Neurofibromin regulates neural stem cell proliferation, survival, and astroglial differentiation in vitro and in vivo

Abstract

Neurofibromatosis 1 (NF1) is a common inherited disease in which affected children exhibit abnormalities in astrocyte growth regulation and are prone to the development of brain tumors (astrocytoma). Previous studies from our laboratory demonstrated that Nf1 mutant mouse astrocytomas contains populations of proliferating nestin+ progenitor cells, suggesting that immature astroglial progenitors may serve as a reservoir of proliferating tumor cells. Here, we directly examined the consequences of Nf1 inactivation on neural stem cell (NSC) proliferation in vitro and in vivo. We found dose-dependent effects of neurofibromin expression on NSC proliferation and survival in vitro, which reflected increased RAS pathway activation and increased bcl2 expression. In addition, unlike wild-type NSCs, Nf1-/- NSCs and, to a lesser extent, Nf1+/- NSCs survive as xenografts in naive recipient brains in vivo. Although Nf1-/- NSCs are multipotent, Nf1-/- and Nf1+/-, but not wild-type, NSCs generated increased numbers of morphologically abnormal, immature astroglial cells in vitro. Moreover, the Nf1-/- NSC growth and survival advantage as well as the astroglial cell differentiation defect were completely rescued by expression of the GAP (RAS-GTPase activating protein) domain of neurofibromin. Finally, the increase in astroglial progenitors and proliferating cells seen in vitro was also observed in Nf1-/- and Nf1+/- embryonic as well as Nf1+/- adult brains in vivo. Collectively, these findings support the hypothesis that alterations in neurofibromin expression in the developing brain have significant consequences for astrocyte growth and differentiation relevant to normal brain development and astrocytoma formation in children.

Figure 1.

Nf1 loss leads to increased proliferation and self-renewal of NSCs in vitro. a, Increased numbers of Nf1-/- NSCs were observed at each time point (p < 0.001) compared with Nf1+/- and Nf1+/+ NSCs. b, The number of secondary neurospheres generated per primary neurosphere (a measure of self-renewal potential) was considerably higher in Nf1-/- neurospheres (p < 0.001). c, Neurospheres of all three genotypes showed clonal origin at low-density culture, with a significantly higher clonogenic incidence of Nf1-/- neurospheres (p < 0.001). The inset shows these clonal GFP+ neurospheres. d, The frequency of NSCs required to form neurospheres (limiting dilution analysis) revealed a much higher frequency of neurosphere formation by Nf1-/- NSCs. e, Nf1-/- neurospheres were significantly larger (p < 0.005) and contained five times more cells per sphere than Nf1+/+ spheres.

Figure 2.

Growth factor-deprived Nf1-/- NSCs have increased proliferative and survival advantages in vitro. a, Although very few growth factor-deprived Nf1+/+ NSCs (top) incorporated BrdU, a significantly higher number of Nf1-/- NSCs (bottom) did so under identical conditions (p < 0.001). The photomicrographs show phase contrast, 4′,6-diamidino-2-phenylindole (DAPI) (nuclear stain; blue), BrdU (green), and colocalization of DAPI and BrdU (merged; yellow), respectively. b, Nf1-/- NSCs exhibit reduced cell-cycle transit time as demonstrated by a shift in CFSE fluorescence 5 d after labeling compared with Nf1+/+ NSCs (open histogram). CFSE fluorescence at day 1 is shown by filled histograms. c, Staining with an antibody to cleaved (activated) caspase-3 showed decreased apoptosis in growth factor-deprived Nf1-/- NSCs compared with Nf1+/+ NSCs. d, Growth factor-deprived Nf1-/- NSCs exhibited increased Bcl2 expression, hyperactivation of RAS, and increased activation of RAS downstream effectors. Increased expression of Bcl2, RAS-GTP, phospho-Akt, and phospho-MAPK in growth factor-deprived Nf1-/- NSCs relative to Nf1+/+ NSCs is shown. These blots were stripped and reprobed with antibodies to total Akt and total MAPK. Total RAS is shown as a control for the RAS activity assay. Tubulin is included as an internal control for equal protein loading.

Figure 3.

Nf1-/- NSCs proliferate and survive as explants and in the brains of immunocompromised mice in vivo. a, A time course cell survival analysis of NSCs in vivo is shown using X-gal staining of representative cortical sections of nu/nu mice brains. Reporter-tagged (LacZ) Nf1+/+, Nf1+/-, and Nf1-/- NSCs were found at the injection site 3 d p.i. Although Nf1+/+ NSCs quickly disappeared from the injection site by 1 month p.i., injected Nf1-/- NSCs were found at and around the injection site. Few Nf1+/- NSCs were observed at the injection tract at 1 month p.i. At 2 months p.i., hardly any Nf1+/+ NSCs could be detected at the injection site. In contrast, Nf1-/- NSCs continued to survive in vivo. b, H&E staining revealed the presence of the injected Nf1-/- cells in the injection tract. Ki67 staining showed proliferating nuclei in the injection tract at 4 and 6 months p.i. c, The injected Nf1-/- NSCs differentiated into GFAP+, BLBP+ astrocytes at 4 and 6 months p.i.

Figure 4.

Nf1-/- NSCs undergo multilineage differentiation. a, No differences in the relative numbers of astrocytes and neurons were observed between Nf1+/+, Nf1+/-, and Nf1-/- cells. However, a 24% increase in O4+ cells was detected in differentiating Nf1-/- NSCs compared with wild-type NSCs. b, Although Nf1+/+ NSCs differentiated into morphologically distinct astrocytes, Nf1-/- NSCs gave rise to an arborous network of fibrillary-appearing astrocytes. Nf1+/- NSCs exhibited an intermediate phenotype. c, Although Nf1+/+, Nf1-/-, and Nf1+/- NSCs differentiated into morphologically similar O4+ oligodendrocytes, a subpopulation of O4-expressing Nf1-/- cells coexpressing GFAP were also detected.

Figure 5.

Loss of Nf1 leads to impaired NSC glial differentiation in vitro. Nf1+/+ NSCs differentiated into mature astrocytes, all of which exclusively or primarily expressed GFAP (green). In contrast, the GFAP+ cells that differentiated from Nf1-/- NSCs robustly coexpressed BLBP, vimentin, and RC2 (markers of immature astrocytes). a, Immunofluorescence staining of Nf1+/+ and Nf1-/- cells expressing GFAP (green), BLBP (red), and colocalization (merged; yellow). b, GFAP (green) and vimentin (red) expression and colocalization (merged; yellow) by Nf1+/+ and Nf1-/- cells. c, Expression of GFAP (green), RC2 (red), and their colocalization (merged; yellow) by Nf1+/+ and Nf1-/- cells. Nuclei in all cases were stained with 4′,6-diamidino-2-phenylindole.

Figure 6.

Ectopic expression of the NF1GRD rescues hyperproliferation and altered glial differentiation of Nf1-/- NSCs. Ectopic expression of the NF1 RASGAP-related domain (NF1GRD; inset), but not a nonfunctional NF1GAP domain (NF1GRD R1276P; inset), significantly reduced proliferation (a; p < 0.005) and self-renewal (b; p < 0.001) of Nf1-/- NSCs. The MEK kinase inhibitor PD98059 (20 μm) also significantly inhibited both hyperproliferation and increased self-renewal of Nf1-/- NSCs to levels comparable with those observed after ectopic NF1GRD expression (A, B). c, Reduction of Bcl2 expression, Akt, and MAPK activation to wild-type levels was observed after ectopic expression of the NF1GRD in Nf1-/- NSCs. No effect on Bcl2, Akt, and MAPK activation was observed in either Nf1-/- or Nf1+/+ NSCs after MSCV-Pac infection. All blots were stripped and reprobed with antibodies to total Akt and total MAPK. Tubulin is included as an internal control for equal protein loading. d, Immunofluorescence staining with antibodies to GFAP (green) and BLBP (red) demonstrated the characteristic morphology of Nf1-/- astrocytes that differentiated from Nf1-/- NSCs (left). Rescue of Nf1-/- NSCs infected with MSCV-NF1GRD (right) demonstrates differentiation into morphologically distinct astrocytes (green) with significantly reduced BLBP expression, comparable with that observed in wild-type cells. Nf1-/- NSCs infected with control virus (MSCV-Pac) showed robust expression of BLBP along with GFAP in astrocytes (left), similar to astrocytes that differentiated from uninfected Nf1-/- NSCs (data not shown). Infection of wild-type NSCs with either control virus or MSCV-NF1GRD did not alter the astrocyte phenotype (data not shown).

Figure 7.

Nf1+/- NSCs exhibit abnormal astroglial differentiation. Wild-type NSCs differentiated into GFAP+ flat astrocytes that mostly lacked BLBP expression (top). In contrast, Nf1+/- NSCs differentiated into a mixture of both morphologically distinct flat GFAP+ cells and filamentous GFAP+ cells (green). Unlike Nf1+/+ GFAP+ cells, Nf1+/- cells expressed varying levels of BLBP (red; bottom). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole.

Figure 8.

Altered neurofibromin expression results in increased neural stem/progenitor cells in the brains of Nf1+/- and Nf1-/- mice in vivo. a, Immunolocalization of neural stem and progenitor cells in the embryonic brains (E12.5) of Nf1+/+, Nf1+/-, and Nf1-/- mice. Increased numbers of Sox2+, BLBP+, Nkx2.2+, and Olig1+ cells were observed in the developing brains of both Nf1+/- and Nf1-/- mice compared with embryonic brains of Nf1+/+ mice. b, Persistent populations of proliferating cells were observed in the brain of adult Nf1+/- mice (arrows). BrdU+ proliferating cells are shown in the subventricular zone (SVZ) and hippocampus (HC) of Nf1+/+ and Nf1+/- mice. The number of BrdU+ cells were calculated for each region in Nf1+/+ and Nf1+/- mice. There were significantly more proliferating cells in the SVZ (p < 0.005) and HC (p < 0.001) of Nf1+/- mice compared with Nf1+/+ mice. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole.