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. Author manuscript; available in PMC: 2021 Aug 23.
Published in final edited form as: Nat Neurosci. 2021 Feb 8;24(3):343–354. doi: 10.1038/s41593-020-00796-z

Figure 5. Tri-culture model of Alzheimer’s Disease shows increased C3 in AD tri-cultures caused by reciprocal signaling from microglia to astrocytes.

Figure 5.

A) IF shows that the day 80 APPSWE+/+ neurons and isogenic control neurons express FOXG1 B) and the cortical layer markers CTIP2 and TBR1. Scale bar = 40 μM in (A) and (B). C) ELISA shows that day 50 APPSWE+/+ neurons secrete more total amyloid than isogenic control neurons. n=3 distinct culture supernatants, two-tailed t-test, ** p = 0.0016, Error bars = SD, center = mean. D) IF shows APPSWE+/+ tri-cultures with d80 APPSWE+/+ neurons (MAP2+), wildtype hPSC-derived microglia (IBA1+) and astrocytes (GFAP+), and isogenic control tri-cultures with d80 isogenic control neurons (MAP2+), wildtype hPSC-derived microglia (IBA1+) and wildtype astrocytes (GFAP+). Scale bar = 100 μM. E) ELISA shows that APPSWE+/+ tri-cultures secrete more C3 than isogenic control tri-cultures. n=3 (distinct culture supernatants), 2-way ANOVA with Bonferroni post-hoc test, **** p<0.0001. Error bars = SD, center = mean. F) ELISA shows that C3KOA tri-cultures with APPSWE+/+ neurons show lower C3 secretion as compared to APPSWE+/+ tri-cultures with wildtype astrocytes. C3KOM APPSWE+/+ tri-cultures secrete low levels of C3. n=3 (distinct culture supernatants), 2-way ANOVA with post-hoc Tukey’s test, **** p<0.0001, F=9.577, df=1. C3KOA tri-cultures with APPSWE+/+ neurons show higher levels of C3 secretion as compared to C3KOA tri-cultures with WT neurons. n=3 separate culture supernatants, 2-way ANOVA with Bonferroni post-hoc test, *** p=0.0007, F=182.9, df=5. Translucent bars (TRI) represent data originally presented in E). Error bars = SD, center = mean. G) Western blot (cropped) shows that APPSWE+/+ cultures which contain microglia show higher levels of C1QA as compared to isogenic control cultures (TRI, M/N, C3KOA, C3KOM). H) Neuroinflammatory loop schematic in an in vitro model of AD where APPSWE+/+ neurons activate microglia which activate reciprocal signaling to astrocytes leading to increased C3 release, as well as cause increased C1Q secretion and deposition.