Figure 4.

KO^R or CTRL MGL rescues the synaptic defects of KO neurons in long-term co-culture experiments

(A) Bright-field images of neuron-MGL (labeled with long-term stable membrane stain PKH26 in red) co-culture on day 2 and 50 of neuronal differentiation, scale bar 400 μm.

(B) Representative images of co-culture neurons (stained with MAP2 and HOMER1) and MGLs (stained with CD68) for 2 months. The last panel shows a magnification of the square area shown. Note that MGL is nearby of MAP2⁺ neurons and synapses, scale bar 50 μm.

(C) Representative images of synaptic puncta co-localization with or without CTRL, KO, or KO^R MGLs, scale bar 20 μm.

(D) Quantification of the number of synaptic puncta (KO vs. CTRL neurons without MGL ^∗∗p = 0.0013; KO neurons without MGL vs. KO neurons with CTRL MGLs ^∗∗p = 0.0023; KO neurons with CTRL MGLs vs. KO neurons with KO MGLs ^∗∗∗p = 0.0007, one isogenic KO/KO^R pair [KO^R results shown in Figure.S5d], and one CTRL MGL line was used [related to KO], synaptic puncta from 10 neurons were counted, the experiment was run in two independent batches).

(E) Quantification of the number of co-localized synaptic puncta (CM, conditioned media from CTRL or KO MGLs) (^∗p = 0.0482 one isogenic KO/KO^R pair and one CTRL MGLs line was used, synaptic puncta from 10 neurons were counted).

(F) Quantification of the number of co-localized synaptic puncta without healthy human primary fibroblasts (Fibro), (^∗∗p = 0.0084, one isogenic KO/KO^R pair, and one CTRL MGLs line was used, synaptic puncta from 10 to 15 neurons were counted).

(G) Bright-field images of spheroids co-cultured with MGL (in red) before and after plating on MEA plates, scale bar 400 μm.

(H) Graph showing the spike rate compared to CTRL spheroids without any MGLs, recorded in 5 min emerging from spheroids with or without CTRL or KO^R MGLs. ^∗∗p = 0.0021, ^∗p = 0.0150, n.s. not significant, one isogenic rescue line and two different CTRL and KO lines were used, two to three independent experiments per genotype were used. Significance is assessed by one-way ANOVA with Tukey’s multiple comparison test for the experiments in D, E, F, and H (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). The number of neurons counted for synaptic puncta is represented by one data point in each graph. All synaptic puncta quantification was calculated as a percentage compared to CTRL neurons. Bars represent mean ± SEM.