Figure 5.

ADH-503 rescues phagocytosis in KO MGLs and synaptic defects in KO spheroids and improves disease score and survival in mouse

(A) Schematic of the compounds used in the screening; agonist compounds are in red and inhibitors in blue.

(B) Drug screening using phagocytosis assay. Phagocytosis percentage of zymosan particles compared to CTRL MGLs. Significance was tested by two-way ANOVA (^∗∗∗∗p < 0.001, ^∗p = 0.0109, ^∗∗p = 0.0041), experiment run in two batches, n = 12 to 24 biological replicates. Bars represent mean ± SEM.

(C) MECP2 binding events to ITGAM (CD11b) promoter, baseline binding events detected (untreated) or at the promoter region of ITGAM. Significance was tested by two-way ANOVA followed by Sidak multiple comparison test (^∗∗∗p = 0.0004, ^∗p = 0.0236, CTRL vs. KO ITGAM -190 bp ^∗∗p = 0.0013, untreated KO^R vs. ITGAM -190 bp ^∗∗p = 0.0019), n = 3 independent experiments. Bars represent mean ± SEM.

(D) Peaks detected in the promoter region of ITGAM in CTRL and KO^R MGLs by MECP2 ChIP-seq.

(E) Quantification of colocalized synaptic (VGLUT/HOMER1) puncta in KO spheroids co-cultured with KO or CTRL MGLs and treated with 1 μM of ADH-503 using Imaris software. The dotted line indicates the number of colocalized synaptic puncta in CTRL spheroids co-cultured with CTRL MGLs. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (KO no MGL vs. KO + CTRL MGLs ^∗p = 0.0163, KO no MGL vs. KO + KO MGLs+ ADH-503 ^∗p = 0.0109, KO + KO MGLs vs. KO + CTRL MGLs ^∗p = 0.0344, KO + KO MGLs vs. KO + KO + ADH-503 ^∗p = 0.0236, KO + CTRL MGLs vs. KO + KO MGLs+ ADH-503, and KO no MGL vs. KO + KO MGLs were non-significant).

(F) Schematic of ADH-503 treatment of MeCP2-KO male mice.

(G) Average total symptom score was calculated in each group at indicated time points. MeCP2-KO mice treated with ADH-503 showed a significant reduction in total score compared with vehicle-treated Mecp2-KO mice. N = KO-vehicle: 11, KO-ADH-503: 10. Significance was tested with Student’s t test, week 8; ^∗∗∗p = 0.0004, week 9, ^∗∗∗p = 0.0007.

(H) Kaplan-Meier survival curves. MeCP2-KO mice treated with ADH-503 survived significantly longer than non-treated control MeCP2-KO mice (^∗p = 0.0244; log rank test), with median survival of 63.5 days (for control mice) and 91 days (for ADH-503-treated mice). N = KO: 20, KO-ADH-503: 10.

(I) Cryosections of WT or MeCP2-KO mouse brains treated with vehicle or ADH-503 stained with NeuN (scale bar 100 μm, on the left) and measurement of soma size was plotted on a bar graph. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗∗p = 0.0078, ^∗∗∗p = 0.0004), n = 6 mice/group.