Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Determination of the interaction affinity between GM1 and APOE. A , schematic illustration of determining the binding affinity between lipid structure and APOE using MST. B , the binding affinity (K D ) between APOE3 or APOE4 and lipid structures containing GM1 or cholesterol (n = 3). C , the binding affinity between APOE3 or APOE4 and lipid structures with different GM1 concentrations. (n = 3). D - H , negative staining images of lipid structures with varying GM1 concentrations. p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( A ) is created with BioRender.com . APOE, apolipoprotein E; MST, microscale thermophoresis.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Binding Assay, Negative Staining, Microscale Thermophoresis

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Analysis of GM1 localization and levels following the cellular uptake of lipid structures. A , schematic illustration of the method used to determine GM1 following the cellular uptake. B , GM1 levels in differentiated PC-12 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 3) ( C ) representative images showing GM1 and APOE localization in differentiated PC-12 cells following cellular uptake, as determined by confocal microscopy. (n ≥ 6) ( D ) GM1 levels in HEK-293 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 6) ( E ) (1) cellular uptake of DiD-labeled cholesterol and GM1 lipid structures in U-87 MG cells, and (2) changes in cholesterol levels in U-87 MG cells after cellular uptake of APOE3 and APOE4-enriched cholesterol lipoproteins, as well as a mixture of APOE3 and APOE4-enriched cholesterol and GM1 lipoproteins. (n ≥ 6). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( A ) is created with BioRender.com . Created in BioRender. Dokholyan, N. (2025) https://BioRender.com/g17z389 . APOE, apolipoprotein E; HEK, human embryonic kidney; PC, l -α-phosphatidylcholine.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Confocal Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: The binding affinity of APOE-enriched lipoprotein and APOE receptor LDLR using MST. A and B , the binding affinity between APOE3-enriched lipoprotein with varying GM1 concentration to LDLR. (n = 3) ( C and D ) the binding affinity between APOE4-enriched lipoprotein varying GM1 concentration to LDLR (n = 3). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). APOE, apolipoprotein E; LDLR, low-density lipoprotein receptor; MST, microscale thermophoresis.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Binding Assay, Concentration Assay, Microscale Thermophoresis

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 cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

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 cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

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 cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

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 cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

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 cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

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 cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

Techniques: Transfection, Flow Cytometry, Comparison

Journal: eLife

Article Title: APOE2 is associated with longevity independent of Alzheimer’s disease

doi: 10.7554/elife.62199

Figure Lengend Snippet: Figure 1. APOE associate with lifespan irrespective of AD in clinical cohorts. (A) Kaplan-Meier survival curve according to APOE genotype. (B) Effects of APOE4 (e3/e4 or e4/e4) or APOE2 (e2/e3 or e2/e2) on survival compared to APOE3 (e3/e3) as a reference in all subjects (‘All subjects’), when stratifying by AD diagnosis at last visit (‘No AD’ or ‘AD’), in subjects with neuropathologically-assessment (‘Patho-assessed’), and subjects with minimal amyloid pathology (‘Minimal amyloid’). HR = hazard ratio. CI = confidence interval. HRs and 95% CIs result from Cox proportional hazards regression models. Models for all subjects were adjusted for sex, race, cognitive status at last visit, presence of AD at last visit, and cardiovascular factors. The models for subjects with neuropathological assessment were adjusted for sex, race, CERAD diffuse plaque score, CERAD neuritic plaque score, Braak NFT stage, presence of vascular pathology, and cardiovascular factors. All other models were adjusted for sex, race, and cardiovascular factors.

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus. musculus) Apoetm1(APOE*2)Mae or apoE2-TR Sullivan et al., 1998 RRID:MGI:3695702 Genetic reagent (Mus. musculus) Apoetm2(APOE*3)Mae or apoE3-TR Sullivan et al., 1997 RRID:MGI:3695698 Genetic reagent (Mus. musculus) Apoetm3(APOE*4)Mae or apoE4-TR Knouff et al., 1999 RRID:MGI:4355228 Genetic reagent (Mus. musculus) Apoetm1Unc or Apoe-KO Piedrahita et al., 1992 RRID:MGI:4358709 Antibody Goat polyclonal anti-ApoE-biotin Meridian Life Science Cat#: K74180B ELISA detection (1:4000) Antibody Goat polyclonal antiApolipoprotein E Antibody Millipore Cat#: AB947 ELISA capture (1:4000) Peptide, recombinant protein ApoE3 Fitzgerald Cat#: 30 R-2381 ELISA standard Commercial assay or kit Cholesterol E Wako Cat. #: 999–02601 Commercial assay or kit L-Type Triglyceride M Wako Cat. #: 994–02891 Commercial assay or kit HDL-Cholesterol E Wako Cat. #: 997–01301 Software, algorithm SAS SAS Institute, Inc version 9.4 Software, algorithm R Statistical Software R Foundation for Statistical Computing version 3.2.3 Software, algorithm JMP Pro SAS Institute, Inc version 12 Software, algorithm AnyMaze software Stoelting Co Animal behavior test Human clinical and neuropathological data The clinical data from NACC, which were collected by the 34 past and present Alzheimer’s Disease Centers (ADCs) from September 2005 to November 2016 as the longitudinal Uniform Data Set (Weintraub, 2009), were assessed in this study.

Techniques: Biomarker Discovery

Journal: eLife

Article Title: APOE2 is associated with longevity independent of Alzheimer’s disease

doi: 10.7554/elife.62199

Figure Lengend Snippet: Figure 3. APOE2 is associated with preserved activity in clinical cohorts. (A) Odds ratio with 95% CIs of APOE2 or APOE4 on ‘dropped activities and interests’, ‘total GDS score’, and ‘memory problem’ compared to APOE3, as calculated by logistic regression models that were adjusted for sex, race, and age at the time of the GDS questionnaire. (B) Kaplan–Meier survival curve of subjects with/without ‘dropped activities and interests’; dropped activities and interests were associated with poorer survival (Hazard ratio = 1.20, p=0.010). *p<0.05; calculated by Cox proportional hazards regression analysis adjusting for sex, race, and APOE genotype group.

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus. musculus) Apoetm1(APOE*2)Mae or apoE2-TR Sullivan et al., 1998 RRID:MGI:3695702 Genetic reagent (Mus. musculus) Apoetm2(APOE*3)Mae or apoE3-TR Sullivan et al., 1997 RRID:MGI:3695698 Genetic reagent (Mus. musculus) Apoetm3(APOE*4)Mae or apoE4-TR Knouff et al., 1999 RRID:MGI:4355228 Genetic reagent (Mus. musculus) Apoetm1Unc or Apoe-KO Piedrahita et al., 1992 RRID:MGI:4358709 Antibody Goat polyclonal anti-ApoE-biotin Meridian Life Science Cat#: K74180B ELISA detection (1:4000) Antibody Goat polyclonal antiApolipoprotein E Antibody Millipore Cat#: AB947 ELISA capture (1:4000) Peptide, recombinant protein ApoE3 Fitzgerald Cat#: 30 R-2381 ELISA standard Commercial assay or kit Cholesterol E Wako Cat. #: 999–02601 Commercial assay or kit L-Type Triglyceride M Wako Cat. #: 994–02891 Commercial assay or kit HDL-Cholesterol E Wako Cat. #: 997–01301 Software, algorithm SAS SAS Institute, Inc version 9.4 Software, algorithm R Statistical Software R Foundation for Statistical Computing version 3.2.3 Software, algorithm JMP Pro SAS Institute, Inc version 12 Software, algorithm AnyMaze software Stoelting Co Animal behavior test Human clinical and neuropathological data The clinical data from NACC, which were collected by the 34 past and present Alzheimer’s Disease Centers (ADCs) from September 2005 to November 2016 as the longitudinal Uniform Data Set (Weintraub, 2009), were assessed in this study.

Techniques: Activity Assay

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: 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: Beilstein Journal of Nanotechnology

Article Title: Serum heat inactivation diminishes ApoE-mediated uptake of D-Lin-MC3-DMA lipid nanoparticles

doi: 10.3762/bjnano.16.57

Figure Lengend Snippet: Schematic representation of the heat treatment and ThT staining of ApoE. Binding of ThT to misfolded or aggregated proteins greatly enhances its fluorescence (A). The fluorescence of solutions containing ApoE3 or BSA following ThT staining as determined by spectrophotometry, after a 30 min incubation at room temperature (rt), 37, 56 and 75 °C ( n = 3) (B). Differences were considered statistically significant at p < 0.05 and were annotated as ns = non-significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and **** = p ≤ 0.0001. The graphics in were generated with images provided by Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 4.0 International License, https://creativecommons.org/licenses/by/4.0/

Article Snippet: Human recombinant ApoE3 was purchased from Fitzgerald Industries.

Techniques: Staining, Binding Assay, Fluorescence, Spectrophotometry, Incubation, Generated

Journal: Beilstein Journal of Nanotechnology

Article Title: Serum heat inactivation diminishes ApoE-mediated uptake of D-Lin-MC3-DMA lipid nanoparticles

doi: 10.3762/bjnano.16.57

Figure Lengend Snippet: Heat-mediated aggregation/denaturation prevents ApoE from binding to LNPs and reduces LNP uptake by cells. The fluorescence of HMEC-1 after uptake of LNP containing alexa fluor 647 labelled siRNA at different concentrations of recombinant ApoE3, BSA or medium supplemented with NHI FCS (A). The concentration of fluorescent siRNA was 1.725 pmol per well and uptake was measured after 4 h ( n = 3). The fluorescence of HMEC-1 cells after uptake of LNPs in medium supplemented with HI or NHI ApoE (B). Schematic representation of the bead capture and staining of ApoE bound LNPs (C). The fluorescence of ApoE3 bound to LNPs that are captured by magnetic beads after incubation in human serum, heat treated human serum and recombinant ApoE3 in PBS, as determined by flow cytometry (D) ( n = 2). Differences were considered statistically significant at p < 0.05 and were annotated as ns = non-significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and **** = p ≤ 0.0001. The graphics in were generated with images provided by Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 4.0 International License, https://creativecommons.org/licenses/by/4.0/

Article Snippet: Human recombinant ApoE3 was purchased from Fitzgerald Industries.

Techniques: Binding Assay, Fluorescence, Recombinant, Concentration Assay, Staining, Magnetic Beads, Incubation, Flow Cytometry, Generated

Journal: Beilstein Journal of Nanotechnology

Article Title: Serum heat inactivation diminishes ApoE-mediated uptake of D-Lin-MC3-DMA lipid nanoparticles

doi: 10.3762/bjnano.16.57

Figure Lengend Snippet: The uptake of MC3 LNPs is ApoE dependent and is affected by heat inactivation of FCS, while the uptake of C12 LNPs is not. Uptake of MC3 or C12 LNPs containing alexa fluor 647 labelled siRNA by HMEC-1, U87 and MDA-MB-231 in medium supplemented with NHI FCS, HI FCS or HI FCS with 1 µg/mL ApoE3. Analysed by flow cytometry ( n = 3). Differences were considered statistically significant at p < 0.05 and were annotated as ns = non-significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and **** = p ≤ 0.0001.

Article Snippet: Human recombinant ApoE3 was purchased from Fitzgerald Industries.

Techniques: Flow Cytometry

Journal: Beilstein Journal of Nanotechnology

Article Title: Serum heat inactivation diminishes ApoE-mediated uptake of D-Lin-MC3-DMA lipid nanoparticles

doi: 10.3762/bjnano.16.57

Figure Lengend Snippet: Heat inactivation of FCS and endogenous ApoE diminishes the firefly luciferase knockdown efficacy of MC3 LNPs, but not of C12 LNPs. Firefly luciferase activity in U87 and MDA-MB-231 48 h after uptake of siLuc loaded MC3 or C12 LNPs in medium supplemented with NHI FCS, HI FCS or HI FCS with 1 µg/mL ApoE3. Luciferase activity was analysed by measuring bioluminescence ( n = 3). Differences were considered statistically significant at p < 0.05 and were annotated as ns = non-significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and **** = p ≤ 0.0001.

Article Snippet: Human recombinant ApoE3 was purchased from Fitzgerald Industries.

Techniques: Luciferase, Knockdown, Activity Assay