Fig 1: Organoids containing either APOE3/3 or APOE4/4 microglia exposed to rApoE4 secrete pro-inflammatory IL-8; however, only organoids containing APOE3/3 microglia exhibit an increase in NfL release, a clinical biomarker of AD. (A) Organoids with either APOE3/3 or APOE4/4 microglia exposed to rApoE4 protein show comparable levels of IL-8 secretion. However, comparison of organoids with either APOE3/3 or APOE4/4 microglia exposed to rApoE4 and Aβ demonstrates that (B) only APOE3/3 MG containing organoids release neuronal cell injury marker NfL into the supernatant, despite comparable levels of IL-8 secretion. GFAP release was also measured (C), though it did not reach the level of significance via ANOVA; direct t-tests between APOE3/3 MG + rApoE3 + Aβ vs APOE3/3 + rApoE4 + Aβ were statistically significant (p = 0.0248). (D) Characterization of the cell-specific contributions to the IL-8 pro-inflammatory response in the APOE3/3 organoids exposed to rApoE4 + Aβ shows that the disease induction model is primarily microglial-driven. Furthermore, (E) microglial lipid accumulation is also induced, as compared to induction with LPS + IFNγ. ELISA measurements includes biological replicates only (n = 3 organoids per condition) 24 h after model induction; IF volumetric quantification was performed using 10× confocal imaging using 2 organoids per condition 3 days after model induction. Statistical analysis was performed using one-way ANOVA and Tukey’s (A–C,E) or Dunnett’s post hoc test (D); p-value levels of significance are as follows: *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.
Fig 2: The rApoE4 AD neuroimmune organoid model demonstrates pharmacological pro-inflammatory properties unique to traditional LPS/IFNγ drug screening, with SB203580 (a p38 MAPK inhibitor) exhibiting both anti-inflammatory and neuroprotective properties while reducing microglial lipid accumulation. Treatment with a selection of anti-inflammatory tool compounds on either (A) LPS/IFNγ-treated organoids or the (B) rApoE4 AD organoids demonstrates adaptability for AD drug screening with readouts for pro-inflammatory microglial activation, (C) clinically relevant neuronal and astrocytic biomarkers NfL and GFAP, and (D) lipid dysfunction by measurement of cell-specific accumulation. ELISA measurements (2–4 organoids per condition), and volumetric IF measurements from confocal 10X images (n = 2 organoids per condition), were performed 3 days after model induction. Organoids were preincubated for 1 h using SB203580 (10 µM), Curcumin (20 µM), IKK16 (1 µM), Celecoxib (10 µM), or TAK-242 (1 µM) prior to induction of rApoE4 AD phenotype. Statistics were performed using one-way ANOVA and Tukey’s post hoc test; p-value levels of significance are as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
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