apoe3 e3  (Jackson Laboratory)

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: bioRxiv

Article Title: XL-MS and De Novo Protein Design Identified a Common Motif for TREM2 Binding

doi: 10.64898/2026.04.23.720433

Figure Lengend Snippet: (a) SDS-PAGE analysis showing successful cross-linking of the heterodimeric TREM2 ECD /Trx-ApoE3 complex. (b) Representative high-quality MS/MS spectra of inter-protein cross-linked peptides. (c) Bar representation showing intra-protein XLs, inter-protein XLs, and inter-protein self-links. Figure was created using xiNET . ApoE3: light blue (N-terminal region, residues 1-167), light yellow (hinge region, residues 168-205), and pink (C-terminal region, residues 206-299).

Article Snippet: 20 μM Trx-ApoE3 (Sino Biological 10817-H30E) and 20 μM TREM2 ectodomain were incubated for 1 h at 4°C.

Techniques: SDS Page, Tandem Mass Spectroscopy

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: APOE4 causes deficits in the Kir4.1. (a) Representative images of retinal slices showing Glutamine synthase (GS) and Kir4.1 staining pattern in APOE3 and APOE4 mice, scale 20 μm ( n : APOE3 = 3, APOE4 = 3). (b) Bar graph showing quantification of immunofluorescence for Kir4.1 and GS ( n : 11–12 images/group). (c) Representative current traces of Kir4.1 from freshly isolated Müller cells from APOE3 and APOE4 mice with and without 1 mM BaCl 2 treatment. Currents were elicited by a 50‐ms hyperpolarization to −140 mV from a holding potential of −60 mV. The dashed line indicates the closed state (zero current), the downward pulses represent channel openings, corresponding to inward K + current. The flickers indicate channel opening and closing. (d) Representative current–voltage (I–V) relationship of whole‐cell voltage‐gated K + currents of Kir4.1 from freshly isolated Müller cells from APOE3 and APOE4 mice with and without 1 mM BaCl 2 treatment. (e) Current densities of Kir4.1 from freshly isolated Müller cells from APOE3 and APOE4 mice collected from +30 mV ( n : APOE3 = 26 cells/9 mice, APOE4 = 33 cells/8 mice). Values are expressed as mean ± SEM. An unpaired t ‐test was used for statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells were then incubated O/N at 4°C with Anti‐HA (Cat. #26183, Invitrogen, 1:200), APOE (Cat. #ab52607, Abcam, 1:100), APOE3 (Cat. #MAB41442‐SP, Novus Biologicals, CO, USA, 1:100) and APOE4 (Cat. #NBP1‐49529SS, Novus Biologicals, 1:100) antibody, followed by washing and a 2‐h incubation with the appropriate secondary antibody the next day.

Techniques: Staining, Immunofluorescence, Isolation

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: Mitochondrial dysfunction in APOE4 . (a) Representative images of retinal slices showing glutamine synthase (GS) and TOMM20 staining pattern in APOE3 and APOE4 mice, scale 20 μm ( n : APOE3 = 3, APOE4 = 3). (b) Bar graph showing quantification of immunofluorescence for TOMM20 and GS ( n : 10–11 images/group). Values are expressed as mean ± SEM. An unpaired t ‐test was used for statistical analysis. * p < 0.05, *** p < 0.001.

Article Snippet: The cells were then incubated O/N at 4°C with Anti‐HA (Cat. #26183, Invitrogen, 1:200), APOE (Cat. #ab52607, Abcam, 1:100), APOE3 (Cat. #MAB41442‐SP, Novus Biologicals, CO, USA, 1:100) and APOE4 (Cat. #NBP1‐49529SS, Novus Biologicals, 1:100) antibody, followed by washing and a 2‐h incubation with the appropriate secondary antibody the next day.

Techniques: Staining, Immunofluorescence

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: APOE4 decreases Kir4.1 and mitochondrial expression in rMC‐1. (a) Schematic showing the generation of rMC‐1 expressing human APOE isoforms. rMC‐1 was transiently transfected with human APOE2 / APOE3 / APOE4 , and EV was used as a control. (b) mRNA expression of Kcnj10 gene for Kir4.1 normalized to a housekeeping gene β‐actin. (c) Representative western blots of Kir4.1 expression and (d) quantification of integrated optical density (IOD) ratio of Kir4.1 and α‐tubulin showing decreased protein expression of Kir4.1 in APOE4 ‐transfected rMC‐1. (e) Representative images of rMC‐1 transfected with human APOE2 / APOE3 / APOE4 /EV showing decreased TOMM20 staining pattern in APOE4 ‐transfected rMC‐1, scale: 20 μm ( n : 3 independent experiments). (f) Quantification of TOMM20 staining intensity per cell area ( n : 15–24 cells/condition). (g) mRNA expression of Mfn1 , Mfn2 , and Dnm1 , showing that APOE4 ‐transfected rMC‐1 reduced Mfn1 , Mfn2 , and Dnm1 gene expression as compared to EV/ APOE2 / APOE3 ‐transfected rMC‐1 ( n : 4 independent experiments). Values are expressed as mean ± SEM. One‐way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The cells were then incubated O/N at 4°C with Anti‐HA (Cat. #26183, Invitrogen, 1:200), APOE (Cat. #ab52607, Abcam, 1:100), APOE3 (Cat. #MAB41442‐SP, Novus Biologicals, CO, USA, 1:100) and APOE4 (Cat. #NBP1‐49529SS, Novus Biologicals, 1:100) antibody, followed by washing and a 2‐h incubation with the appropriate secondary antibody the next day.

Techniques: Expressing, Transfection, Control, Western Blot, Staining, Gene Expression, Comparison

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: APOE4 impairs mitochondrial respiration and reduces metabolic flexibility in rMC‐1. (a) OCR traces in rMC‐1 expressing EV/ APOE2 / APOE3 / APOE4 in response to sequential addition of oligomycin (oligo), FCCP, and rotenone/antimycin A (Rot/AA). APOE4 expressing rMC‐1 showed consistently lower OCR across conditions. (b) Quantification of basal respiration, maximal respiration, and non‐mitochondrial respiration, with APOE4 expressing rMC‐1 showing significantly reduced maximal and non‐mitochondrial respiration. (c) Quantification of spare respiratory capacity, ATP‐linked respiration, and proton leak. APOE4 ‐expressing rMC‐1 exhibited a marked reduction in spare respiratory capacity, while ATP‐linked respiration showed a downward trend. (d) ECAR profile in rMC‐1 expressing EV/ APOE2 / APOE3 / APOE4 in response to oligomycin (oligo), FCCP, and rotenone/antimycin A (Rot/AA) shows comparable basal rates across groups. (e) Quantification of glycolytic reserve, basal, and maximal ECAR. APOE4 rMC‐1 displayed a significantly reduced glycolytic reserve compared to EV, APOE2 , and APOE3 ‐transfected rMC‐1. (f) Quantification of glycolytic capacity and non‐glycolytic ECAR showing no significant changes across groups. (g) PPR traces in rMC‐1 expressing EV/ APOE2 / APOE3 / APOE4 in response to oligomycin (oligo), FCCP, and rotenone/antimycin A (Rot/AA) show overall comparable levels across groups. (h) Quantification of basal and maximal PPR confirms no significant APOE isoform differences. (i) Quantification of glycolytic PPR and non‐glycolytic PPR also showing no significant differences across groups ( n : 3 independent experiments, with 3–4 technical replicates per condition). Values are expressed as mean ± SEM. One‐way ANOVA with Tukey's test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were then incubated O/N at 4°C with Anti‐HA (Cat. #26183, Invitrogen, 1:200), APOE (Cat. #ab52607, Abcam, 1:100), APOE3 (Cat. #MAB41442‐SP, Novus Biologicals, CO, USA, 1:100) and APOE4 (Cat. #NBP1‐49529SS, Novus Biologicals, 1:100) antibody, followed by washing and a 2‐h incubation with the appropriate secondary antibody the next day.

Techniques: Expressing, Transfection

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: MitoQ restores Kir4.1 gene and protein expression in rMC‐1 transfected with APOE4 . (a) mRNA expression of Kcnj10 gene for Kir4.1 normalized to housekeeping gene for β‐actin after treating rMC‐1 with 1 μM MitoQ and vehicle. mRNA expression of Kir4.1 was significantly increased in APOE4 ‐transfected rMC‐1 upon treatment with 1 μM MitoQ compared to the vehicle. (b) Representative western blots of Kir4.1 expression and quantification of IOD ratio of Kir4.1 and α‐tubulin showing comparable protein expression of Kir4.1 in APOE4 ‐transfected rMC‐1 as compared to EV/ APOE2 /APOE3‐transfected rMC‐1 after treating with 1 μM MitoQ. Values are expressed as mean ± SEM. Two‐way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis. * p < 0.05, ** p < 0.01. ( n : 3–4 independent experiments).

Article Snippet: The cells were then incubated O/N at 4°C with Anti‐HA (Cat. #26183, Invitrogen, 1:200), APOE (Cat. #ab52607, Abcam, 1:100), APOE3 (Cat. #MAB41442‐SP, Novus Biologicals, CO, USA, 1:100) and APOE4 (Cat. #NBP1‐49529SS, Novus Biologicals, 1:100) antibody, followed by washing and a 2‐h incubation with the appropriate secondary antibody the next day.

Techniques: Expressing, Transfection, Western Blot, Comparison

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: MitoQ decreases mitochondrial ROS in APOE4 ‐transfected rMC‐1. Representative images of unstained rMC‐1 and rMC‐1 transfected with EV/ APOE2 / APOE3 / APOE4 and treated with (a) vehicle or (b) MitoQ (1 μM). Cells were analyzed on a flow cytometer with 610/20 nm bandpass emission filter. (c) Bar graph showing quantification of % of MitoSox Red positive cells. Mitochondrial reactive oxygen species (ROS) was decreased upon treating APOE4 ‐transfected rMC‐1 with 1 μM MitoQ. Values are expressed as mean ± SEM ( n : 3 independent experiments). One‐way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis. * p < 0.05, ** p < 0.01.

Article Snippet: The cells were then incubated O/N at 4°C with Anti‐HA (Cat. #26183, Invitrogen, 1:200), APOE (Cat. #ab52607, Abcam, 1:100), APOE3 (Cat. #MAB41442‐SP, Novus Biologicals, CO, USA, 1:100) and APOE4 (Cat. #NBP1‐49529SS, Novus Biologicals, 1:100) antibody, followed by washing and a 2‐h incubation with the appropriate secondary antibody the next day.

Techniques: Transfection, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: High-density lipoprotein mimetic peptide 4F ameliorates APOE4-associated lipid dysfunction in primary and iPSC-derived astrocytes and cerebral organoids

doi: 10.64898/2025.12.16.694774

Figure Lengend Snippet: Primary astrocytes from humanized APOE3 or APOE4 mice were incubated in serum-free media for 24 hours. SDS-PAGE and NDGGE were performed to determine the relative secretion and lipidation state of APOE. ( A ) Representative image and quantification from SDS-PAGE performed on media and cell lysates from primary astrocytes. ( B ) Representative image and quantification from NDGGE performed on fresh media from primary astrocytes. Tubulin was used as a loading control. Data was normalized to APOE3 and expressed as a percent. Data represents Mean ± SEM of 7-8 replicates from 3 independent experiments. Welsch’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Homozygous humanized APOE KI mice expressing APOE3 (E3) or APOE4 (E4) were purchased from Jackson laboratory (Stock # 029018 and 027894).

Techniques: Incubation, SDS Page, Control

Journal: bioRxiv

Article Title: High-density lipoprotein mimetic peptide 4F ameliorates APOE4-associated lipid dysfunction in primary and iPSC-derived astrocytes and cerebral organoids

doi: 10.64898/2025.12.16.694774

Figure Lengend Snippet: (A) Representative image from SDS-PAGE performed on media and cell lysates from iAstrocytes. ( B ) Representative image from NDGGE performed on fresh media from primary astrocytes. ( C,D ) Quantification of secreted and lipidated APOE from iAstrocytes. Data was normalized to APOE3 and expressed as a percent. ( E,F ) Quantification of secreted and lipidated APOE from iAstrocytes treated with 4F. Tubulin was used as a loading control. Data was normalized to vehicle for each genotype and expressed as a percent of vehicle treatment. Data represents Mean ± SEM of 7-12 replicates from 3 independent experiments. Welsch’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Homozygous humanized APOE KI mice expressing APOE3 (E3) or APOE4 (E4) were purchased from Jackson laboratory (Stock # 029018 and 027894).

Techniques: SDS Page, Control

Journal: bioRxiv

Article Title: High-density lipoprotein mimetic peptide 4F ameliorates APOE4-associated lipid dysfunction in primary and iPSC-derived astrocytes and cerebral organoids

doi: 10.64898/2025.12.16.694774

Figure Lengend Snippet: APOE3 and APOE4 iPSC-derived astrocytes were treated with 30 μM oleic acid with or without 5 μM 4F for 24 hours and stained with BODIPY to measure lipid droplets. Number of puncta were quantified and normalized to DAPI. Data represents average number of puncta/cell per field of view from 3 culture wells from 1 representative experiment. Scale bar, 50 μm. ( A ) Representative images and ( B ) quantification of APOE3 or APOE4 iAstrocytes treated with vehicle or 4F +/- oleic acid. Two-way ANOVA with Tukey’s post-hoc correction. * p < .05, ** p < .01, **** p <.0001

Article Snippet: Homozygous humanized APOE KI mice expressing APOE3 (E3) or APOE4 (E4) were purchased from Jackson laboratory (Stock # 029018 and 027894).

Techniques: Derivative Assay, Staining

Journal: bioRxiv

Article Title: High-density lipoprotein mimetic peptide 4F ameliorates APOE4-associated lipid dysfunction in primary and iPSC-derived astrocytes and cerebral organoids

doi: 10.64898/2025.12.16.694774

Figure Lengend Snippet: iPSCs were used to generate cerebral organoids. APOE3 and APOE4 cerebral organoids were treated with 5 μM 4F with or without 5 μM aggregated Aβ42 peptide for 24 hours. ( A ) Media and cell lysates were subjected to SDS-PAGE and immunoblot analysis. ( B ) Fresh media was analyzed for APOE lipidation using non-denaturing gradient gel electrophoresis (NDGGE). Data was normalized to vehicle for each genotype and expressed as a percent of vehicle treatment, Data represents Mean ± SEM of 7-8 replicates from 2 independent experiments. One-way ANOVA with Tukey’s post-hoc correction, # p = .06,* p < .05, **p < .01, ***p < .001.

Article Snippet: Homozygous humanized APOE KI mice expressing APOE3 (E3) or APOE4 (E4) were purchased from Jackson laboratory (Stock # 029018 and 027894).

Techniques: SDS Page, Western Blot, Denaturing Gradient Gel Electrophoresis