Journal: Science Advances

Article Title: DKK3-LRP1 complex and a chemical inhibitor regulate Aβ clearance in models of Alzheimer’s disease

doi: 10.1126/sciadv.adz2099

Figure Lengend Snippet: ( A and B ) Subcellular colocalization of fluorescently labeled Aβ 42 (FAM-Aβ 42 ; green) with LysoTracker (red) in SH-SY5Y neuroblastoma cells and C8-D1A astrocytic cells following treatment with CM as indicated. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 50 μm. ( C ) Schematic of the experimental protocol used for culturing and differentiating neural stem cells derived from a patient with AD (AD-NSCs). ( D ) Images showing internalization of FAM-Aβ 42 (green) in differentiated AD-NSCs, colabeled with Tuj1 for neurons (red; top) or glial fibrillary acidic protein (GFAP) for astrocytes (red; bottom) under various CM treatments. Scale bar, 50 μm. Tuj1, β-III tubulin. ( E ) ELISA quantification of Aβ 42 uptake in cells treated with CTL, DKK1, or DKK3 CM containing soluble Aβ 42 . ( F ) Similar setup as in (E) but with Aβ 42 preincubated for 2 hours to allow for internalization, followed by a wash and an additional 8-hour incubation in fresh medium. ELISA quantified the decrease in internalized Aβ 42 as an estimate of cellular Aβ clearance (see Materials and Methods for details). Data are presented as mean ± SD. Statistical significance determined by Student’s t test, with ** P < 0.01, *** P < 0.001, and n.s. (not significant).

Article Snippet: Human iPSC-derived NSCs (Axol Bioscience, ax0111) were maintained according to the manufacturer’s protocols on Laminin-coated chamber slides (Thermo Fisher Scientific, 154526).

Techniques: Labeling, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Science Advances

Article Title: DKK3-LRP1 complex and a chemical inhibitor regulate Aβ clearance in models of Alzheimer’s disease

doi: 10.1126/sciadv.adz2099

Figure Lengend Snippet: ( A ) Differentiated AD-NSCs were incubated with 1 μM Aβ 42 and various concentrations of purified DKK1-Flag or DKK3-Flag for 30 min, followed by IP of LRP1 and immunoblotting using specific antibodies. Five percent of the total lysate was used as input, with IgG serving as a negative CTL. ( B ) SH-SY5Y cells stably expressing mLRPIV treated as in (A) but with an expanded range of DKK1 or DKK3 concentrations. Levels of Aβ 42 following HA-tag IP were quantified using ELISA. ( C ) SPR analysis depicting the competitive binding dynamics of Aβ 42 to mLRPIV in the presence of 35.5 nM DKK3. The assay was conducted with varying concentrations of Aβ 42 . ( D ) IF imaging demonstrating the localization of DKK3-Flag (red) and mLRPIV-HA (green) in SH-SY5Y cells at 0, 30, and 60 min posttreatment with DKK3-Flag CM. Arrow indicates cell membrane localization; arrowhead points to intracellular localization. Nuclei are counterstained with DAPI (blue). Scale bar, 50 μm. ( E ) Immunoblot analysis showing levels of Mem and Cyt mLRPIV in SH-SY5Y cells after treatment with 50 nM recombinant DKK1 or DKK3 at specified time points. Na + - and K + -dependent ATPase (Na + ,K + -ATPase) and ACTIN were used as loading controls for membrane and cytoplasmic proteins, respectively. ( F ) Quantification of band density for proteins shown in (E). Membrane and cytoplasmic proteins were normalized to Na + ,K + -ATPase or ACTIN, respectively. Error bars represent mean ± SD from biological triplicates.

Article Snippet: Human iPSC-derived NSCs (Axol Bioscience, ax0111) were maintained according to the manufacturer’s protocols on Laminin-coated chamber slides (Thermo Fisher Scientific, 154526).

Techniques: Incubation, Purification, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Imaging, Membrane, Recombinant