Figure 4. Microglia expressing FAD-linked PS1 variants impair proliferation of NPCs in in vitro co-culture assays.

(A) Proliferation achieved by NPCs, expressing PS1hWT, PS1ΔE9 or PS1M146L, grown in SFM supplemented with BrdU (mean ± SEM from a minimum of three experiments per group).

(B - D) IB-4+ microglial cells (B, green) and nestin+ NPCs (C, red) under co-culture conditions. The merged image is shown in D. Fibrillar staining of the intermediate filament protein, Nestin, is evident on NPCs (arrow head) but not on microglial cells (arrow). Scale bar: 10 μm.

(E - F) Proliferation of PS1hWT NPCs when co-cultured with AraC-treated resting (E) or IL4-activated (F) microglia (MG) expressing PS1hWT, PS1ΔE9 or PS1M146L as measured by BrdU labeling. ** indicates significant difference from PS1hWT MG at P < 0.01 on day 3 or 6.

(G - J) Confocal z-series reconstructed image of cells stained with IB-4 (G), anti-nestin (H), anti-BrdU antibody (I) and DAPI (J), following 6 days under co-culturing conditions.

(K - N) Confocal images of a single z-plane within nestin+ neural progenitor cluster (K; red, arrow head) reveals several BrdU+ progenitors (K; cyan blue, enclosed within circle) and an IB-4+ MG (K; green, arrow). Insert shows the zoom-in area marked within dotted square (L). Scale bar: 50 μm.

(M - N) Zoomed images of cells that are co-labeled with BrdU/nestin (M) or BrdU/IB-4 (N). Scale bar: 10 μm.

(O - P) Quantification of BrdU and nestin (O) or BrdU and IB-4 (P) co-labeled cells in the co-culture assay. The asterisk indicates significant difference from PS1hWT MG at * P < 0.05 and ** P < 0.01.