Journal: Journal of Neuroinflammation

Article Title: The Alzheimer’s disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway

doi: 10.1186/s12974-017-0835-4

Figure Lengend Snippet: Signal transduction mechanism and detection principle of the BWZ reporter assays. In the BWZ cells, TREM2 (shown in blue ) is expressed in a single chimeric protein containing CD8 transmembrane domain (shown in green ) and a CD3ζ domain (shown in purple and pink ), with an extracellular FLAG-tag (shown in brown ). When a TREM2 ligand, here ApoE, binds to TREM2 ( a ), the CD3ζ ITAM motifs (shown in purple ) are phosphorylated ( b ), which leads to an intracellular signaling cascade resulting in an increased intracellular Ca 2+ concentration. This induces the NFAT promoter, so that β-galactosidase is expressed from the lacZ gene ( c ). β-galactosidase is released from the cells using a lysis buffer containing saponin ( d ). The yellow β-galactosidase substrate, CPRG, is also present in the lysis buffer, and when cleaved by β-galactosidase, it forms a red product which is measured by absorbance ( e )

Article Snippet: Saponin (#558255) and polyclonal goat anti-ApoE antibody (#178479) were from Merck Millipore (Darmstadt, Germany).

Techniques: Transduction, FLAG-tag, Concentration Assay, Lysis

Journal: Journal of Neuroinflammation

Article Title: The Alzheimer’s disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway

doi: 10.1186/s12974-017-0835-4

Figure Lengend Snippet: Dose-dependent ApoE ε3-induced signal transduction in BWZ reporter cells expressing human TREM2. A dose-dependent stimulation of signal transduction in BWZ-hTREM2 cells was observed when cells were incubated in wells coated with an increasing concentration of recombinant ApoE ε3. Stimulation of BWZ-hTREM2 cells was compared to stimulation of BWZ cells instead transfected with TIM2 (BWZ-TIM2). All cells were stimulated with PMA (3.3 nM). As a positive control of BWZ-hTREM2 stimulation, cells were incubated with a rat anti-TREM2 antibody (“α-T2”) in solution in wells coated with an anti-rat antibody. As a positive control for TIM2 stimulation, a rat anti-TIM2 antibody (“α-TIM2”) was used in conjunction with coating wells with anti-rat antibody similarly as for the BWZ-hTREM2 cells. Negative controls for both cell lines included cells stimulated only with PMA (“PMA only”), or cells growing in anti-rat coated wells (“coating only”). The columns depict mean ± SEM, and reflect data from three individual experiments with triplicates

Article Snippet: Saponin (#558255) and polyclonal goat anti-ApoE antibody (#178479) were from Merck Millipore (Darmstadt, Germany).

Techniques: Transduction, Expressing, Incubation, Concentration Assay, Recombinant, Transfection, Positive Control

Journal: Journal of Neuroinflammation

Article Title: The Alzheimer’s disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway

doi: 10.1186/s12974-017-0835-4

Figure Lengend Snippet: The three human ApoE isoforms serve as agonists for human and murine TREM2. Human ApoE ε2 ( inverted green triangle ), ε3 ( light blue diamond ), and ε4 ( red triangle ) all stimulated human ( a ) and murine ( b ) TREM2 receptor signal transduction in a dose-dependent manner. EC 50 for human ApoE was 27, 33, and 34 nM for ApoE ε2, ε3, and ε4, respectively. EC 50 for murine TREM2 was 40, 54, and 39 for ApoE ε2, ε3, and ε4, respectively. The points depict mean ± SEM of all responses measured in triplicates from five (human TREM2) and three (murine TREM2) independent experiments. Data from each ApoE isoform was normalized individually as described in the “ ” section. The scale of the x -axis is logarithmic and shows concentration of human ApoE in M

Article Snippet: Saponin (#558255) and polyclonal goat anti-ApoE antibody (#178479) were from Merck Millipore (Darmstadt, Germany).

Techniques: Transduction, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: The Alzheimer’s disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway

doi: 10.1186/s12974-017-0835-4

Figure Lengend Snippet: High-affinity binding of recombinant human ApoE ε2, ε3, and ε4 to recombinant human TREM2. In an ELISA-based binding assay with recombinant human TREM2 and recombinant human ApoE, ApoE ε2 ( inverted green triangle ), ApoE ε3 ( light blue diamond ), and ApoE ε4 ( red triangle ) isoforms bound to human TREM2 in vitro with K d of 13, 16, and 9.5 nM, respectively. The points depict mean ± SEM of all responses that were measured in triplicates from three independent experiments. Data from each ApoE isoform was normalized individually as described in the “ ” section. The x -axis shows the ApoE concentrations (in M) on a logarithmic scale

Article Snippet: Saponin (#558255) and polyclonal goat anti-ApoE antibody (#178479) were from Merck Millipore (Darmstadt, Germany).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Journal of Neuroinflammation

Article Title: The Alzheimer’s disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway

doi: 10.1186/s12974-017-0835-4

Figure Lengend Snippet: An ApoE-mimetic peptide (amino acids 130–149) reduced binding of human ApoE to human TREM2. An ApoE-mimetic (amino acids 130–149) or a scrambled peptide was allowed to compete with ApoE for binding to immobilized recombinant human TREM2 in the ELISA-based assay. This led to reduced binding of ApoE, isoforms ε2 ( inverted green triangle ), ε3 ( light blue diamond ), and ε4 ( red triangle ), to human TREM2 with K i of 827, 475, and 1537 nM for ApoE ε2, ε3, and ε4, respectively. The points depict mean ± SEM of binding measured in duplicates from four independent experiments. The data was normalized to maximum (100%), which is binding of ApoE in the absence of a competing peptide, and minimum (0%), which is signal in the absence of ApoE as well as a TREM2-coat. The x -axis shows ApoE-mim149 or scrambled peptide concentration (in M) on a logarithmic scale

Article Snippet: Saponin (#558255) and polyclonal goat anti-ApoE antibody (#178479) were from Merck Millipore (Darmstadt, Germany).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Apolipoprotein E4 Effects a Distinct Transcriptomic Profile and Dendritic Arbor Characteristics in Hippocampal Neurons Cultured in vitro

doi: 10.3389/fnagi.2022.845291

Figure Lengend Snippet: Schema of the experimental design. (A) Harvesting of native apoE lipoproteins. Immortalized Apoe –/– astrocytes, and astrocytes homozygous for human APOE ε 2 , ε 3 , and ε 4 alleles are grown in the serum free DMEM/F-12 medium supplemented by 25-hydroxycholesterol for 48 h. Conditioned media are collected and 10-fold concentrated using 10 kDa MWCO Amicon Centrifugal Filter Units. ApoE level is verified by ELISA and balanced out by adding 10-fold concentrated media from Apoe –/– astrocytes if needed. ApoE lipoprotein preparations or control media (10-fold concentrated Apoe –/– conditioned media) are added to cultured neurons. (B) Treatment of primary hippocampal neurons with apoE lipoproteins. Primary hippocampal neuronal cultures are established from P0–P2 pups of CD1 mice in aseptic conditions. At 10 DIV and again at 13 DIV the control media or apoE lipoproteins are added. At 17 DIV the experiment is concluded and the neurons are subjected to morphological, biochemical, or transcriptomic analyses. The gradual development of dendritic arbor during in vitro maturation is schematically depicted in (B) . The diagram was created with the aid of BioRender.com .

Article Snippet: ApoE level was determined by sandwich ELISA utilizing HJ15.7 mouse monoclonal antibody (mAb) anti human apoE ( ) as the capture antibody (0.1 μg/mL) (gift of D. M. Holtzman) and biotinylated goat anti-human apoE polyclonal antibody (1:5,000) (Meridian Life Science, Inc., Memphis, TN, United States) as the detection antibody ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Cell Culture, In Vitro

Journal: Frontiers in Aging Neuroscience

Article Title: Apolipoprotein E4 Effects a Distinct Transcriptomic Profile and Dendritic Arbor Characteristics in Hippocampal Neurons Cultured in vitro

doi: 10.3389/fnagi.2022.845291

Figure Lengend Snippet: Characterization of the native apoE lipoprotein particles and their uptake by hippocampal neurons in vitro . Shown are Western blot analysis (A) and densitometric quantification (B) of apoE lipoproteins from astrocytic conditioned media, which are resolved under native conditions. The control denotes conditioned media from Apoe –/– astrocytes. The left and the right panel in (B) represent densitometric quantification of apoE protein band optical densities above and below the 242 kDa molecular weight marker, which is arbitrary used to discern large and small apoE lipoprotein particles, respectively. Whilst apoE2 and apoE3 signal dominates above the 242 kDa marker the apoE4 signal is localized mainly below it, what indicates reduced lipidation of apoE4 compared to apoE2 and apoE3. Shown is Western blot analysis of the apoE protein from the conditioned media of primary hippocampal neurons (C) and from the neuronal lysate (D) , which are resolved under reducing conditions. Neurons were treated with the control media (from Apoe –/– astrocytes) or indicated apoE lipoproteins. The conditioned media were sampled approximately 20 min. after adding the first dose of apoE lipoproteins, while neuronal lysates are from 17 DIV neurons collected at the conclusion of the experiment. Included also is Western blot analysis of GAPDH, used as the loading control for the Western blotting analysis of neuronal lysate. Shown are ELISA measures of apoE level in the neuronal media (E) and in the neuronal lysates (F) , which were sampled at the beginning and at the conclusion of the experiment, respectively. Consistently with the Western analysis, the ELISA measurements show comparable level of all three apoE isoforms in the neuronal media at the beginning of the experiment and elevated level of intraneuronal apoE4 compared to apoE2 and apoE3 at the conclusion of the experiment. Values in (B) , represent mean and SEM from five independent experiments, while those in (E,F) represent mean and SEM from eight independent experiments. (B,E,F) p < 0.0001 (ANOVA); * p < 0.05, *** p < 0.001, and **** p < 0.0001 (Holm’s–Sidak’s post hoc test).

Article Snippet: ApoE level was determined by sandwich ELISA utilizing HJ15.7 mouse monoclonal antibody (mAb) anti human apoE ( ) as the capture antibody (0.1 μg/mL) (gift of D. M. Holtzman) and biotinylated goat anti-human apoE polyclonal antibody (1:5,000) (Meridian Life Science, Inc., Memphis, TN, United States) as the detection antibody ( ).

Techniques: In Vitro, Western Blot, Control, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay