Thrombospondin 1--a key astrocyte-derived neurogenic factor

Abstract

Thrombospondin 1 (TSP1), an oligomeric matrix protein, is known for its antiangiogenic activity. Recently, TSP1 has been shown to regulate synaptogenesis in the developing brain. In this study, we examine another role of TSP1 in the CNS, namely, in proliferation and differentiation of neural progenitor cells (NPCs). We found that adult mice deficient in TSP1 exhibit reduced proliferation of NPCs in vivo [13,330+/-826 vs. 4914+/-455 (mean+/-se wt vs. TSP1(-/-)); P<0.001, Student's t test] and impaired neuronal differentiation (1382+/-83 vs. 879+/-79; P<0.001). In vitro, NPC obtained from adult TSP1(-/-) mice display decreased proliferation in BrdU assay (48+/-8 vs. 24+/-3.5%; P<0.01) and decreased neuronal fate commitment (8+/-0.85 vs. 4.6+/-0.5%; P<0.05) in contrast to wild-type NPCs. Both proliferation and neuronal differentiation deficits are remediable in vitro by exogenous TSP1. Notably, conditioned medium from TSP1(-/-) astrocytes, unlike that from control astrocytes, fails to promote neurogenesis in wild-type NPCs, suggesting that TSP1 is one of the key molecules responsible for astrocyte-induced neurogenesis. Our data demonstrate that TSP1 is a critical participant in maintenance of the adult NPC pool and in neuronal differentiation.

Figure 1

TSP1 deficiency impairs NPC proliferation and neuronal differentiation in vivo. A–H) To examine proliferation of neural progenitors in TSP1^−/− and WT mice, exogenous (BrdU; A–D) and endogenous (Ki-67; E–H) proliferation markers were used. Labeled cells were quantified in SGL (A, B, E, F) and SVZ (C, D, G, H) areas of the brain. A significant reduction in proliferating cells was found in both SVZ and SGL of TSP1^−/− mice compared to WT counterparts. I–L) To examine neuronal differentiation in TSP1^−/− and WT mice, brain slices containing SGL (I, J) and SVZ (K, L) were double-labeled with DCX and BrdU at 7 d after BrdU injection. Significantly less DCX/BrdU double-positive cells were found in TSP1^−/− as compared to WT mice. Quantification data are from ≥4 animals/group. Confocal micrographs are representative. ***P < 0.001; Student’s t test. Scale bars = 50 μm (B, D, F, H, L); 20 μm (J).

Figure 2

TSP1 deficiency impairs NPC proliferation in vitro. A) Phase contrast representative images showing primary neurosphere cultures derived from SVZ of WT and TSP1^−/− mice at different time points in vitro. B–E) Quantification of average number (B), diameter (C), self-renewal (neurospheres/plated cells×100) (D), and amplification (live cells from neurospheres/plated cells) of P1 neurospheres obtained from WT and TSP1^−/− mice (E). Data are means ± se from ≥3 independent experiments; ≥4 mice/group. F) TSP1^−/− NPC proliferation and sphere formation were terminally impaired at P7, as compared with WT counterparts. G) Phase-contrast representative images showing primary neurosphere cultures derived from SVZ of WT and TSP1^−/− p14 pups during P3 in vitro. H) No difference in self-renewal capacity was observed for NPCs obtained from TSP1^−/− and WT p14 mice. I) FACS histograms for dissociated WT or TSP1^−/− NPCs from P5 labeled for CD133 and CD24. J) Significant reduction in CD133^hiCD24^lo early progenitor cells is evident in TSP1^−/−-derived NPCs. K) To examine proliferation of NPCs, neurospheres from WT and TSP1^−/− mice were incubated with BrdU (10 μM) for 8 h and labeled with anti-Nestin (red) and anti-BrdU (green) antibodies. L) Average percentage of BrdU⁺ cells of total Nestin⁺ cells from 3 independent experiments; ≥40 neurospheres/group. M) To examine apoptosis, neurospheres from WT and TSP1^−/− mice were TUNEL labeled. Micrographs are representative. N) Average percentage of TUNEL⁺ cells of total cells from ≥3 independent experiments; ≥40 neurospheres/group. O) To determine whether TSP1 affects cell cycle progression, dissociated WT and TSP1^−/− NPCs were analyzed by FACS for DNA content using DRAQ-5. Cell cycle histograms are representative. P) Average percentages of G1-, S-, and G2-phase cells from 3 independent experiments. Q–T) P3 neurospheres derived from TSP1^−/− mice were treated with 2 μg/ml of purified TSP1 for 72 h. Q) Representative phase-contrast images showing primary neurospheres from TSP1^−/− mice at P3 in vitro with and without treatment with purified TSP1. R–T) Quantification of average number (R), diameter (S), and amplification (T) (live cells from neurospheres/plated cells) of TSP1^−/− P3 neurospheres treated with purified TSP1 or vehicle. All data are means ± se from ≥3 independent experiments; ≥4 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001; Student’s t test. Scale bars = 200 μm (A); 100 μm (F, G, Q); 20 μm (K, M).

Figure 3

TSP1 deficiency impairs NPC neuronal differentiation in vitro and could be rescued by purified TSP1. A) P4 NPCs from WT or TSP1^−/− mice were differentiated in culture for 7 d and immunolabeled with βIII-tubulin (neurons), GFAP (astrocytes), and NG2 (OPCs). Decreased neurogenesis with increased oligodendrogenesis was observed in NPCs from TSP1^−/−, as compared to WT mice. B–D) P4 NPCs from WT or TSP1^−/− mice were cultured with or without purified TSP1 (2 μg/ml; every 12 h for 6 d). Immunolabeling for differentiated cells revealed increase in neuronal differentiation (B) and a decrease in oligodendrocyte differentiation (C) after incubation of TSP1^−/− NPCs with purified TSP1. D) P4 NPCs from WT mice treated similarly with purified TSP1 exhibited increased neuronal differentiation. Data are means ± se of ≥3 independent experiments. *P < 0.05; Student’s t test. E) Representative micrographs of WT and TSP1^−/− NPC differentiation under different conditions. F) P4 NPCs from WT or TSP1^−/− mice were differentiated in culture for 7 d and immunolabeled for DCX/TUNEL or NG2/TUNEL to detect neuronal (DCX) and oligodendrocyte (NG2) precursor death. Less than 1% of either DCX⁺ or NG2⁺ cells were TUNEL positive; no significant changes were observed between WT and TSP1^−/− groups. Scale bars = 30 μm (F); 10 μm (E).

Figure 4

TSP1 deficiency increases mature oligodendrocyte differentiation. P4 NPCs from WT or TSP1^−/− mice were differentiated in culture for 14 d and immunolabeled with βIII-tubulin, GFAP, NG2, and RIP (mature oligodendrocytes). Fewer neurons but more OPCs and mature oligodendrocytes were differentiated from TSP1^−/− NPCs as compared to WT counterparts. A) Average percentages of βIII-tubulin⁺, GFAP⁺, NG2⁺, and RIP⁺ cells of total seeded cells were obtained from 4 independent experiments. B) Representative micrographs of differentiated WT and TSP1^−/− NPCs immunolabeled for above-mentioned markers. Data are averages ± se. *P < 0.05, ***P < 0.001; Student’s t test. Scale bar = 20 μm.

Figure 5

TSP1 is required for astrocyte-induced neuronal differentiation. A–D) P4 NPCs from TSP1^−/− (A, B) and WT (C, D) mice were cultured for 7 d in the presence of ACM obtained from either TSP1^−/− or WT astrocytes. Control cells were kept in regular differentiation medium. Incubation of TSP1^−/−-derived NPCs with WT-ACM increased neuronal (A) and decreased oligodendrocyte (B) differentiation. TSP1^−/−-ACM did not affect differentiation of TSP1^−/− NPCs. Incubation of WT NPCs with WT-ACM resulted in increased neuronal (C) and decreased oligodendrocyte (D) differentiation. TSP1^−/−-ACM did not affect differentiation of WT NPCs. E) Representative micrographs of WT and TSP1^−/− NPCs differentiated under different conditions.. Data are means ± se of ≥3 independent experiments. *P < 0.05; Student’s t test. Scale bar = 10 μm.