Non-cell-autonomous effects of presenilin 1 variants on enrichment-mediated hippocampal progenitor cell proliferation and differentiation

Abstract

Presenilin 1 (PS1) regulates environmental enrichment (EE)-mediated neural progenitor cell (NPC) proliferation and neurogenesis in the adult hippocampus. We now report that transgenic mice that ubiquitously express human PS1 variants linked to early-onset familial Alzheimer's disease (FAD) neither exhibit EE-induced proliferation, nor neuronal lineage commitment of NPCs. Remarkably, the proliferation and differentiation of cultured NPCs from standard-housed mice expressing wild-type PS1 or PS1 variants are indistinguishable. On the other hand, wild-type NPCs cocultured with primary microglia from mice expressing PS1 variants exhibit impaired proliferation and neuronal lineage commitment, phenotypes that are recapitulated with mutant microglia conditioned media in which we detect altered levels of selected soluble signaling factors. These findings lead us to conclude that factors secreted from microglia play a central role in modulating hippocampal neurogenesis, and argue for non-cell-autonomous mechanisms that govern FAD-linked PS1-mediated impairments in adult hippocampal neurogenesis.

Figure 1. Expression of FAD-linked PS1 variants impairs enrichment-induced proliferation of hippocampal neural progenitors

(A - F) Photomicrographs of BrdU (green or yellow)+ cells in the DG of PS1hWT (A, D), PS1ΔE9 (B, E) and PS1M146L (C, F). SH, standard housing conditions (A, B, C); EE, enriched conditions (D, E, F). NeuN, red. Scale bar: 100 μm. (G) Quantification of BrdU+ cells (mean ± SEM; N = 10 - 15 per group). * P < 0.05. (H) Quantification of granule cell number. * P < 0.05. (I) Photomicrograph of primary neurospheres. Scale bar: 50 μm. (J) Image of nestin (green)+ neurosphere. Scale bar: 25 μm. (K) Nuclei were stained with DAPI (blue). (L) Percentage of multipotent neurospheres obtained in clonogenic neurosphere assay (mean ± SEM, N = 5 animals per group). ** P < 0.01.

Figure 2. FAD-linked PS1 mutants alter differentiation pattern of neural progenitors

(A - E) Images of BrdU+ cells that are colabeled with either DCX (A), βIII-tubulin (B), NeuN (C), GFAP (D) or s100β (E). Scale bar: 50 μm (A, B, D, and E) or 100 μm (C). (F, H, J, L, N) The fraction of BrdU+ cells that expressed DCX (F), βIII-tubulin (H), NeuN (J), GFAP (L), or s100β (N). (G, I, K, M, O) The number of new DCX (G)-, βIII-tubulin (I)-, NeuN (K)-, GFAP (M)-, or s100β (O)-cells. * P < 0.05; ** P < 0.01. N = 8 per group.

Figure 3. FAD-linked PS1 mutant mice fail to exhibit high activity-induced increase in microglial cell numbers

(A - L) Images of microglia in DG of standard PS1hWT (A, B, and C), highly active PS1hWT (D, E, and F), highly active PS1M146L (G, H, and I), and highly active PS1hWT (high magnification, J, K, and L). Microglia are labeled with IB-4 (green; A, D, G, and J) and Iba1 (red; B, E, H, and K). Merged images are shown in (C, F, I, and L). Scale bars: 100 μm in A; 50 μm in J. (M) Quantification of Iba1+ cells in standard or enriched PS1hWT and FAD-linked PS1 variants (mean ± SEM; N = 10 - 15 per group). (N) Quantification of Iba1+ cells in highly active or low active PS1hWT and FAD-linked PS1 variants. * P < 0.05.

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.

Figure 5. Conditioned media from microglia expressing FAD-linked PS1 variants impair proliferation of neural progenitors

(A - B) Proliferation achieved by PS1hWT NPCs following treatment with conditioned media collected from resting (A) or IL4-activated (B) microglia (MG) expressing mentioned PS1 transgenes, as measured by BrdU labeling. ** indicates significant difference from PS1hWT MG media at P < 0.01 on day 3 or 6.

Figure 6. Differentiation pattern of cultured NPCs is modulated by FAD-linked PS1 mutant expressing microglia

(A) Progeny cell types generated by NPCs in the multipotency assay were quantified by sampling a minimum of 300 cells as marked by DAPI or propidium iodide+ nuclei (mean ± SEM of the results obtained from independent cultures established from 6 animals per group). (B) Fold changes in the number of βIII-tubulin+ neuronal or GFAP+ astrocyte progeny cell type derived from PS1hWT NPCs exposed to CM collected from resting or IL4-activated MG, over differentiated PS1hWT NPC cultures in the absence of MG CM (mean ± SEM; N = 6). The asterisk indicates significant difference from PS1hWT MG CM at * P < 0.05 and ** P < 0.01.

Figure 7. Quantitative changes in signaling factors secreted by PS1hWT versus FAD-linked PS1 variant expressing microglia

(A) Images of biotin-label based protein antibody arrays probed with CM collected from resting or IL4-activated microglia (MG). (B) Soluble factors whose levels were consistently altered (up or down ‘-’; mean ± SEM of fold changes observed across three experiments). - denotes, no detectable change in mean signal intensity.