene Knockout Kit (ABCA1-sgRNAs and Cas9 recombinant protein, Synthego).

Techniques: Expressing, Purification, Immunoprecipitation, Staining, Clinical Proteomics, Membrane, Transfection, Labeling, Recombinant, Cell Culture, Marker, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: ABCA1 mediates mTORC1 activation and cellular senescence. ( A ) ABCA1 knockdown astrocytes were treated with MβCD (5 mg/mL) for 2 h, followed by incubation with control medium (Med) or LDL (100 µg/mL) for 2 h. ABCA1, total and phosphorylated mTOR, and S6K were detected by WB ( n = 3 biological replicates). ( B ) BHK cells were treated with mifepristone (0.1 nM) for 16 h to induce ABCA1 expression, followed by treatment with LDL (100 µg/mL) for 24 h. ABCA1, total and phosphorylated mTOR, and S6K were detected by WB ( n = 5 biological replicates). ( C ) The cortex of 10-month-old ABCA1 brain conditional knockout mice was homogenized in RIPA buffer. ABCA1, total and phosphorylated mTOR, and S6K were detected by WB. ( n = 6 for ABCA1 WT mice, n = 8 for ABCA1 knockout mice of both sexes). ( D ) Phosphorylated and total mTOR, and S6K in the cortex of 22-month-old APOE3 and APOE4-TR mice were detected by WB ( n = 7 for each genotype, both sexes). ( E ) Astrocytes were loaded with medium (control) or LDL (100 µg/mL) for 24 h. ABCA1 and p21were detected by WB ( n = 3 biological replicates). ( F ) Astrocytes were loaded with medium (Ctrl), 7α-OHC (7α-hydroxycholesterol) or 7β-OHC (7β-hydroxycholesterol) for 24 h. p21 was detected by WB ( n = 2 biological replicates). ( G ) Representative images of p21 staining by immunofluorescence in the ABCA1 WT and ABCA1 brain conditional knockout mouse brain slides. ( n > 300 nuclei from 6 mice in each group). ( H ) Representative images of p21 staining by immunofluorescence in the brain of APOE3 and APOE4-TR mice ( n > 90 nuclei from 5 mice in each group). ( I ) Representative images of LAMP1 and ABCA1 staining by immunofluorescence in the APOE3 and APOE4-TR mouse (14-month-old) brain slides ( n = 9–15 LAMP1 + ROIs from 3 mice in each group). ( J ) Lysosomes were isolated from brains of ApoE3 and ApoE4-TR mice (14-month-old). ABCA1 protein levels in lysosome were measured by WB. ( n = 4–5 each group, mixed gender). Data are represented as the mean ± SD and analyzed using a two-tailed t-test ( B-J ) or one-way ANOVA followed by Tukey’s test ( A ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: An ABCA1 knockdown astrocyte cell line was created by transfection with ABCA1 CRISPR Gene Knockout Kit (ABCA1-sgRNAs and Cas9 recombinant protein, Synthego).

Techniques: Activation Assay, Knockdown, Incubation, Control, Expressing, Knock-Out, Staining, Immunofluorescence, Isolation, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Reduction of oxysterols by HPCD increases ABCA1 and endo-lysosome recycling in APOE4-TR mice. ( A ) Study design and timeline for mouse experiments. APOE4-TR mice were injected with (2-Hydroxypropyl)-β-cyclodextrin (HPCD) for 2 months, followed by behavioral and pathological tests ( n = 14, 5 females and 9 males for PBS group; n = 15, 5 females and 10 males for the HPCD group). ( B ) Total cholesterol levels in mouse brain homogenates. ( C-E ) Oxysterols including 7β-hydroxycholesterol ( C ), 25-hydroxycholesterol ( D ) and 7α,25-dihydroxycholesterol ( E ) levels in mouse brain were measured by mass spectrometry. ( F ) Total membrane protein levels of ABCA1 and caveolin-1 in the mouse cortex detected by WB. ( G ) Lysosomes were isolated from mouse brains. ABCA1 protein levels in lysosomes were detected by WB. ( H ) Representative confocal images of LAMP1 and ABCA1 staining in mouse brain sections. Quantification of ABCA1 intensity in LAMP1 + area. ( n = 150 LAMP1 + ROI from 5 mice in each group; A.U.). ( I ) Protein levels of EEA1 (early endosome marker), Rab9 (late endosome marker), and LAMP1 (lysosome marker) in the membrane fraction (TBSX) and the intracellular fraction (GnHCl) were detected by WB. ( n = 10, five females and five males in each group for B-G, and I). Data are represented as mean ± SD and analyzed using a two-tailed t-test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001. Females are indicated with empty dots and males are indicated with filled dots for all data plots

Article Snippet: An ABCA1 knockdown astrocyte cell line was created by transfection with ABCA1 CRISPR Gene Knockout Kit (ABCA1-sgRNAs and Cas9 recombinant protein, Synthego).

Techniques: Injection, Mass Spectrometry, Membrane, Isolation, Staining, Marker, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Oxysterol accumulation promotes cellular senescence and neuroinflammation in APOE4 and AD through the aggregation of ABCA1 in lysosomes. Compared to healthy cells, a higher oxysterol burden in AD, particularly in APOE4 carriers, leads to increased ABCA1 and caveolin-1 expression, and lysosomal dysfunction marked by increased lipofuscin staining. This sequestration reduces ABCA1 endosome-lysosome recycling, and exacerbates cholesterol and lipid accumulation within lysosomes, activating mTORC1, and contributing to cellular senescence and neuroinflammation. Treatment with cyclodextrin mitigates these effects by lowering oxysterols. Illumination is created by BioRender. CD: Cyclodextrin

Article Snippet: An ABCA1 knockdown astrocyte cell line was created by transfection with ABCA1 CRISPR Gene Knockout Kit (ABCA1-sgRNAs and Cas9 recombinant protein, Synthego).

Techniques: Expressing, Staining