Journal: Alzheimer's & Dementia

Article Title: APOE Christchurch‐mimetic therapeutic antibody reduces APOE‐mediated toxicity and tau phosphorylation

doi: 10.1002/alz.13436

Figure Lengend Snippet: Anti‐ApoE3‐GAG antibody screening for top candidates. (A‐D) Screening of hybridoma cell supernatants via ELISA to test binding to peptides representing HSPG‐binding domains of ApoE3 (A), full‐length (FL) ApoE3 (B), ApoE3Ch peptide of ApoE3‐HSPG domain (C), and FL ApoE3Ch (D). Data show that all clones bind to HSPG region (ApoE peptide) with similar affinity (A) compared to binding between clones to the FL protein, in which clone 7C3.Ab (light purple) was the strongest, followed by 7C11.Ab (cyan), 1D6.Ab (green), 1H4.Ab (red), and 3A6.Ab (purple). Conversely, none of the antibodies tested showed binding for ApoE3Ch peptide (C) or FL ApoE3Ch (D), confirming the selectivity for the HSPG‐binding region of ApoE3. Binding expressed as averaged binding percentage normalized to the maximum intensity of absorbance as a function of logarithm of dilution factor ± SEM of three repeated measures. (E and F) Binding analysis via ELISA of purified monoclonal antibodies 7C11.mAb (E) and 1H4.mAb (F), using either FL protein (ApoE3 in purple and ApoE3Ch in red) or the amino acid sequence of the HSPG‐binding region (ie, peptide) of both ApoE3 (in green) and ApoE3Ch (in cyan). Data show differential binding for clones for either full‐length or small peptides of ApoE3 and ApoE3Ch. Binding analyses shown in panels (E) and (F) are expressed as averaged binding percentage normalized to the maximum intensity of absorbance over logarithmic nanomolar concentration (Log C) ± SEM of n = 3 repeated measurements.

Article Snippet: Clear polystyrene microplates (R&D Systems, Minneapolis, MN, USA; Catalog No. DY990) were coated with 0.0025 μg/μL of recombinant ApoE variants (ApoE2, ApoE3, ApoE4, ApoE3Ch; Innovagen) prepared in 2 mM CaCl 2 20 mM Tris Buffer saline (TBS‐C) and incubated for 16 h at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Clone Assay, Purification, Sequencing, Concentration Assay

Journal: Alzheimer's & Dementia

Article Title: APOE Christchurch‐mimetic therapeutic antibody reduces APOE‐mediated toxicity and tau phosphorylation

doi: 10.1002/alz.13436

Figure Lengend Snippet: Assaying selectivity of anti‐ApoE‐GAG antibodies for ApoE variants of the novel ApoE antibodies. Titration of anti‐ApoE3 antibodies 7C11 (A), 1H4 (B), a commercially available anti‐ApoE antibody clone D6E10 (C), and a commercially available antibody anti‐His tag antibody (D). The assay was performed using a constant concentration of recombinant His‐tagged ApoE variants (ApoE2 in black; ApoE3Ch in cyan; ApoE3 in red; ApoE4 in purple). Data are expressed as binding percentage normalized to the maximum absorbance over logarithmic nM concentration and suggests that the 7C11 and 1H4 anti‐ApoE3‐HSPGs antibodies are more selective for ApoE2 and ApoE4 variants. (A‐D) All data are expressed as average binding percentage obtained from three independent experiments. (E‐H) Biolayer interferometry (BLI) binding analysis of ApoE3 (E), ApoE4 (F), ApoE2 (G), ApoE3Ch (H), and to immobilized 7C11 antibody to an AMC chip showing that 7C11 has the highest binding affinity for ApoE4, whereas it was not possible to measure binding affinity to ApoE3Ch. n.m. ≡ not measurable.

Article Snippet: Clear polystyrene microplates (R&D Systems, Minneapolis, MN, USA; Catalog No. DY990) were coated with 0.0025 μg/μL of recombinant ApoE variants (ApoE2, ApoE3, ApoE4, ApoE3Ch; Innovagen) prepared in 2 mM CaCl 2 20 mM Tris Buffer saline (TBS‐C) and incubated for 16 h at 4°C.

Techniques: Titration, Concentration Assay, Recombinant, Binding Assay

Journal: Alzheimer's & Dementia

Article Title: APOE Christchurch‐mimetic therapeutic antibody reduces APOE‐mediated toxicity and tau phosphorylation

doi: 10.1002/alz.13436

Figure Lengend Snippet: Affinity and in silico analysis of 7C11.mAb antibody. (A, B) Representative chromatograms of ApoE3 1.47 μM (magenta) and ApoE3 1.47 μM incubated with 7C11.mAb 0.6 μM (blue, A) or ApoE4 1.47 μM (purple) and ApoE4 1.47 μM incubated with 7C11.mAb 0.6 μM (light blue, B). Data show that 0.6 μM concentration of 7C11.mAb has a strong inhibitory effect on the heparin binding of ApoE3 and ApoE4, as confirmed by the shift of pmax at lower retention times and reduction of the peak intensity as compared to ApoE alone. All chromatograms are expressed as normalized intensities to the maximum intensity of emission over time in minutes (min) and are representative of n = 3 independent measurements. To compare the changes in ApoE, the 7C11.mAb chromatographic profile was subtracted. The percentage of 0.8 M NaCl over the salt gradient is represented by the dotted line. (C) BLI binding analysis of ApoE4 to immobilized 7C11.chIgG1 antibody to an anti‐human IgG Fc (AHC) chip showing that 7C11.chIgG1 binding affinity for ApoE4 with a K D < 10 ‐12 M. (D) Binding analysis via surface plasmon resonance (SPR) of the Fab fragment of the 7C11.mAb antibody expressed as response difference over time of increasing concentrations of ApoE4 tested with immobilized Fab from antibody 7C11.mAb. (E, F) Representative in silico docking analysis of 7C11‐Fab antibody (in purple) to ApoE3 (E) or ApoE3Ch (F) N‐terminal region (in magenta). Polar contacts between the antibody and ApoE are represented in cyan. ApoE sequence for both ApoE3 and ApoE3Ch used for in silico analysis is reported next to the structures and highlighted in magenta; polar contacts within the sequence are highlighted in cyan.

Article Snippet: Clear polystyrene microplates (R&D Systems, Minneapolis, MN, USA; Catalog No. DY990) were coated with 0.0025 μg/μL of recombinant ApoE variants (ApoE2, ApoE3, ApoE4, ApoE3Ch; Innovagen) prepared in 2 mM CaCl 2 20 mM Tris Buffer saline (TBS‐C) and incubated for 16 h at 4°C.

Techniques: In Silico, Incubation, Concentration Assay, Binding Assay, SPR Assay, Sequencing

Journal: Alzheimer's & Dementia

Article Title: APOE Christchurch‐mimetic therapeutic antibody reduces APOE‐mediated toxicity and tau phosphorylation

doi: 10.1002/alz.13436

Figure Lengend Snippet: Introducing the Christchurch (Ch) mutation in ApoE4 or mimicking reduced GAG binding rescues cytotoxicity in vitro and tau pathology in vivo. (A) Cytotoxicity percentage of increasing concentrations of ApoE4 (purple) and ApoE4Ch (blue) used to treat for 24 h SH‐SY5Y cells showing the loss of toxicity via LDH assay in the presence of the Ch mutation. (B) Cytotoxicity assay of increasing doses of 7C11 human chimeric antibody (7C11.chIgG1) showing that it is not cytotoxic compared to IgG1 control. (C) Cytotoxicity assay comparing 1 μM ApoE4‐treated cells to 1 μM ApoE4 co‐administered with either 7C11 human chimeric antibody (7C11.chIgG1 6.7 nM) or heparin 13 nM showing that ApoE4‐derived cytotoxicity is significantly reduced in vitro (* p = .0147, unpaired two‐tailed t ‐test, n = 4 biological replicates). Neither heparin nor 7C11 were cytotoxic. Data were normalized by considering ApoE4 cytotoxicity as 100%. (D) Representative far‐western blotting of ApoE3 and ApoE4 showing that in the absence of heparin (left blots), 7C11 binds more to ApoE compared to blots probed in the presence of heparin (right blots). Total levels of ApoE were not affected when membranes were probed with anti‐ApoE antibody (botton blots). (E) Pictographic representation of experimental design of in vivo intravitreal injections of ApoE variants or vehicle or ApoE3 + 7C11.mAb performed on day 1, euthanasia and retina dissection from the collected eyes on day 3, and staining on day 4 after injection. (F) Representative immunofluorescence staining of dissected retina injected intravitreally with 2 μL of vehicle (PBS), ApoE3 1.47 μM, or ApoE3 1.47 μM + 7C11.mAb 6 μM, showing reduction of retinal damage and inflammation, as shown by increased levels of isolectin staining (green) and AT8‐tau staining (in red) in the presence of 7C11.mAb antibody. For all images, the top panel is a composite of staining obtained with DAPI to detect nuclei (blue) and AT8‐tau to detect hyperphosphorylated tau accumulation (AT8, red), isolectin B4 (green) to detect vasculature. The red channel is also displayed in the bottom panel. Scale bar = 50 μm. (G) Quantification of the relative AT8‐positive tau levels normalized to retinal ganglion cell (RGC) area in ApoE3 and ApoE3 + 7C11.mAb‐treated retinas showing a significantly reduced AT8‐tau staining in the presence of 7C11.mAb compared to ApoE3 alone (** p = .0042, unpaired two‐tailed t ‐test, n = 3, 4 biological replicates).

Article Snippet: Clear polystyrene microplates (R&D Systems, Minneapolis, MN, USA; Catalog No. DY990) were coated with 0.0025 μg/μL of recombinant ApoE variants (ApoE2, ApoE3, ApoE4, ApoE3Ch; Innovagen) prepared in 2 mM CaCl 2 20 mM Tris Buffer saline (TBS‐C) and incubated for 16 h at 4°C.

Techniques: Mutagenesis, Binding Assay, In Vitro, In Vivo, Lactate Dehydrogenase Assay, Cytotoxicity Assay, Derivative Assay, Two Tailed Test, Far Western Blot, Dissection, Staining, Injection, Immunofluorescence

Journal: Alzheimer's & Dementia

Article Title: APOE Christchurch‐mimetic therapeutic antibody reduces APOE‐mediated toxicity and tau phosphorylation

doi: 10.1002/alz.13436

Figure Lengend Snippet: In vivo and human neuropathology characterization of new anti‐ApoE‐HSPG antibodies. (A) Pictographic representation of experimental design of intraperitoneal (ip) injections of WT and APOE4 KI mice to determine brain penetration of Alexa‐647‐labeled antibodies (day 1, 50 mg/kg) or WT and APOE4 KI mice to determine changes in pTau levels (days 1 to 4). (B) Representative staining of hippocampal regions of APOE4 KI mice ip injected with either vehicle (PBS), ms.IgG‐Alexa647, or ms.7C11‐Alexa647 for 24 h, 50 mg/kg. Nuclei are stained with DAPI in blue. Scale bar = 200 μm. (C) Image analysis of injected brains shown in panel (B) was performed by measuring fluorescence intensity (FI) on 647‐nm channel. Higher FI of both ms.IgG1‐Alexa647 ( p < .001, one‐way ANOVA) and 7C11.mAb‐Alexa647 ( p < .001, one‐way ANOVA) injected groups was observed compared to vehicle. (D) Representative images of DAPI and pTau (S396) staining in APOE4 KI mice injected ip with either ms.IgG1 or 7C11.mAb, compared to negative control (secondary only antibody). Scale bar = 50 μm. (E) Representative coronal section stained with DAPI showing with a red square the anatomical region selected for the analysis presented in panel (F). Scale bar = 1 mm. (F) APOE4 KI mice treated with 7C11 antibody showed a reduced number of pTau (S396)‐positive cells ( p = .0286, one‐tailed Mann‐Whitney test). (B, D, E) Nuclei are stained with DAPI in blue. (G) Representative immunohistochemistry staining of ApoE levels on paraffin‐embedded temporal cortex specimens from PSEN1 E280A carriers with different APOE genotypes: APOEε3/ε3 (first row), APOE ε3/ε3Ch (second row), APOE ε3Ch/ε3Ch (third row), APOE ε4/ε4 (last row). ApoE staining performed (from left to right) with anti‐ApoE‐E6D7, 1H4.mAb, 7C11.mAb, and 19G.mAb. Comparison of different staining patterns suggesting that, unlike the anti‐ApoE‐E6D7, which homogeneously stains ApoE variants throughout the tissue, 1H4.mAb preferentially stains ApoE3 and ApoE4 located either in the neuronal tissue or surrounding the vessels; 7C11.mAb preferentially detects ApoE3 and ApoE4 localized in deposits and surrounding the vessels, and 19G.mAb selectively stains ApoE3Ch. For each specimen analyzed, the region of the cerebral cortex used for the staining is reported in the bottom left boxes. Scale bar = 100 μm. Regions of interest in panels (B), (D), (G) are pointed with triangles.

Article Snippet: Clear polystyrene microplates (R&D Systems, Minneapolis, MN, USA; Catalog No. DY990) were coated with 0.0025 μg/μL of recombinant ApoE variants (ApoE2, ApoE3, ApoE4, ApoE3Ch; Innovagen) prepared in 2 mM CaCl 2 20 mM Tris Buffer saline (TBS‐C) and incubated for 16 h at 4°C.

Techniques: In Vivo, Labeling, Staining, Injection, Fluorescence, Negative Control, One-tailed Test, MANN-WHITNEY, Immunohistochemistry, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Expression Pattern of the Aspartyl-tRNA Synthetase DARS in the Human Brain

doi: 10.3389/fnmol.2018.00081

Figure Lengend Snippet: DARS expression in the adult human brain. (A,B) Levels of DARS (A) and DARS2 (B) mRNA across different brain regions [CX, motor cortex; HC, hippocampus; BS, brainstem (pons); CB, cerebellum; ST, striatum; CC, corpus callosum] determined by qRT-PCR. DARS and DARS2 mRNA were normalized to the housekeeping gene β-Actin ( n = 5). (C) Western-blot detection of DARS in lysates from HEK 293 cells ectopically expressing human DARS including an N-terminal FLAG-HA-tag or a control plasmid. DARS protein was detected with a monoclonal mouse antibody from SantaCruz (mαDARS), or a polyclonal rabbit antibody from Novus Biologicals (rbαDARS), or an HA antibody. β-Actin served as internal standard. (D) Representative Western-blot depicting the expression of DARS, β-Actin, and GAPDH across different brain regions. (E) Levels of DARS protein (detected with mαDARS) normalized to β-Actin ( n = 5). Data represent mean ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA).

Article Snippet: Slides were incubated overnight at 4°C with the following primary antibodies: mαDARS (1:50; SantaCruz no. sc-393275), rbαNeuN (1:40; Cell Signaling no. 12943S), rbαASPA (1:300; ), rbαGFAP (1:300; DAKO no. Z0334), and rbαIba1 (1:300; Wako no. 019-19741).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation

Journal: eLife

Article Title: Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

doi: 10.7554/eLife.86920

Figure Lengend Snippet:

Article Snippet: Antibody , α-ApoE4 (mouse monoclonal) , Cell Signaling, Danvers, MA , (4E4) #2208 RRID: AB_2238543 , Detects HDL (mobile protein) (1:1000).

Techniques: Variant Assay, Expressing, Knock-Out, Control, Transfection, Construct, Plasmid Preparation, Recombinant, Functional Assay, Software, Western Blot, Derivative Assay, Binding Assay, Immunoprecipitation