Functions of fibroblast growth factor (FGF)-2 and FGF-5 in astroglial differentiation and blood-brain barrier permeability: evidence from mouse mutants

Abstract

Multiple evidence suggests that fibroblast growth factors (FGFs), most prominently FGF-2, affect astroglial proliferation, maturation, and transition to a reactive phenotype in vitro, and after exogenous administration, in vivo. Whether this reflects a physiological role of endogenous FGF is unknown. Using FGF-2 and FGF-5 single- and double mutant mice we show now a region-specific reduction of glial fibrillary acidic protein (GFAP), but not of S100 in gray matter astrocytes. FGF-2 is apparently the major regulator of GFAP, because in mice deficient for FGF-2, GFAP is distinctly reduced in cortex and striatum, whereas in FGF-5-/- animals only a reduction in the midbrain tegmentum can be observed. In FGF-2-/-/FGF-5-/- double mutant animals, GFAP-immunoreactivity is reduced in all three brain regions. Cortical astrocytes cultured from FGF-2-/-/FGF-5-/- double mutant mice revealed reduced levels of GFAP, but not S100 as compared with wild-type littermates. This phenotype could be rescued by exogenous FGF-2 but not FGF-5 (10 ng/ml). Electron microscopy revealed reduced levels of intermediate filaments in perivascular astroglial endfeet. This defect was accompanied by enhanced permeability of the blood-brain barrier (BBB), as detected by albumin extravasation. Levels of the tight junction proteins Occludin and ZO-1 were reduced in blood vessels of FGF-2-/-/FGF-5-/- double mutant mice as compared with wild-type littermates. Our data support the notion that endogenous FGF-2 and FGF-5 regulate GFAP expression in a region-specific manner. The observed defect in astroglial differentiation is accompanied by a defect in BBB function arguing for an indirect or direct role of FGFs in the regulation of BBB permeability in vivo.

Figure 1.

Protein expression of GFAP in different brain regions of mice deficient for FGF-2, as revealed by indirect fluorescence immunocytochemistry. A, Abundant GFAP-immunoreactive astrocytes can be observed in the frontal cortex of wild-type mice. B, In the same region of FGF-2^-/- animals, GFAP immunoreactivity has nearly disappeared. C, GFAP immunoreactivity is also abundant in the corpus callosum of wild-type mice, but in the knock-out (D) there is no overt reduction (D). E, GFAP immunoreactivity in astrocytes of the striatum in wild-type mice is distinctly reduced in FGF-2^-/--deficient mice (F). No overt differences in GFAP immunoreactivity between wild-type and FGF-2/FGF-5 double mutant mice can be observed in the midbrain tegmentum (G, H). Scale bar, 40 μm.

Figure 2.

Immunohistochemical detection of GFAP in the midbrain tegmentum and Western blot analysis of GFAP in different brain regions of FGF-5^-/- mice. A, Abundant GFAP-immunoreactive astrocytes can be observed in the midbrain tegmentum of wild-type mice. B, GFAP immunoreactivity in the same brain region of FGF-5^-/- mice has largely disappeared. C, Western blot. In FGF-5^-/- mice, immunoreactivity for GFAP is only reduced in the mesencephalon (Mes), whereas in frontal cortex (FCort), parietal cortex (PCort), and striatum (Striat) of FGF-5^-/--deficient mice (ko), no apparent alterations as compared with material from wild-type littermates (wt) can be observed. Scale bar, 40 μm.

Figure 3.

Immunocytochemical double staining for the astroglial marker proteins GFAP (A, B) and S100 (C, D) in the frontal cortex of FGF-2^-/-/FGF-5^-/- double mutant mice (B, D) and wild-type littermates (A, C). Note the apparent decrease of GFAP immunoreactivity in the frontal cortex of FGF-2^-/-/FGF-5^-/- double mutant mice, whereas S100 levels are unchanged. E, Statistical evaluation of numbers of S100-positive cells in a given area (500 × 500 μm) in frontal cortex (FCort), corpus callosum (CCall), striatum (Striat), and midbrain tegmentum (MBT) of FGF-2^-/-/FGF-5^-/- double mutant mice (ko) and their wild-type littermates (wt). Seven consecutive sections with a section interval of 50 μm were evaluated for each of six wild-type and knock-out animals. Scale bar, 40 μm.

Figure 4.

Western blot analysis of the astroglial marker proteins GFAP and S100 in different brain regions of FGF-2^-/-/FGF-5^-/- double mutant mice and wild-type littermates. GFAP immunoreactivity is clearly decreased in all brain areas studied: frontal cortex (FCort), parietal cortex (PCort), striatum (Striat), and mesencephalon (Mes); note that levels of S100 are not overtly affected.

Figure 5.

GFAP mRNA expression as revealed by semiquantitative RT-PCR. A, PCR products for GFAP derived from whole-cell RNA of frontal cortex (FCort), parietal cortex (PCort), striatum (Striat), and mesencephalon (Mes) of FGF-2^-/-/FGF-5^-/- double mutant mice are clearly reduced as compared with wild-type littermates. B, Parallel amplification of the housekeeping gene GAPDH revealed no apparent differences in the intensities of PCR products.

Figure 6.

GFAP (A, B) and S100 (C, D) immunoreactivities in primary cultures of cortical astrocytes from FGF-2^-/-/FGF-5^-/- double mutant mice (B, D) and wild-type littermates (A, C). Note reduction of GFAP immunoreactivity in cultures established from knock-out mice, with no apparent reduction in S100 immunoreactivity. E-G, Reduced levels of GFAP revealed by Western blot analysis can be rescued by application of exogenous FGF-2 alone or in combination with FGF-5. Note that FGF-5 failed to rescue the GFAP phenotype in cortical astrocytes (compare Fig. 2). Scale bar, 20 μm.

Figure 7.

A, B, Reduced levels of GFAP immunoreactivity in perivascular astroglial endfeet in the striata of FGF-2/FGF-5 double mutant mice (B) as compared with wild-type littermates (A). Scale bar, 20 μm. C, D, Electron microscopic images of representative areas showing capillary endothelial cells (EC) with adjacent basal lamina (^*) and astroglial endfeet (AC). Note the reduced number of intermediate filaments (arrowhead) and a disrupted cytoskeleton in the astroglial endfoot in the striatum of an FGF-2/FGF-5 double mutant mouse. MI, Mitochondrion. Scale bar, 1 μm. E, Frequency distribution of five distinct categories (Cat I-V) of perivascular astroglial endfeet distinguished by the density of intermediate filaments, as depicted in C and D. Comparison of FGF-2/FGF-5 double mutant mice and wild-type littermates: 100 capillaries were evaluated for each of five wild-type and knock-out mice. Data are given as the mean percentage of all vessels evaluated. ^*p < 0.05; ^**p < 0.01.

Figure 8.

Increased albumin extravasation in the striatum of FGF-2^-/-/FGF-5^-/- double mutant mice (B) as compared with wild-type littermates (A). Note that the strong albumin-specific immunoreactivity in the parenchyma adjacent to striatal blood vessels (arrowheads) in FGF-2^-/-/FGF-5^-/- double mutant mice (B) is lacking in wild-type littermates (A). Scale bar, 20 μm. C, Statistical evaluation of the proportion of brain capillaries with apparent albumin extravasation in frontal cortex (FCort), corpus callosum (CCall), striatum (Striat), and midbrain tegmentum (MBT) of FGF-2^-/-/FGF-5^-/- double mutant mice (ko) and their wild-type littermates (wt). Seven consecutive sections with a section interval of 50 μm were evaluated for each of six wild-type and knock-out animals.

Figure 9.

Immunofluorescence showing decreased levels of tight junction proteins ZO-1 and Occludin (arrowheads) in striatal blood vessels of FGF-2^-/-/FGF-5^-/- double mutant mice (B, D) as compared with wild-type littermates (A, C). Scale bar, 10 μm. E, Western blot analysis revealed decreased levels of Occludin in all brain areas studied. FCort, Frontal cortex; PCort, parietal cortex; Striat, striatum; Mes, mesencephalon. Scale bar, 10 μm.