Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation

Abstract

Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1(GFAP)CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1(GFAP)CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1(GFAP)CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.

FIG. 1.

Generation and characterization of GFAP-Cre transgenic mice. (A) Schematic diagram of the GFAP-Cre-IRES-LacZ transgene. The transgene contains the 2.2-kb fragment of the human GFAP promoter (hGFAP) and cDNAs encoding the nucleus-targeted proteins Cre recombinase (Cre) and β-galactosidase (nLacZ). The IRES sequence allows cotranslation of the Cre and LacZ proteins from the same mRNA. (B) Southern blot analysis of DNA from mouse tail biopsies digested with BglII and NotI to excise the GFAP-Cre-IRES-LacZ transgene (first five lanes) and of the original GFAP-Cre-IRES-LacZ construct used for pronuclear injection (lane +) with a 1-kb Cre probe. Founder 8 carries an 8-kb transgene identical to the original construct (lane +). The GFAP-Cre transgenic mice showed strong LacZ expression in the brain (C), while no LacZ expression was observed in the brain of wild-type littermates (D). Cre immunohistochemistry demonstrated Cre expression in the CA1 region of the hippocampus of the GFAP-Cre mice (E), whereas no Cre expression was detected in wild-type littermates (F). Double immunofluorescence with anti-β-galactosidase antibodies (H; green) and anti-Cre antibodies (I; red) demonstrated colocalization of the Cre and LacZ nuclear protein expression in the same cells in the CA1 region of the hippocampus (J; yellow). DAPI staining of nuclei is shown in panel G.

FIG. 2.

β-Galactosidase is expressed only in astrocytes in the brains of GFAP-Cre mice. LacZ is specifically expressed in GFAP-immunoreactive astrocytes (arrows point to representative astrocytes expressing LacZ) in the hippocampus, corpus callosum, and cerebellum. Note that Bergmann glia in the cerebellum show high LacZ expression. APC-immunoreactive oligodendrocytes (stars) in the corpus callosum and MAP2-immunoreactive neurons (arrowheads denote the neuronal cell bodies) in the hippocampus and cerebellum of the GFAP-Cre transgenic mice do not demonstrate LacZ activity. Magnification, ×400.

FIG.3.

LacZ reporter expression during embryonic development of GFAP-Cre transgenic mice. β-Galactosidase activity was detected by embryonic day 14.5 in the brain of the GFAP-Cre transgenic mice. In E18.5 GFAP-Cre embryos, robust LacZ activity is detected in the brain, spinal cord, and optic nerves. No LacZ expression is observed in a control littermate (E18.5 control). The intestines of both GFAP-Cre and control mice show endogenous LacZ activity. Mouse embryos were photographed at ×8 magnification at E11.5 and E13.5, ×5 magnification at E15.5, and ×3 magnification at E18.5.

FIG. 4.

Astrocyte-specific Nf1 conditional knockout mice demonstrate moderate increases in astrocyte number. Low-power photomicrographs of GFAP immunohistochemistry of 40-μm brain sections (microtome) were aligned from rostral to caudal, with the right side of each panel representing Nf1^GFAPCKO brain sections (CKO) and the left side of each panel representing brain sections from control mice (C). Nf1^GFAPCKO mice demonstrate increased GFAP immunoreactivity throughout the brain. Higher magnification of the boxed areas in the brains of control mice (C) on the left side of the upper panels (a, b, and c) and of the boxed areas in the brains of Nf1^GFAPCKO mice (CKO) on the right side of the upper panels (d, e, and f) revealed increased numbers of astrocytes in the corpus callosum, hippocampus, and cortex of Nf1^GFAPCKO mice. Increased numbers of astrocytes without major changes in morphology were also observed in Nf1^GFAPCKO hippocampal sections by GFAP and eosin staining (panels j, k, and l) compared to control mice (panels g, h, and i).

FIG. 5.

The number of GFAP-immunoreactive astrocytes is increased in the hippocampus CA1 region of astrocyte-specific Nf1 conditional knockout mice compared to that of control littermates. Quantitation of the GFAP-positive astrocytes in the CA1 region of the hippocampus of the mutant and control mice demonstrated 1.6-fold more astrocytes in 2-month-old Nf1^GFAPCKO mice than in GFAP-Cre and Nf1flox/flox controls (A) and a slight increase to 1.8-fold in 6-month-old (B) and to 2.2-fold in 12-month-old (C) Nf1^GFAPCKO mice compared with Nf1flox/flox and Nf1flox/wt controls. Data are presented as mean values ± SD and were analyzed by analysis of variance followed by the Bonferroni t test, with significance set at P < 0.05.

FIG. 6.

Increased astrocyte number in the brains of Nf1^GFAPCKO mice is the result of increased astrocyte proliferation. PCNA immunofluorescence identifies proliferating cells (red) in the CA1 region of the hippocampus of adult Nf1^GFAPCKO (CKO) mice. White arrows point to representative PCNA-labeled cells (panel a). Almost no PCNA-immunoreactive proliferating cells were present in the hippocampus of control (C) mice (panel d). GFAP immunofluorescence (green) of astrocytes in the CA1 region of the hippocampus is shown for CKO mice (panel b) and for control mice (panel e). Note that there are increased numbers of astrocytes in panel b, corresponding to the mutant mice, compared to panel e (control mice). Merged PCNA/GFAP/DAPI images are shown for CKO (panel c) and control (panel f) mice. The nuclei of the cells in these images are stained with DAPI and appear dark blue. The white arrows in the merged PCNA/GFAP/DAPI (panel c) for the Nf1^GFAPCKO mice denote several double-labeled GFAP-positive astrocytes with PCNA-labeled nuclei (pink). No GFAP/PCNA/DAPI-immunolabeled cells are observed in the hippocampus of control mice (panel f).

FIG. 7.

Inactivation of Nf1 influences the growth but not the tumorigenic properties of astrocytes derived from Nf1^GFAPCKO mice in vitro. (A) Cultured astrocytes were tested by PCR with specific primers to detect the presence of the Cre transgene, the conditional Nf1flox allele, and the recombined Nf1 allele. The recombined Nf1 allele (R-Nf1), as a result of Cre-mediated recombination of the conditional allele (floxNf1), is detected only in astrocytes that contain the Cre transgene (lanes 1 and 2) derived from astrocyte-specific Nf1 knockout mice. (B) Neurofibromin protein expression is lost in astrocytes in culture derived from the brains of GFAP-Cre Nf1flox/flox (Nf1^GFAPCKO) mice and reduced in astrocytes derived from GFAP-Cre Nf1flox/wt mice compared with astrocytes from the Nf1flox/flox littermates. (C) Cultured astrocytes derived from Nf1^GFAPCKO mice (Nf1CKO) show a growth advantage in comparison with control Nf1flox/flox (Nf1flox) astrocytes.

FIG. 8.

Embryonic day 12.5 Nf1^−/− astroglial cells show a significant increase in growth in vitro but lack tumorigenic properties. (A) Western blotting of astroglial cell extracts with an antineurofibromin antibody demonstrated loss of neurofibromin in Nf1^−/− astroglial cells. (B) Growth curves of Nf1^−/−, Nf1^+/−, and Nf1^+/+ astrocytes showed a significant growth advantage for Nf1^−/− astroglial cells at 3 and 6 days in culture. (C) When stimulated with fetal bovine serum, Nf1^−/− astroglial cells showed increased [³H]thymidine incorporation in comparison with Nf1^+/− astrocytes. (D) The growth advantage observed by cell counting and [³H]thymidine incorporation was also demonstrated by flow cytometry analysis of the cell cycle of Nf1^−/− and Nf1^+/− cells. (E) Nf1^−/− and Nf1^+/− astroglial cells do not form colonies in soft agar. C6 glioma cells were used as a positive control in the soft agar assay.

FIG. 9.

Postnatal day 2 Nf1^−/− astroglial cells show a significant increase in growth in vitro but lack tumorigenic properties. (A) Cre-adenovirus (Ad-Cre) treatment of astrocytes in culture results in Cre-mediated recombination of the Nf1flox allele (floxNf1) and formation of the recombined allele (R-Nf1), whereas no recombination is observed in astrocytes infected with a control LacZ adenovirus (Ad-LacZ). (B) No expression of the Nf1 protein (neurofibromin) was detected in Nf1flox/flox astrocytes after Ad5-Cre infection. The protein levels are not affected when Nf1flox/flox astrocytes are infected with Ad5-LacZ virus. (C) Ad5-Cre-infected Nf1flox/flox astrocytes (Ad-Cre) show a growth advantage compared with Ad5-LacZ-infected Nf1flox/flox astrocytes (Ad-LacZ).