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: The Journal of Biological Chemistry

Article Title: Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles *

doi: 10.1074/jbc.M115.669226

Figure Lengend Snippet: Factor H fragment FH5–7 interacted with serum apoE. A, in a pulldown assay, 0.2 mg of purified histidine-tagged FH5–7 protein (input) was first coupled on a HisTrap HP 1-ml column. After washing, the column was eluted with a salt gradient. An assay without FH5–7 coupling was used to control nonspecific binding of serum proteins to the column (Control). The collected fractions were run into SDS-PAGE gels under reducing conditions. An ∼34-kDa band in fractions eluted from the FH5–7 coupled column is seen in the sample but not in the control assay (0.8–1.8 m NaCl). This protein was identified as apoE by mass spectrometry analysis. B, for microscale thermophoresis analysis FH5–7 was incubated with labeled apoE2. The concentration of labeled ligand was 0.0013 μm, and the starting concentration for the unlabeled FH5–7 was 5 μm. The average of two experiments suggested a Kd value of 0.202 μm.

Article Snippet: Microscale Thermophoresis ApoE2 protein was labeled with an N -hydroxysuccinimide-reactive Red dye (catalog no. L001) following the manufacturer's instructions (NanoTemper).

Techniques: Purification, Control, Binding Assay, SDS Page, Mass Spectrometry, Microscale Thermophoresis, Incubation, Labeling, Concentration Assay