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: Cell reports

Article Title: APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release

doi: 10.1016/j.celrep.2023.113252

Figure Lengend Snippet:

Article Snippet: Mouse: PS19-E3 APOE3-KI crossbred to Tau-P301S (PS19 line) (Jax #008169) , This paper , N/A.

Techniques: Recombinant, Protease Inhibitor, Blocking Assay, Plasmid Preparation, Stripping Membranes, Electron Microscopy, Saline, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Extraction, Software, Generated, Microscopy, Laser-Scanning Microscopy, Imaging, Spectrophotometry

Journal:

Article Title: A Synthetic Peptide Blocking the Apolipoprotein E/?-Amyloid Binding Mitigates ?-Amyloid Toxicity and Fibril Formation in Vitro and Reduces ?-Amyloid Plaques in Transgenic Mice

doi:

Figure Lengend Snippet: A: Thioflavin-T assay demonstrates the modest ability of Aβ12–28 to form fibrils which is significantly lower than Aβ1–40 and Aβ1–42 (P < 0.0001, repeated measures analysis of variance; P < 0.001 Aβ12–28 versus Aβ1–40 and Aβ12–28 versus Aβ1–42 Tukey HSD post-hoc test). No fibrils were formed by Aβ12–28P. B: Aβ1–42 was incubated in the presence of either apo E3 or apo E4 (solid red and blue lines) that significantly increased amount of fibrils formed over time compared with Aβ1–42 alone (green line; P < 0.0001, repeated measures analysis of variance; P < 0.01 and P < 0.05 Tukey HSD post-hoc test for specific comparison of Aβ1–42+apo E4 and Aβ1–42+apo E3 versus Aβ1–42 alone, respectively). Chaperoning effect of apo E4 and apo E3 on Aβ1–42 fibril formation was significantly reduced if apo E was preincubated with equimolar concentrations of Aβ12–28P (dashed red and blue lines; P < 0.01 and P < 0.05 Tukey HSD post-hoc test for specific effect of Aβ12–28P on Apo E4 and apo E3, respectively). There were no significant differences between fibrillization curves of Aβ1–42 incubated with apo E4 and Aβ12–28P or apo E3 and Aβ12–28P versus Aβ1–42 incubated alone. Aβ12–28P alone had no significant effect on Aβ1–42 fibril formation.

Article Snippet: Recombinant apo E3 and apo E4, were purchased from Calbiochem Corp. (San Diego, CA).

Techniques: ThT Assay, Incubation

Journal: Viruses

Article Title: HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles

doi: 10.3390/v13050929

Figure Lengend Snippet: Effect of Myr47 and its mutants on the ApoE3-LP interaction. ( A ) Effect of ApoE3 on the cellular LP uptake activity in the presence of Myr47 or its mutants. Hep G2 cells were incubated with DiD-LPs (5 μg/mL) in serum-free DMEM containing Myr47 or its mutants (500 nM) with/without ApoE3 (25 μg/mL) for 3 h at 37 °C, and then analyzed with flow cytometry. Data are shown as geometric means ± S.D. ( n = 3). ( B ) Effect of Myr47 or its mutants on the interaction of LPs and ApoE3 was evaluated with BLItz. Data are shown as means. ( n = 3).

Article Snippet: Human apolipoprotein E3 (ApoE3) was purchased from Peprotech, Rocky Hill, NJ, USA.

Techniques: Activity Assay, Incubation, Flow Cytometry