Ephrins as negative regulators of adult neurogenesis in diverse regions of the central nervous system

Abstract

In the central nervous system (CNS) of adult mammals, neurogenesis occurs in only two restricted areas, the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ). Isolation of multipotent progenitor cells from other CNS regions suggests that their neurogenic potential is dictated by local environmental cues. Here, we report that astrocytes in areas outside of the SGZ and SVZ of adult mice express high levels of ephrin-A2 and -A3, which present an inhibitory niche, negatively regulating neural progenitor cell growth. Adult mice lacking both ephrin-A2 and -A3 display active ongoing neurogenesis throughout the CNS. These findings suggest that neural cell replacement therapies for neurodegeneration or injury in the adult CNS may be achieved by manipulating ephrin signaling pathways.

Fig. 1.

Isolation of neural progenitors from nonneurogenic brain regions. (a) Niche astrocytes induced neurosphere formation from adult cortical cells. Many neurospheres (GFP⁺) developed in neocortical cell cultures prepared from adult GFPtg mice that were plated in the same well with niche astrocytes (GFP⁻), but not in those cocultured with NIH 3T3 fibroblasts (NIH 3T3). (Scale bar: 50 μm.) (b) Numbers of neurospheres developed in dissociated cortical (Cx), cerebellar (CB), and spinal cord (SC) cell cultures plated in the presence or absence of niche astrocytes (Ast). Neurospheres >50 cells were counted (*, P < 0.001; Student's t test). (c) Numbers of neurospheres counted in progenitor cell cultures plated in the same well with niche astrocytes (Ast) or adult cortical astrocytes (AstCx) shown as relative to that obtained from control cultures (without feeder cells).

Fig. 2.

Ephrin-A2 and -A3 are negative regulators of neural progenitor cell growth in vitro. (a) Representative Western blot of triplicate experiments and quantification of ephrin-A2 and -A3 expression show high levels of ephrin-A2 and -A3 expression in the nonneurogenic neocortex (Cx) or cortical astrocytes of adult mice (AstCx) as compared with adult SGZ or P0 astrocytes (Ast). (b) Epifluorescence photomicrographs showing double immunolabeling of cortical sections of adult mice with anti-ephrin-A2 and -A3 with primary antibodies against astrocyte (GFAP), neuron (NeuN), or oligodendrocyte (RIP) markers. Note that ephrin-A2⁺ and -A3⁺ cells were colocalized with anti-GFAP, but not immunostaining for other cell markers. (Scale bar: 20 μm.) (c) Relative number of neurospheres developed in dissociated cultures of neural progenitors isolated from the neocortex and SGZ of adult mice in the presence of control Fc (cont) or clustered ephrin-A2 (A2c) or -A3 (A3c). (d and e) Number of BrdU⁺ (d) and TUNEL⁺ (e) cells counted from neurosphere-derived cell cultures that were treated with control Fc (cont) or clustered ephrin-A2 (A2c) or -A3 (A3c) and shown as relative to control Fc treated cultures. Note the significant decreases in BrdU incorporation, but not TUNEL⁺ cells, in A2c- and A3c-treated group as compared with controls. Data represent mean value ± SD (*, P < 0.01 compared with the corresponding control group; Student's t test).

Fig. 3.

Robust neural progenitor cell proliferation in the CNS of adult A2^−/−A3^−/− mice. (a) Montage views of sagittal brain sections show the cortical morphology and anti-BrdU labeling in adult WT (a′′) and A2^−/−A3^−/− mice (a′). (Insets) Higher power views of BrdU labeling. Note numerous BrdU⁺ cells in cortical areas of adult A2^−/−A3^−/−, but not WT, mice. [Scale bars: 1 mm (a); 100 μm (a′ and a′′)]. (b–d) Quantification of BrdU⁺ cells in the gray matters of the neocortex (b) and SGZ (d) and measurement of cortical areas (c) in brain sections of adult WT (n = 5), A2^−/− (n = 4), A3^−/− (n = 4), and A2^−/−A3^−/− mice (n = 6). (e and f) Numbers of neurospheres developed from neocortical or SGZ cell cultures derived from adult WT (e and f) and A2^−/−A3^−/− (f) mice in the absence (e) or presence (f) of ephrin-A2 or -A3. (g) Number of TUNEL-positive cells counted from brain sections of WT, A2^−/−, A3^−/−, and A2^−/−A3^−/− mice. No significant differences were noted between mice. Data represent mean value ± SD (*, P < 0.01 compared with WT; Student's t test and ANOVA).

Fig. 4.

Ongoing neurogenesis in nonneurogenic CNS regions of adult A2^−/−A3^−/− mice. (a–h) Fluorescence confocal images taken from cortical areas (corresponding to a′′ of Fig. 3) of adult WT (a, e, and g) and A2^−/−A3^−/− (b–d, f, and h) mice that are double-labeled by anti-DCX (green) and BrdU (red) (a–d) or by Tuj-1 (green) and DAPI (blue) (e–h). Note numerous DCX⁺ neuroblasts and their neurites that are labeled by Tuj1 in cortical sections of adult A2^−/−A3^−/− (b–d and f–h), but not WT (a and e), mice. Arrows (c and d) point to cells that are double-labeled. [Scale bars: 50 μm (a, b, e, and f) and 10 μm (c, d, g, and h).] (i and j) Counts of DCX⁺ (i) and Tuj-1⁺ (j) cells per 1,000 nuclei in cortical sections of adult WT (n = 5) and A2^−/−A3^−/− mice (n = 6). (k) Percentage of DCX⁺ cells double-labeled with primary antibodies against BrdU and DCX from A2^−/−A3^−/− mice that received daily injection of BrdU for 14 consecutive days. (l) Percentage of BrdU⁺ cells double-labeled with primary antibodies against BrdU and early neuronal markers HuD, DCX, nestin, Tuj1, or mature neuronal and glial markers MAP2, NeuN, GFAP, O4, and nestin in the cortical sections of adult A2^−/−A3^−/− mice that received BrdU pulse-labeling. Data represent mean value ± SD (*, P < 0.01 over WT or PBS control group; Student's t test and ANOVA).

Fig. 5.

Generation of neurons and glial cells by cortical neural progenitors in adult A2^−/−A3^−/− mice. Shown are single optical sections and orthogonal confocal images taken from cortical sections of adult A2^−/−A3^−/− mice that were double labeled for BrdU (red) and primary antibodies (green) against either MAP2, HuD, NeuN, and GFAP. Arrows point to cells that are double-labeled for BrdU and the primary antibodies above. (Scale bars: 15 μm.)