Journal: bioRxiv

Article Title: Development of a high-throughput, quantitative platform using human cerebral organoids to study virus-induced neuroinflammation in Alzheimer’s disease

doi: 10.1101/2024.03.21.585957

Figure Lengend Snippet: (A) UMAP plots showing the normalized fluorophore intensities for Aβ monomers using Solanezumab (red), HSV-1 (green) and Zombie fluorophore (blue) across uninfected, HSV-1 infected and ACV-treated cells. (B) Pairwise adjusted correlation heatmaps using the intensities for HSV-1, Zombie fluorophore and Solanezumab (Aβ monomers) across uninfected, HSV-1 infected and ACV-treated cells, shown for 2 sets of replicates (Replicate 1 and Replicate 2). (C) Boxen plots showing the normalized Aβ42 fluorophore intensities for uninfected cells (HSV-1 - cells) versus infected cells (HSV-1 + cells) within the same infected sample, as well as uninfected cells versus infected cells within the same ACV-treated sample. (D) Boxen plots showing the normalized Solanezumab fluorophore intensities for uninfected cells (HSV-1 - cells) versus infected cells (HSV-1 + cells) within the same infected sample, as well as uninfected cells versus infected cells within the same ACV-treated sample. (E) Concentrations of Aβ42/40/38 in pg/mL, Aβ42/40 ratios and Aβ42/38 ratios detected from conditioned media that had undergone heat inactivation for uninfected (control) dcOrgs, HSV-1 infected (HSV-1+) dcOrgs, HSV-1 infected and ACV-treated (Treated) dcOrgs, and dcOrgs with UV-inactivated HSV-1 (UV-HSV-1). P -values shown were calculated using 1-sided Wilcoxon ranked sum test with comparison to control uninfected dcOrgs. (F) Concentrations of Aβ42/40/38 in pg/mL, β42/40 ratios and Aβ42/38 ratios detected from conditioned media that had undergone heat inactivation for uninfected (control) dcOrgs and IAV infected (IAV+) dcOrgs. P -values shown were calculated using 1-sided Wilcoxon ranked sum test with comparison to control uninfected dcOrgs.

Article Snippet: The antibodies used in our study were: Alexa Fluor 647-conjugated Aβ1-42 (Bioss Antibodies bs-0107R-BF647) at a 1:50 dilution, Alexa Fluor 647-conjugated Tau (Thr212) (Bioss Antibodies bs-5420R-BF647) at a 1:50 dilution, Alexa Fluor 647-conjugated Solanezumab (Novus Biologicals FAB9919R) at a 1:50 dilution, Alexa Fluor 647-conjugated TRA-1-60 (BioLegend 330605) at a 1:20 dilution, Alexa Fluor 647-conjugated Nestin (Novus Biologicals IC1259R-100UG) at a 1:50 dilution, Alexa Fluor 647-conjugated EOMES (Novus Biologicals IC6166R-100UG) at a 1:50 dilution, APC-conjugated TuJ1 (Biolegend 801219) at a 1:20 dilution, Alexa Fluor 647-conjugated NeuN (Novus Biologicals NBP1-92693AF647) at a 1:50 dilution, Alexa Fluor 647-conjugated VMAT2 (R&D Systems FAB8327R) at a 1:20 dilution, Alexa Fluor 647-conjugated GFAP (BioLegend 644706) at a 1:20 dilution, APC-conjugated GLAST (Miltenyi 130-123-555) at a 1:50 dilution, Alexa Fluor 647-conjugated Iba1 (Novus Biologicals 603102) at a 1:50 dilution, APC-conjugated P2RY12 (BioLegend 392113) at a 1:20 dilution, Alexa Fluor 647-conjugated CD4 (Biolegend 300520) at a 1:20 dilution, Alexa Fluor 647-conjugated Olig1/2/3 (Bio-techne FAB2230R-MTO) at a 1:50 dilution, Alexa Fluor 647-conjugated O4 (R&D Systems FAB1326R) at a 1:20 dilution and Alexa Fluor 647-conjugated O1 (R&D Systems FAB1327R) at a 1:20 dilution.

Techniques: Infection, Control, Comparison

Journal: BMJ Neurology Open

Article Title: Altered amyloid plasma profile in patients with disabling headaches after SARS-CoV-2 infection and vaccination

doi: 10.1136/bmjno-2024-001013

Figure Lengend Snippet: Plasma levels of (A) amyloid precursor protein (APP), (B) cathepsin L, (C) pregnancy zone protein (PZP) and (D) serum amyloid A (SAA1) in healthy controls (HC) (n=16), participants with persistent headache after SARS-CoV-2 vaccine (COvax) (n=31) and participants with persistent headache after COVID-19 ( C19 ) (n=29).

Article Snippet: Plasma levels of APP (Cat# DY850), PZP (Cat# DY8280-05), CTSL (Cat# DY952) and SAA1 (Cat# DY3019-05) were measured by ELISA using commercially available antibodies (R&D Systems, Minneapolis, Minnesota, USA) in a 384-format using a combination of a SELMA pipetting robot (Analytik Jena AG, Jena, Germany) and a BioTek dispenser/washer (BioTek Instruments, Winooski, VT).

Techniques: Clinical Proteomics

Journal: Molecular therapy. Nucleic acids

Article Title: miRNA-31 Improves Cognition and Abolishes Amyloid-β Pathology by Targeting APP and BACE1 in an Animal Model of Alzheimer's Disease.

doi: 10.1016/j.omtn.2020.01.010

Figure Lengend Snippet: Figure 1. Expression of miR-31 Decreases APP and Bace1 Expression Levels (A) Schematic representation of the predicted binding sites of the miRNAs in the 30 UTR of genes of interest. miR-17-3p, miR-31-5p, miR-200c-3p, and miR-497-3p are predicted to bind the 30 UTR of human APP mRNA, while miR-31-5p and miR-497-3p are also predicted to bind the 30 UTR of mouse Bace1 mRNA. Additionally, miR-31-5p has a putative binding site in the CDS of human APP mRNA encoding the APP695 protein isoform. (B–D) Biochemical validation of putative binding sites was performed employing the luciferase assay. (B) miR-17-3p, miR-31-5p, miR-200c-3p, and miR-497-3p reduced luciferase activity upon co-transfection with the human 30 UTR APP plasmid in HEK293 cells. NMC, miR-17, and miR-200c, n = 4; miR-31 and miR-497, n = 2. (C and D) miR-31-5p was also able to reduce luciferase activity in HT-22 and

Article Snippet: The luciferase reporter plasmids encoding the 30 UTR of human APP (#SC212695) and mouse BACE1 (#MmiT030538) were purchased from OriGene (USA) and GeneCopoeia (USA), respectively.

Techniques: Expressing, Binding Assay, Biomarker Discovery, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

doi: 10.1371/journal.pone.0077972

Figure Lengend Snippet: ( A ) GST and GST-Tat were purified on glutathione-Sepharose beads. The beads were boiled to elute the bound proteins, which were then run on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (left panel). GST pulldown assay with SK-N-MC neuroblastoma cell lysates shows a strong interaction between APP and GST-Tat (right panel). SK-N-MC neuroblastoma cell extracts incubated with GST- or GST-Tat-coated beads for 3 hours. The beads were washed three times with PBS and the eluted proteins were analyzed by western blotting with an anti-APP antibody (22C11). ( B ) Coimmunoprecipitation of Tat and APP in HEK 293FT cells transfected with Tat and/or Myc-tagged APP695 vectors. Proteins were precipitated with anti-Tat or anti-APP (6E10) antibodies and immunoblotted with anti-Tat or anti-Myc antibodies. ( C ) Coimmunoprecipitation of U-87 MG cell lysates transduced with mock, Lenti-Tat, or Lenti-mTat virus. APP was precipitated with an APP antibody (6E10), and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (22C11) or anti-Tat antibodies. Reciprocally, Tat was precipitated with anti-Tat antibody, and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (A8717) or anti-Tat antibody. ( D ) Purified recombinant APP interacts with GST-Tat. Purified recombinant APP (500 ng) was incubated with GST- or GST-Tat-coated beads and the eluted proteins were analyzed by western blotting with an APP antibody. A large amount of recombinant APP bound to the GST-Tat beads. ( E ) Tat interacts strongly with APP. SK-N-MC neurobalstoma cell lysates were incubated with GST, GST-Tat, or GST-Tat beads, and washed three times in buffer containing 137, 200, 300, 400 or 500 mM NaCl. APP remained associated with GST-Tat under high-salt conditions. ( F ) The cysteine-rich domain of Tat is important for association with APP. Deletion mutants were produced as GST-fusion proteins and subjected to GST-pulldown assays with SK-N-MC cell lysates. L, load; B; bound.

Article Snippet: Purified recombinant APP (cat. no APP-526H, Creative BioMart, NY, USA) was resuspended in PBS supplemented with 1% NP-40 to yield a final concentration of 1 ng/μl, and 500 μl was incubated with GST- or GST-Tat-coated beads for 3 hours at 4°C.

Techniques: Purification, SDS Page, Staining, GST Pulldown Assay, Incubation, Western Blot, Transfection, Transduction, Virus, Recombinant, Produced

Journal: Neurotherapeutics

Article Title: Discovery of an APP-selective BACE1 inhibitor for Alzheimer's disease

doi: 10.1016/j.neurot.2025.e00610

Figure Lengend Snippet: Inhibitory activity and brain permeability of FAH65E(-) . Shown are (A) dose-response curves for FAH65 racemate and the FAH65E(+) and (-) enantiomers in the P5-P5′ assay and (B) sAPPβ and (C) Aβ1-42 in CHO-7W cells after treatment with increasing concentrations of FAH65 racemate and enantiomers. Legend in B also applies to C. Data graphed as the mean and SEM. (D) FAH65E(-) (black line) and sAPP β (blue dashed line) levels in brain from ApoE4TR-5XFAD mice after oral delivery of 30 ​mg/kg FAH65E(-) doses are shown. PK-PD study design did not include 0 h timepoint untreated mice but included time points 1, 2, 4 and 6 ​h after last dose on Day 2. N ​= ​3 mice per time point.

Article Snippet: Media was assayed using an AlphaLISA for Aβ1-42 (Perkin Elmer catalog # AL276C), sAPPα (R&D Systems catalog # AF1168 conjugated with Perkin Elmer acceptor beads catalog # 6772001 + 2B3 antibody from IBL catalog # 11088 biotinylated), and sAPPβ (Perkin Elmer AL276-acceptor + IBL catalog # 18957 biotinylated).

Techniques: Activity Assay, Permeability