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: bioRxiv

Article Title: Alzheimer’s disease risk factor APOE4 exerts dimorphic effects on female bone

doi: 10.1101/2025.10.16.682922

Figure Lengend Snippet: A ) Liquid chromatography coupled to data-independent acquisition-mass spectrometry (LC-DIA-MS) was performed on decalcified, guanidine (Gu-HCl) extracted, trypsin digested cortical bone samples from aged (15-month-old) female APOE2/3/4 mice. Protein groups were identified (1892) with 2+ unique peptides detected per protein. B ) PLS-DA analysis of the APOE bone proteomes showed separation of groups by APOE allele status. C ) Differential pair-wise regulation of proteins by APOE allele (absolute Log 2 (FC) > 0.58, Q < 0.05) showed that APOE4 drove the largest differences in protein expression when comparing genotypes. D ) The volcano plots depict the largest number of differentially regulated proteins occurring between APOE4 and APOE3 where 380 proteins were significantly upregulated in APOE4 animals and only 13 proteins were downregulated. E ) Proteomic analysis performed on the hippocampal tissue from these same animals resulted in far fewer pairwise regulated proteins, but APOE4 consistently upregulated ECM and glycoprotein binding proteins hippocampus F ) IPA of differentially regulated bone proteins from each pairwise comparison resulted in one predicted regulated pathway, neutrophil extracellular trap signaling, in APOE2 vs. APOE3 , and a mix of predicted enriched activated and repressed pathways from APOE4 vs. APOE2 including activation of neutrophil pathways. However, predicted pathways in APOE4 vs . APOE3 comparison were almost uniformly activated, including metabolic functions such as oxidative phosphorylation, electron transport, and the citric acid cycle, except the notable downregulation of mitochondrial dysfunction.

Article Snippet: 15-month-old male and female humanized APOE knock-in ( APOE2 , APOE3 , APOE4 ; Taconic Biosciences, B6.129P2-Apoetm1(APOE*2/3/4)MaeN9) carrying homozygous human ε2, ε3, or ε4 alleles were maintained at the Buck Institute for Research on Aging (IACUC protocol #2023-0018).

Techniques: Liquid Chromatography, Data-independent acquisition, Mass Spectrometry, Expressing, Binding Assay, Comparison, Activation Assay, Phospho-proteomics