Journal: Nature

Article Title: Astrocyte-derived interleukin-3 reprograms microglia and limits Alzheimer’s disease

doi: 10.1038/s41586-021-03734-6

Figure Lengend Snippet: a. Representative immunofluorescence images of the cortex of control subjects (C) or Alzheimer’s disease (AD) patients stained for IL-3 and GFAR b. Measurement of IL-3 levels in cortex tissue homogenates from control and AD patients (n=15 controls, n=23 AD patients, see Extended Data Table S6 for characteristics). c. Representative immunofluorescence images of the cortex of humans with or without Alzheimer’s disease (AD) stained for IL-3Rɑ and Iba1. d. qPCR analysis of fold change in IL3Rɑ expression in the frontal cortex of control non-demented individuals and AD patients (n=28 controls, n=30 AD patients, see Extended Data Table S7 for characteristics). e. Assessment of brain IL3Rɑ expression with APOE genotype (n=30 AD patients). f. Correlation of mean IL3Rɑ expression with years (yrs) of disease duration (n=30 AD patients). Correlation of IL3Rɑ expression with formic acid (FA)-soluble Aβ40 (g) and Aβ42 (h) in the frontal cortex of AD patients (n=23 AD patients). *p<0.05, **p<0.01, ***p<0.001. Open circles represent control subjects and red circles represent AD patients. Error bars indicate mean ± SEM.

Article Snippet: Quantitative real-time TaqMan PCR was performed using the following FAM labelled TaqMan primers (Applied Biosystems): Il3 (Mm00439631_m1), Il3rα (Mm00434273_m1), Ccl2 (Mm00441242_m1), Complement C3 (Mm01232779_m1), Gfap (Mm01253033_m1), Ccl7 (Mm00443113_m1), Ccl5 (Mm01302427_m1), Il1β (Mm00434228_m1), Tnfα (Mm00443258_m1), Il6 (Mm00446190_m1), IL10 (Mm01288386_m1), Ccl12 (Mm01617100_m1), Trem2 (Mm04209424_g1), Syk (Mm01333032_m1), Tyrobp (Mm00449152_m1), Cd33 (Mm00491152_m1), Cd36 (Mm00432403_m1), Tlr4 (Mm00445273_m1), Sra (Mm00491755_m1), Cd206 (Mm01329362_m1), Mpp9 (Mm00442991_m1), Spp1 (Mm00436767_m1), Clec7a (Mm01183349_m1), Lyz2 (Mm04214174_uH), Apoe (Mm01307192_m1), Itgax (Mm00498708_g1), Itgam (Mm00434455_m1), Ptprc (Mm01293577_m1), Ctsg (Mm00456011_m1), Igf1 (Mm00439560), Cd68 (Mm03047343_m1).

Techniques: Immunofluorescence, Control, Staining, Expressing

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 1. APOE accumulates in the MAMs of cultured hepatocytes, equally evident during APOE3 and APOE4 overexpression: (a) subcellular fractions were isolated from Huh7 cells by Percoll density gradient ultracentrifugation and analyzed by Western blotting. Representative images show the accumulation of the APOE protein in pure MAMs. Respective marker proteins were detected to visualize the purity of mitochondria (COX), MAMs (CANX) and cytosol (TUB); (b) APOE protein levels were quantified in the whole cell sample and crude and pure MAM fractions and normalized to the APOE level in the whole cell sample. In the pure MAM, the APOE protein level was significantly higher compared to the whole cell (p < 0.05, unpaired t-test) as indicated by an asterisk (*). The data are means ± SEM (n = 3); (c) no APOE isoform-dependent difference was observed in the accumulation of APOE in the pure MAM fraction in APOE3- and APOE4-transfected Huh7 cells. Data are means ± SEM (n = 2) and the accumulation of APOE in MAMs is related to the APOE protein level in the whole cell samples; subcellular fractions: c. mito., crude mitochondria; p. mito., pure mitochondria; c. MAM, crude mitochondria ER-membranes (MAMs), p. MAM, pure MAM.

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Over Expression, Isolation, Western Blot, Marker, Transfection

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 2. The number of visualized MERCs in cultured hepatocytes and MAM-assembling protein levels in the mouse liver are similar in presence of APOE3 and APOE4: (a) representative images of MERC-PLA experiments in APOE-transfected Huh7 cells that were incubated with 1 or 5 g/L glucose for six hours. VDAC1-IP3R1 PLA signals are shown in green, APOE was additionally stained in red to identify successfully transfected cells, and cell nuclei appeared blue by staining with DAPI. 400× magnification; scale bar 5 µm; (b) PLA signals from at least 100 cells per sample from three independent experiments were quantified showing a significant reduction of PLA signals per cell in all samples in response to the glucose challenge. No difference was observed comparing APOE3-, APOE4- and mock- transfected cells. Significance was accepted at p < 0.05, indicated with an asterisk (*); a two-way ANOVA was performed followed by the Šídák’s multiple comparisons test; (c) MAM-assembling as well as ER and mitochondrial marker proteins were analyzed in the livers of APOE-targeted replacement mice by Western blotting and representative images are shown; (d) densitometric analysis revealed no significant differences between APOE3 and APOE4 mice (unpaired t-test). Target band intensity was normalized by total protein load and related to the mean of APOE3 mice. Data are means ± SEM (n = 5–6).

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Transfection, Incubation, Staining, Marker, Western Blot

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 3. Mitochondrial APOE-interacting proteins are remotely connected to MAMs, with no apparent difference comparing APOE3- and APOE4-transfected cells. (a) Visualization of the eleven higher abundant protein–protein interactions (PPIs) shared by APOE3- and APOE4-transfected cells compared to unmodified Huh7 cells. PPIs were categorized by their main cellular localization identified according to The Human Gene Database GeneCards. Hitherto unknown PPIs (7/11) are highlighted by the greater thickness and blue color of the borderline. (b) Representative Western blot images of the presence of the APOE-interacting proteins LONP1, TOMM40 and VDAC1 in subcellular fractions isolated from Huh7 cells. Respective marker proteins for mitochondrial and ER-related MAM proteins were detected (GRP75, mitochondria, MAM; and PEMT, ER and MAM). (c) Representative Western blot images showing APOE, LONP1, TOMM40 and VDAC1 as well as the ER/MAM marker CANX in subcellular fractions of APOE3- and APOE4-transfected cells. (d) Target band intensities were quantified and normalized by total protein load per lane. The target protein level in the pure MAM fraction was related to the whole cell sample showing similar results in APOE3- and APOE4-transfected cells. Data are means ± SEM (n = 2). Subcellular fractions: c. mito., crude mitochondria; p. mito., pure mitochondria; c. MAM, crude mitochondria ER membranes (MAMs), p. MAM, pure MAM.

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Protein-Protein interactions, Western Blot, Isolation, Marker

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 4. Presence in MAMs and extent of the PPI of selected candidates and APOE in ER-stressed cultured hepatocytes. (a) Western blot images of LONP1, TOMM40, VDAC1 and APOE detection in subcellular fractions of unmodified Huh7 cells. Thapsigargin treatment (50 µM, 24 h) provoked the accumulation of LONP1 and APOE in MAMs relative to the whole cell sample and compared to untreated control cells. GRP75 served as a marker for MAMs and the positive control for thapsigargin induced MAM protein translocation. c. MAM, crude mitochondria ER membranes (MAMs), p. MAM, pure MAM; (b) representative Western blot images of LONP1, TOMM40 and VADC1 in APOE co-IP samples from unmodified Huh7 cells treated with thapsigargin (50 µM, 24 h). No signals were visible in the IP negative control (no antibody used, no AB). S, IP supernatant, inp., input control; (c) target band intensity was normalized by the corresponding APOE band intensity. Relative target protein levels in thapsigargin-stressed cells were related to the mean of untreated cells (expressed as thapsigargin-induced change). Data are means from three individual cell culture experiments and co-IP (n = 3).

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Western Blot, Control, Marker, Positive Control, Translocation Assay, Co-Immunoprecipitation Assay, Negative Control

Journal: Cell

Article Title: Tissue Repair Signals and In Vitro Culture: Inflammatory Cytokine TNFα Promotes the Expansion of Primary Hepatocytes in 3D Culture

doi: 10.1016/j.cell.2018.11.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Taqman Probe Apoe , Applied Biosystem , Cat# 4331182 Mm01307193_g1.

Techniques: Virus, Recombinant, RNAscope, Reverse Transcription, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Expressing, Plasmid Preparation, Software, Cell Analysis

Journal: Cell

Article Title: Tissue Repair Signals and In Vitro Culture: Inflammatory Cytokine TNFα Promotes the Expansion of Primary Hepatocytes in 3D Culture

doi: 10.1016/j.cell.2018.11.012

Figure Lengend Snippet: Taqman™ q-PCR probes.

Article Snippet: Taqman Probe Apoe , Applied Biosystem , Cat# 4331182 Mm01307193_g1.

Techniques: Amplification

Journal: iScience

Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

doi: 10.1016/j.isci.2024.110006

Figure Lengend Snippet: Characterization of apoE particles (A) Schematic illustration of apoE particle purification by apoE antibody column. (B) Schematic illustration of apoE particle analysis by size exclusion chromatography (SEC). (C) ApoE particle ratios in different elution fractions were determined by ELISA. (D) ApoE particle sizes were analyzed by dynamic light scattering (DLS). (E) Representative images of astrocytic apoE particles (A-ApoE) and microglial apoE particles (M-ApoE) visualized with a transmission electron microscopy. Scale bar, 50 nm. (F) Quantification of size of apoE particles/group. At least 50 apoE particles were used for the analysis. Data are presented as mean ± SEM. Statistical significance was determined with unpaired Student’s t test. ∗∗∗, p < 0.001.

Article Snippet: ApoE particles absorbed grids were incubated with Rabbit apoE antibody (Cat# 68587, CST) for 40 min and then incubated with 6 nm Colloidal Gold AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson) for 20 min.

Techniques: Purification, Particle Size Analysis, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Alzheimer's disease : JAD

Article Title: The APOE-TOMM40 Humanized Mouse Model: Characterization of Age, Sex, and PolyT Variant Effects on Gene Expression

doi: 10.3233/JAD-230451

Figure Lengend Snippet: A) A comparison of the human and mouse homologous genomic regions encompassing TOMM40 and APOE and surrounding genes and intergenic sequences. The schematic describes the chromosomal location of the genes cluster in the mouse Chr. 7 (left) and the human chromosome 19 (right); Ideograms on the sides of the figure; a zoom-in views of the respective genomic regions containing the PVRL2, TOMM40, APOE, and APOC1 genes. Note the opposite orientations of the respective chromosomes. B) A genome browser view (GRCh37/hg19) of the human TOMM40-APOE locus overlapping the transgene sequence (humanized mouse insert sequence, track highlighted in green). Lower track, the position and gene structure, exon (blue boxes) and introns (blue lines) of the human TOMM40 and APOE.

Article Snippet: ABI MGB probe and primer set assays were used to amplify the human TOMM40 cDNA (ID Hs01587378_mH, 146bp) and APOE cDNA (ID Hs00171168_m1, 108bp); and the two RNA reference controls of the mouse endogenous, Gapdh (ID Mm99999915_g1, 109bp) and Ppia (ID Mm02342429_g1, 112bp) (Applied Biosystems).

Techniques: Comparison, Sequencing

Journal: Journal of Alzheimer's disease : JAD

Article Title: The APOE-TOMM40 Humanized Mouse Model: Characterization of Age, Sex, and PolyT Variant Effects on Gene Expression

doi: 10.3233/JAD-230451

Figure Lengend Snippet: A) The genomic region of mouse chromosome 7 encompassing Tomm40, Apoe and intergenic sequences (20.1 kb). B) The mouse genomic region after the exchange with an acceptor cassette containing Pgk-driven hygromycin and HSV-thymidine kinase genes flanked by heterologous loxP sites, wildtype loxP (red, located just downstream of the polyA site of Pvrl2 gene) and mutant lox2272 (magenta, located ~1 kb downstream of the last exon of the mouse Apoe gene). The cassette was flanked by a long arm at the 5’ end and a short arm at the 3’ end, and a CMV-driven diphtheria toxin gene (DT-TA) upstream of the long arm. C) The ‘humanized’ mouse genomic region resulted from successful targeted replacement carrying the hTOMM40-hAPOE genes, and the native human sequences upstream and downstream of these genes and the intergenic region. D) PCR analysis of ES cell clones after cassette exchange between targeting vector and acceptor cassette. ES of mouse wild type (lane A), ES with the acceptor cassette (lane B), ES heterozygotes for the ‘523’ polyT S allele (lanes C-F, upper band) together with the mouse native allele (lanes C-F, lower band).

Article Snippet: ABI MGB probe and primer set assays were used to amplify the human TOMM40 cDNA (ID Hs01587378_mH, 146bp) and APOE cDNA (ID Hs00171168_m1, 108bp); and the two RNA reference controls of the mouse endogenous, Gapdh (ID Mm99999915_g1, 109bp) and Ppia (ID Mm02342429_g1, 112bp) (Applied Biosystems).

Techniques: Mutagenesis, Clone Assay, Plasmid Preparation

Journal: Journal of Alzheimer's disease : JAD

Article Title: The APOE-TOMM40 Humanized Mouse Model: Characterization of Age, Sex, and PolyT Variant Effects on Gene Expression

doi: 10.3233/JAD-230451

Figure Lengend Snippet: Fold levels of human (h)APOE and hTOMM40 mRNA were assayed by real-time RT-qPCR and calculated relative to endogenous mouse Gapdh and Ppia expression using the 2−ΔΔCt method. A) The homozygous ‘523’ polyT S genotype was associated with significant elevated hAPOE-mRNA levels compared to the VL/VL genotype (p = 3.2x10−10; NVL =30; NS =51). B) Age was significantly associated with hAPOE-mRNA levels (p = 0.0040). Mice at 18 months (N18-month =14) expressed significantly more hAPOE than other age groups (N10-wks=13, N6-month=24, N12-month=24, N15-month=6). C) Mice homozygous for the ‘523’ polyT S allele demonstrated significantly higher hAPOE-mRNA levels at 18 months (N18-mooth=14) than other age groups (p = 0.042, 0.046 and 0.077; N10-wks=13, N6-month =12, N12-month=12, respectively). D) Mice homozygous for the ‘523’ polyT VL allele demonstrated a non-significant increase in hAPOE-mRNA levels with age (N6-month=12, N12-month =12, N15-month=6). E) The homozygous ‘523’ polyT S genotype was associated with significant elevated hTOMM40-mRNA levels compared to the VL/VL genotype (p = 6.9x10−5).

Article Snippet: ABI MGB probe and primer set assays were used to amplify the human TOMM40 cDNA (ID Hs01587378_mH, 146bp) and APOE cDNA (ID Hs00171168_m1, 108bp); and the two RNA reference controls of the mouse endogenous, Gapdh (ID Mm99999915_g1, 109bp) and Ppia (ID Mm02342429_g1, 112bp) (Applied Biosystems).

Techniques: Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Different risk and protective factors predict change of planning ability in middle versus older age

doi: 10.1038/s41598-024-76784-1

Figure Lengend Snippet: Overview of the assessed parameters and used instruments.

Article Snippet: Genotyping and imputation of Single Nucleotide Polymorphisms , KIBRA rs17070145 BDNF rs6265 DBH rs1611115 DRD1 rs4532 ANKK1 rs1800497 DRD2 rs6277 COMT rs4680 COMT rs4818 APOE rs429358 APOE rs7412 , Affymetrix Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) . APOE SNPs (rs429358 and rs7412) were aggregated accordingly to include the epsilon 2, 3 and 4 variants in the analyses. .

Techniques: Infection, Biomarker Discovery, Genome Wide

Journal: Scientific Reports

Article Title: Different risk and protective factors predict change of planning ability in middle versus older age

doi: 10.1038/s41598-024-76784-1

Figure Lengend Snippet: Overview of the SNPs of interest with the respective chromosomes, genes and the corresponding allele variants and the absolute number of observations.

Article Snippet: Genotyping and imputation of Single Nucleotide Polymorphisms , KIBRA rs17070145 BDNF rs6265 DBH rs1611115 DRD1 rs4532 ANKK1 rs1800497 DRD2 rs6277 COMT rs4680 COMT rs4818 APOE rs429358 APOE rs7412 , Affymetrix Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) . APOE SNPs (rs429358 and rs7412) were aggregated accordingly to include the epsilon 2, 3 and 4 variants in the analyses. .

Techniques: