Journal: bioRxiv

Article Title: APOE regulates the transport of GM1

doi: 10.1101/2024.04.02.587789

Figure Lengend Snippet: (a) Schematic illustration of determining the binding affinity between lipid structure and APOE using MST. (b) The binding affinity Kd between APOE3 and lipid structures with different compositions. (n>=4) (c) The binding affinity between APOE3 and lipid structures with different GM1 concentrations. (n>=3) (d) The binding affinity between APOE4 and lipid structures with different compositions. (n>=5) (e) The binding affinity between APOE4 and lipid structures with different GM1 concentrations. (n>=5) 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.

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

Journal: bioRxiv

Article Title: APOE regulates the transport of GM1

doi: 10.1101/2024.04.02.587789

Figure Lengend Snippet: (a) The expression of LDLR on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=4). (b) The expression of VLDLR on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=4). (c) The expression of LRP1 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=3). (d) The expression of ApoER2 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=3). (e) Schematic illustration of determining the changes of APOE secondary structures. (f) The CD results of APOE 3 under the effect of different GM1 content on the lipid structures (n>=3). (g) The CD results of APOE 4 under the effect of different GM1 content on the lipid structures (n=3). (h) Schematic illustration of determining the binding affinity of APOE-enriched lipoprotein and APOE receptor LDLR using MST. (i) The binding affinity between APOE3 and lipid structures with different compositions. (n>=4) (j) The binding affinity between APOE4 and lipid structures with different compositions. (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). (e) and (h) are created with BioRender.com.

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: Expressing, Binding 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 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: 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: 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: 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: The lysates were subjected to SDS–PAGE using anti-human ApoE3 antibody at a 1:150 000 dilution (R&D Systems).

Techniques: Recombinant, Incubation, Western Blot, Transfection

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Human apolipoprotein E. The model structure illustrates the structural regions where deletions were made (deletion of helix 1, residues 1–40; helices 1 and 2, residues 1–71; C-terminal domain, 192–299; helices 6 and 7, 232–299; and helix 7, 272–299) and also shows the polymorphic site (residue 112) that distinguished apoE3 from apoE4. The structure was modified from apoE299_20K (S. Y. Sheu, unpublished data).

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Modification

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: SDS-PAGE analysis of the recombinant human apolipoprotein E. Proteins were analyzed on a gradient (4–12%) gel and stained with Coomassie blue. Lane 1, molecular mass markers; lane 2, apoE3; lane 3, apoE4; lane 4, apoE341-299; lane 5, apoE372-299; lane 6, apoE441-299; lane 7, apoE472-299; lane 8, apoE31-191; lane 9, apoE31-231; lane 10, apoE31-271; lane 11, apoE41-191; lane 12, apoE41-231; lane 13, apoE41-271.

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: SDS Page, Recombinant, Staining

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Sedimentation velocity experiments of apoE3. A fixed initial protein concentration at 0.50 mg/ml was used for the proteins. Analytical ultracentrifugation was performed at a rotor speed of 40,000 rpm, rotor temperature of 20°C in PBS (pH 7.3), and A280nm was scanned. The radial data collecting interval was 0.002 cm and time interval of scans was 480 s. (A) Circles represent the observed spectrum and solid lines are computer-generated results by fitting the experimental data to the Lamm equation with the SEDFIT program. (B) Grayscale of residual bitmap. (C) Residuals plotted as a function of radial position. (D and E) Continuous sedimentation coefficients distribution of the apoE3 proteins at the regularization of p = 0.95 and 0.68. (F) Continuous molar mass distribution of the apoE3 protein at the regularization of p = 0.95.

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Sedimentation, Protein Concentration, Generated

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Sedimentation analysis of full-length and N-terminal truncated apoE3 and apoE4 proteins

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Sedimentation

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Continuous distribution analysis of the apoE3 (A), apoE4 (B), apoE341-299 (C), apoE441-299 (D), apoE372-299 (E), and apoE472-299 proteins (F) at different concentrations. Experiments were conducted at an initial protein concentration of 0.15 (dotted line), 0.50 mg/ml (A and B, solid line; C–F, dashed line), or 1.00 mg/ml (solid line) in PBS (pH 7.3) at 20°C, rotor speed 40,000 rpm. Data were collected at time intervals of 480 s. The confidence level of the regularization after fitting was p = 0.68. (Insets) Grayscale of residual bitmap.

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Protein Concentration

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Size distribution analysis of full-length and N-terminal truncated apoE3 and apoE4 proteins by serial integration

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Concentration Assay

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Continuous distribution analysis of C-terminal truncated apoE3 (A and B) and apoE4 fragments (C and D). Experiments were conducted in PBS (pH 7.3) at 20°C, rotor speed 40,000 rpm. The left panels, continuous sedimentation coefficient distribution, c(s), and the right panels, continuous molar mass distribution, c(M), of the truncated apoE3 and apoE4 proteins (0.15 mg/ml). Solid lines, apoE1-191; dashed lines, apoE1-231; and dotted lines, apoE1-271. The confidence level of the regularization for 1–191 and 1–231 fragments was 0.95 and for 1–271 was 0.68. (Insets) Grayscale of residual bitmap.

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Sedimentation

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: CD spectra of full-length and truncated apoE proteins. The spectra were measured in PBS (pH 7.3). In panel A, closed circles, apoE3; open circles, apoE4; closed triangles, apoE341-299; open triangles, apoE441-299; closed squares, apoE372-299; open squares, apoE472-299. In panel B, closed triangles, apoE31-191; open triangles, apoE41-191; closed diamonds, apoE31-231; open diamonds, apoE41-231; closed hexagons, apoE31-271; open hexagons, apoE41-271.

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques:

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Secondary structures of full-length and truncated apoE3 and apoE4 proteins analyzed by CD spectroscopy

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques:

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: Binding of full-length and truncated apoE isoform proteins with ANS as a fluorescence probe. A fixed concentration of ANS (250 μM) and proteins (3 μM) was used. Excitation wavelength was set at 395 nm. The minimum fluorescence of ANS in PBS (pH 7.3) at 25°C was shown by dotted lines. In panel A, closed circles, apoE3; open circles, apoE4; closed squares, apoE341-299; open squares, apoE441-299; closed triangles, apoE372-299; open triangles, apoE472-299. In panel B, closed triangles, apoE31-191; open triangles, apoE41-191; closed diamonds, apoE31-231; open diamonds, apoE41-231; closed hexagons, apoE31-271; open hexagons, apoE41-271.

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Binding Assay, Fluorescence, Concentration Assay

Journal: Biophysical Journal

Article Title: Structural Variation in Human Apolipoprotein E3 and E4: Secondary Structure, Tertiary Structure, and Size Distribution

doi: 10.1529/biophysj.104.046813

Figure Lengend Snippet: The ANS fluorescence emission spectrum analysis

Article Snippet: The apoE3 41-299 , apoE3 72-299 , apoE3 1-191 , apoE3 1-231 , and apoE3 1-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega).

Techniques: Fluorescence