Journal: Theranostics

Article Title: Why 11β-HSD1 inhibitors show variable efficacy in Alzheimer's therapy: an APOE4-dependent HSD11B1 mechanism

doi: 10.7150/thno.126244

Figure Lengend Snippet: APOE4 protein enhances HSD11B1 expression and increases cortisol levels in neuronal cells. A. Cortisol levels were measured in HT-22 (left) and SH-SY5Y (right) cells treated with recombinant APOE4 (E4) or APOE3 (E3) recombinant proteins. Cells were co-treated with VLDL (25 μg/mL) or HDL (25 μg/mL) plus either APOE4 or APOE3 (10 μg/mL) for 3 days, followed by cortisone (0.4 μg/mL) treatment for an additional 24 hours. Cortisol in the culture supernatants was quantified using an ELISA kit. B. Schematic representation of local cortisol regulation by HSD11B enzymes. C. qRT-PCR analysis of HSD11B1 mRNA expression in HT-22 cells. Cells were treated under the same conditions as in (A), and HSD11B1 mRNA levels were quantified using GAPDH as the internal control. D. APOE4/HDL induces HSD11B1 expression and cortisol activation in primary EC neurons. Primary neurons derived from the EC were treated with recombinant APOE3 or APOE4 proteins in combination with HDL for 3 days, followed by cortisone for an additional 24 hours. Left: HSD11B1 mRNA levels were quantified by qRT-PCR and normalized to GAPDH. Right: Cortisol levels in culture supernatants were measured by ELISA. E. Predicted docking models of cortisone (left) and carbenoxolone (Cbxl; right) with human HSD11B1 using SwissDock. Cortisone, the natural substrate of HSD11B1, binds within a defined pocket on the enzyme surface. Cbxl, a known HSD11B1 inhibitor, occupies a similar docking site, suggesting competitive inhibition by blocking cortisone access. Binding sites are indicated by red dashed circles. F. Dose-dependent reduction in cortisol levels following HSD11B1 inhibition. HT-22 cells were co-treated with APOE4 and increasing concentrations of the HSD11B1 inhibitor carbenoxolone (Cbxl; 5, 10, 15 μM) for 24 hours in the presence of cortisone (0.4 μg/mL). Cortisol levels were measured by ELISA. G. HSD11B1 knockdown attenuates APOE4-induced cortisol activation in HT-22 cells. HT-22 cells were transfected with siRNA ( siHSD11B1 ) targeting Hsd11b1 or a non-targeting control siRNA ( siCtrl ), followed by treatment with recombinant APOE4 in the presence of HDL for 3 days and cortisone for an additional 24 hours. Cortisol levels in culture supernatants were quantified by ELISA.

Article Snippet: Mouse hippocampal neuronal cell line HT-22 (Sigma-Aldrich, SCC129) and the APOE3 (E3/E3) genotype human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266) were cultured in Dulbecco's Modified Eagle Medium (Gibco) and Minimum Essential Medium (Gibc), respectively, at 37°C in a 5% CO2 humidified atmosphere.

Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Activation Assay, Derivative Assay, Inhibition, Blocking Assay, Binding Assay, Knockdown, Transfection

Journal: Theranostics

Article Title: Why 11β-HSD1 inhibitors show variable efficacy in Alzheimer's therapy: an APOE4-dependent HSD11B1 mechanism

doi: 10.7150/thno.126244

Figure Lengend Snippet: C/EBPβ mediates APOE4-induced HSD11B1 expression in neuronal cells. A. Identification of potential transcriptional regulators of HSD11B1. The top 50 genes co-expressed with HSD11B1 were identified using the ARCHS4 RNA-seq database. Enrichment analysis was performed with the Enrichr database and UCSC Genome Browser PWMs to pinpoint transcription factors potentially involved in HSD11B1 regulation. B. Schematic representation of two putative C/EBPβ ( CEBPB ) binding motifs within the HSD11B1 promoter, as identified through chromvatin immunoprecipitation (ChIP) analysis. Data were obtained from the ReMap ChIP-seq database via the UCSC Genome Browser. C. Proposed model illustrating how APOE4 activates C/EBPβ transcriptional activity, thereby promoting HSD11B1 transcription. D. ChIP assay demonstrating C/EBPβ binding to the HSD11B1 promoter in SH-SY5Y cells. Cells were co-treated with HDL and recombinant APOE3 or APOE4 for 3 days. ChIP-qPCR analysis quantified the enrichment of HSD11B1 promoter fragments immunoprecipitated with anti-C/EBPβ compared to IgG controls. The promoter sequence is shown with the transcription start site (+1) marked in dark red, putative C/EBPβ binding sites underlined and highlighted in brown, and the ChIP primer sites indicated in blue. E. qRT-PCR analysis of HSD11B1 mRNA levels following CEBPB (C/EBPβ) knockdown in SH-SY5Y cells. Cells transfected with siCEBPB or siCtrl were co-treated with HDL and APOE4 proteins, and mRNA levels for HSD11B1 and C/EBPβ were quantified. F. Western blot analysis showing the effect of C/EBPβ knockdown on HSD11B1 and C/EBPβ protein levels in SH-SY5Y cells. Cells transfected with siC/EBPβ or siControl were co-treated with HDL and either APOE4 or APOE3 proteins. Total cell lysates were probed with antibodies against HSD11B1, phosphorylated C/EBPβ (Thr235), total C/EBPβ, and GAPDH. G. ELISA quantification of cortisol levels in SH-SY5Y cells after C/EBPβ knockdown. Following transfection with siCEBPB or siCtrl , cells were co-treated with HDL and APOE4, then treated with cortisone for 24 hours. Cortisol levels in the culture supernatants were measured using a Cortisol ELISA Kit.

Article Snippet: Mouse hippocampal neuronal cell line HT-22 (Sigma-Aldrich, SCC129) and the APOE3 (E3/E3) genotype human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266) were cultured in Dulbecco's Modified Eagle Medium (Gibco) and Minimum Essential Medium (Gibc), respectively, at 37°C in a 5% CO2 humidified atmosphere.

Techniques: Expressing, RNA Sequencing, Binding Assay, Immunoprecipitation, ChIP-sequencing, Activity Assay, Recombinant, ChIP-qPCR, Sequencing, Quantitative RT-PCR, Knockdown, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 1. Primary pericytes from ApoE (apolipoprotein E)-targeted replacement mice abundantly secrete lipidated apoE. A, Representative images of pericytes stained for NG2 (neural/glial antigen 2; left) and PDGFRβ (platelet-derived growth factor receptor- β, right) are shown. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). B, The amount of apoE in the conditioned media from primary cultures of endothelial cells (EC), pericytes (PC), and astrocytes (AS) was measured by ELISA and normalized against the total protein concentrations in cell lysates. C, Conditioned media from primary cultures of PC and AS were concentrated and subjected to size- exclusion chromatography run by FPLC using a Superose-6 column. The amount of apoE in each fraction was determined by ELISA. Values of 3 independent experiments were averaged and plotted against fraction numbers. D, Conditioned media from primary cultures of PC and AS were concentrated and subjected to an immunoprecipitation using an apoE-specific antibody. The amount of apoE-associated cholesterol was determined by Amplex Red cholesterol assay after immunoprecipitation with anti-apoE antibody. Data in (B) and (D) are presented as mean±SEM (N=4). Each dot in (B) and (D) represents a measurement from one independent primary cell culture prepared from brains of 4 male and 4 female mice. *P<0.05, apoE3-EC vs apoE3-PC, Mann-Whitney U test (B, top). *P<0.05, apoE4-EC vs apoE4-PC, Mann-Whitney U test (B, bottom). The amount of apoE in the conditioned media from primary AS is not included in the statistical analysis (B). *P<0.05, apoE3- PC vs apoE3-AS, Mann-Whitney U test (D, top); *P<0.05, apoE4-PC vs apoE4-AS, Mann-Whitney U test (D, bottom).

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Staining, Derivative Assay, Enzyme-linked Immunosorbent Assay, Size-exclusion Chromatography, Immunoprecipitation, Amplex Red Cholesterol Assay, Cell Culture, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 2 Continued. (G) in EC were determined by qRT-PCR and compared between EC monoculture, EC cocultured with apoE3-PC or apoE4-PC. Data in (B–G) are presented as mean±SEM (N=4). Each dot in (B–G) represents a measurement from one independent primary cell culture prepared from brains of 4 male and 4 female mice. **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; *P<0.05, EC cocultured with apoE3-PC vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (B, left). Kruskal- Wallis test followed by Dunn multiple comparison tests (B, right). ***P<0.001, EC monoculture vs EC cocultured with apoE3-PC; *P<0.05, EC cocultured with apoE3-PC vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (C, left). ***P<0.001, EC monoculture vs EC cocultured with apoE3-PC; EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (C, right). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; Kruskal-Wallis test followed by Dunn multiple comparison tests (D, left). Kruskal-Wallis test followed by Dunn multiple comparison tests (D, right). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; Kruskal-Wallis test followed by Dunn multiple comparison tests (E, left). ****P<0.0001, EC monoculture vs EC cocultured with apoE3-PC; ***P<0.001, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (E, right). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; Kruskal-Wallis test followed by Dunn multiple comparison tests (F, left). ***P<0.001, EC monoculture vs EC cocultured with apoE3-PC; *P<0.05, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (F, right). ****P<0.0001, EC monoculture vs EC cocultured with apoE3-PC; **P<0.05, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (G, left). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (G, right). N.S. indicates not significant.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Quantitative RT-PCR, Cell Culture, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 5. Reduced collagen-IV deposition along cortical capillaries in ApoE4-targeted replacement (apoE4-TR) mice. A, Collagen IV, CD31, claudin-5, occludin, CD13, and AQP4 were stained in frozen cortical sections from apoE3-TR (male; N=4, female; N=4) or apoE4-TR mice (male; N=4, female; N=4) at the age of 22 mo. B, Total fluorescence intensity of collagen IV in cortical sections from those apoE-TR mice were quantified by ImageJ software (apoE, P=0.0034; sex, P=0.0011, apoE×sex, P=0.7080). C–F, The % of coverage against CD31-positive endothelial by claudin-5 (C, apoE, P=0.9304; sex, P=0.6093, apoE×sex, P=0.1227), occludin (D, apoE, P=0.2478; sex, P=0.1107, apoE×sex, P=0.3657), CD13 (E, apoE, P=0.1683; sex, P=0.0897, apoE×sex, P=0.3923) or AQP4 (F, apoE, P=0.1698; sex, P=0.9376, apoE×sex, P=0.3703) was quantified in cortical sections from the mice. Data in (B–F) are presented as mean±SEM. Each dot in (B–F) represents a measurement from one mouse. *P<0.05, ***P<0.001 by Tukey-Kramer post hoc analysis of 2-way ANOVA. N.S. indicates not significant among groups.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Staining, Fluorescence, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 6. Increased plasma protein leakage in the cortex of ApoE4-targeted replacement (apoE4-TR) mice. A–E, The levels of collagen IV (A; apoE [apolipoprotein E], P<0.0001; sex, P=0.4401, apoE×sex, P=0.7307), claudin-5 (B; apoE, P=0.4365; sex, P=0.5057, apoE×sex, P=0.4332), occludin (C, apoE, P=0.7529; sex, P=0.8528, apoE×sex, P=0.9210), fibrinogen (D, apoE, P=0.0002; sex, P=0.9759, apoE×sex, P=0.5057), and IgG (E, apoE, P=0.0428; sex, P=0.5767, apoE×sex, P=0.6582) were determined by ELISA in apoE3-TR (male; N=8, female; N=9) and apoE4-TR mice (male; N=8, female; N=8) at 22 mo of age. The measurement was normalized by protein concentration in each of the samples. Data in (A–E) are presented as mean±SEM. Each dot represents a measurement from one mouse. *P<0.05, **P<0.01 by Tukey-Kramer post hoc analysis of 2-way ANOVA. F and G, The correlations between the levels of leaked plasma protein (fibrinogen and IgG) and that of collagen IV calculated across the male or female apoE3-TR (red circle) and apoE4-TR (blue circle) mice were assessed through nonparametric Spearman correlation analysis. The correlation coefficient (R2) and P value are shown in each panel. N.S. indicates not significant among groups.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Immunity

Article Title: An Exhausted-Like Microglial Population Accumulates in Aged and APOE4 Genotype Alzheimer’s Brains

doi: 10.1016/j.immuni.2023.12.001

Figure Lengend Snippet: Key Resources Table

Article Snippet: Mice bearing knock-in of human APOE2 , APOE3 , or APOE4 in the murine Apoe locus [B6.129P2- Apoe tm1(APOE*2)Mae N9 ( APOE2 ), B6.129P2- Apoe tm2(APOE*3)Mae N8 ( APOE3 ) and B6.129P2- Apoe tm3(APOE*4)Mae N8 ( APOE4 )] were purchased from Taconic Biosciences and maintained in our facilities.

Techniques: Recombinant, Control, Gene Expression, Sequencing, Imaging, Software

Journal: Journal of Molecular Endocrinology

Article Title: Upregulation of hepatic LRP1 by rosiglitazone: a possible novel mechanism of the beneficial effect of thiazolidinediones on atherogenic dyslipidemia

doi: 10.1530/jme-12-0119

Figure Lengend Snippet: Figure 3 The effect of rosiglitazone on ApoE uptake in HepG2 cells. HepG2 cells were treated with indicated concentrations of rosiglitazone for 48 h. Human recombinant ApoE3 was added to culture media and cells were incubated for 1 h. ApoE3 was reconstituted with lipid using DMPC before the treatment on HepG2 cells. Three independent experiments were performed for the representative figures. (A) Western blot analysis of ApoE3 in HepG2 cells incubated with or without added ApoE3. (B) Western blot analysis of ApoE3 in HepG2 cells incubated with added ApoE3. HepG2 cells were transfected with siRNA targeting human LRP1 (siLRP1) and non-targeting negative siRNA (siCTRL) before the rosiglitazone treatment and adding ApoE3.

Article Snippet: After 48 h, cells were washed once with PBS and then incubated with 25 mg/ml human recombinant ApoE3 (R&D Systems) for 1 h. ApoE3 was reconstituted with lipid using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) before the treatment on HepG2 cells using the method of previous studies (Innerarity et al. 1979).

Techniques: Recombinant, Incubation, Western Blot, Transfection