Fig 1: Focused small-molecule screen identifies DLK-JNK-cJun pathway inhibition protects human neurons from Aß oligomer toxicity.a–d Neurons and astrocytes (a, c) or neurons, astrocytes, and microglia (b, d) were treated with 5 µM sAß42s and small molecules from a focused screen of known neuroprotective agents at multiple concentrations (50, 25, 12.5, and 6.25 µM (double culture), 50, 12.5, 3.1, and 0.78 µM (triple culture). Results were graphed as Synapse % rescue versus MAP2 % rescue (a, b) and Beta III tubulin % rescue versus MAP2 % rescue (c, d). Small molecules that prevented toxicity in dendrites (MAP2), synapses (Synapsin 1/2), cell count (CUX2), or axons (NFL-H) at or above 30% were considered hits (red dotted line); anti-Aß antibody used as a positive control that prevented all types of toxicity. e–g Further validation of top hits DLKi (e), Indirubin-3'-monoxime (f), and AZD0530 (g) from the focused screen by IC50 curves against MAP2 (blue), Synapsin 1/2 (green), CUX2 (red), and NFL-H (purple). h sAß42s treatment induced expression of p-cJun (green) in the nucleus (HuCD, red). i Quantification of MAP2 (blue), HuC/D (red), p-cJun (green) staining indicated an increase in cJun phosphorylation with prolonged sAß42s treatment. j 22-week-old iPSC neuron culture treated with sAß42s showed dose-dependent, sustained phosphorylation of cJun as shown by western blot. GAPDH served as the loading control. k Western blot quantification of p-cJun induction normalized to GAPDH. l–o Inhibition of known components of DLK-JNK-cJun pathway using small molecules VX-680 (l), GNE-495 (m), PF06260933 (n), JNK-IN-8 (o) prevents sAß42s-induced neural toxicity in all measured markers in a dose-dependent manner. Data are presented as mean values +/- SEM and n = 4 wells (e–g, I, k–o). IC50 curves fitted by Prism software (e–g, m–o).
Fig 2: An early window of efficacy for anti-Aß antibody revealed by disease progression modeling.a–c Time-course comparison of 12-week-old iPSC neurons treated with a single dose of sAß42s (solid lines) versus repeated dose of sAß42s at the same concentration (dotted lines) at the indicated concentration. MAP2 area (a), Synapse count (b), and p-Tau 396–404 induction fold (c) were quantified. d Repeated dosing schedule of 12-week-old neuron with 0.6 µM of Aß. Anti-Aß antibodies dosing regimen was started at indicated time point. All cells were treated in the same plate and fixed at 21 days post first dose. MAP2 area (e), synapsin count (f), and p-Tau induction fold (g) were quantified. Anti-gD antibodies were dosed similarly as control (blue bars) along with anti-Aß antibody (red bar). h Time-course study design of Anti-Aß antibodies repeated dosing. sAß42s are added at every indicated timepoints. Anti-Aß antibodies were added at day 0 (red) as a protection model or at day 7 (green) as an intervention model. Anti-gD antibodies were used as control (blue). i Representative images from the indicated experimental treatments. Neurons were stained for dendrite marker MAP2 (red) and nuclear marker CUX2 (green) at 7DIV and 21DIV. The lower panel shows Aß plaque staining (X04, white) and p-Tau S235 (red) staining. Quantification of MAP2 area over time (j) and plaque area (k) illustrated that the anti-Aß intervention model is capable of slowing down neuron degeneration and plaque formation. Scale bars = 50 µm. Data are presented as mean values +/- SEM and n = 4 wells. Two-way ANOVA with Tukey’s multiple comparisons test.
Fig 3: A high-throughput, automated human iPSC-derived neuron differentiation and culturing platform.a Schematic workflow of human iPSC neuron differentiation, plating, maintenance, and maturation with automated media change using Fluent liquid handler (Tecan). Mature culture (12 weeks+) is ready for various experimental treatments and conditions. At the end of the experiments, fixed cells are processed for immunostaining using automated plate washers and then quantified with high content image analysis via IN Cell Analyzer 6000 (GE). b Representative images of unsynchronized, heterogeneous WT iPSC-derived NSC differentiation (red arrows indicate differentiated neurons; green arrows indicate undifferentiated NSCs). Scale bar 50 µm. c Stable expression of cumate-inducible NAG construct and treatment with cell cycle inhibitors synchronizes and homogenizes human iPSC neuron differentiation. Scale bar 50 µm. d Representative image of 20 culture plate media change using Fluent workstation (Tecan). e Fluent 384 tip liquid handler head consistently and systematically removes old media and adds new media across all wells per plate. f Integrated incubator and barcoded plates enable automated plate tracking and care. g Automated plate ejection from incubator and h gripper arm retrieves plate then i places it on plate deck for subsequent media change. j Gripper arm removes the lid and places it on plate-lid hotel during media change. k Differentiated NAG neurons express dendritic marker MAP2 (red), layer II/III cortical marker CUX2 (green), with a small subpopulation expressing layer V/VI marker CTIP2 (blue) indicated by white arrows. Scale bar = 50 µm. l–r Mature NAG neurons express dendritic marker MAP2 (blue), synaptic markers VGLUT2 (red), Shank (green), scale bar = 20 µm. l Synapsin (red), PSD95 (green), scale bar = 10 µm (m), Pan SHANK (green) (n), PanSAPAP (green) (o), GluR1 (green) (p), GluR2 (green) (q), and NR1 (green) (r). s High content image analysis are made from 9 fields/well in a 384-well plate covering 70% well area. t–y Examples of image analysis using IN Cell Developer toolbox companion software to quantitate phenotypes such as dendrites (t, u), synapses (v, w), and axons (x, y) in an automated, systematic, and unbiased way. Careful scripts were developed to isolate exact regions of interest are shown in red on the right panels. Multiple measurements such as total area, total intensity, and count are made for each marker. z Z-factors are calculated from results using a neuron culturing platform and high content image analysis software. Z-factors are in the range from 0.5 to 0.75 and averaged from 10 to 20 different experiments using different batches of neurons. Each experiment with four wells, 1000+ neurons/well quantified. Data are presented as mean values +/- SEM and n = 4 wells.
Fig 4: Human iPSC-derived neuronal model of Alzheimer’s disease recapitulates key hallmark AD pathologies.a, b Differentiated NAG neurons (12 weeks+) show loss of dendrites (MAP2, green) and cell bodies (CUX2, red) when treated with soluble Aß species for 7 days (b) in comparison to no treatment condition (a). c Anti-Aß antibody co-treatment with soluble Aß species blocks Aß-induced cell death. Scale bar 50 µm. d Dose-dependent, progressive cell death as quantified by the percentage of cell body (CUX2) numbers in Aß-treated normalized to untreated control. e Dose-dependent, progressive dendritic (MAP2) loss as quantified by the percentage of MAP2 area in Aß-treated normalized to untreated control. f, g Aß42 treatment induces phosphorylation of Tau (p-Tau 396–404, white) and mislocalization to the cell body. h Anti-Aß antibody co-treatment with sAß42s blocks Aß-induced Tau hyperphosphorylation. Scale bar 50 µm. i Dose-dependent and time course of phosphorylation of Tau at S396/404. Phospho-Tau induced increase at 5 µM Aß treatment before decrease associated with cell death occurred as quantified by fold p-Tau 396/404 staining in Aß-treated normalized to untreated control. j, k Aß42 treatment causes synapse loss in neurons (synapsin, green). l Anti-Aß antibody co-treatment with sAß42s blocks synapse loss phenotype. Scale bar = 5 µm. m Dose–response and time course of synapse (synapsin 1/2) loss in Aß-treated culture normalized to untreated control. n, o sAß42s treatment induces axon fragmentation (beta-3 tubulin Tuj1, white). p Anti-Aß antibody co-treatment blocks axon fragmentation. Scale bar = 50 µm. q Dose–response and time course of axon fragmentation as quantified by percentage of the axon (NFL-H) area in Aß-treated normalized to untreated control. r Anti-Aß antibody rescues all three markers in a dose-dependent manner and IC50 curves can be drawn and calculated (IC50 curve fitted by Prism software). Data are presented as mean values +/- SEM and n = 4 wells. All scale bars = 50 µm.
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