Astrocyte activation is suppressed in both normal and injured brain by FGF signaling

Abstract

In the brain, astrocytes are multifunctional cells that react to insults and contain damage. However, excessive or sustained reactive astrocytes can be deleterious to functional recovery or contribute to chronic inflammation and neuronal dysfunction. Therefore, astrocyte activation in response to damage is likely to be tightly regulated. Although factors that activate astrocytes have been identified, whether factors also exist that maintain astrocytes as nonreactive or reestablish their nonreactive state after containing damage remains unclear. By using loss- and gain-of-function genetic approaches, we show that, in the unperturbed adult neocortex, FGF signaling is required in astrocytes to maintain their nonreactive state. Similarly, after injury, FGF signaling delays the response of astrocytes and accelerates their deactivation. In addition, disrupting astrocytic FGF receptors results in reduced scar size without affecting neuronal survival. Overall, this study reveals that the activation of astrocytes in the normal and injured neocortex is not only regulated by proinflammatory factors, but also by factors such as FGFs that suppress activation, providing alternative therapeutic targets.

Keywords: astrogliosis; brain damage.

Fig. 1.

Removal of FGF signaling in adult cortical astrocytes. (A) Sections of brains were collected and stained 2 wk after TM treatment of Nestin-CreER mice carrying a universal Cre-reporter allele, Rosa26^{lox-stop-lox-EGFP}. More than 99% of recombined cells (GFP⁺, green) are astrocytes (s100⁺, red) with a characteristic bush-like morphology; fewer than 1% are neurons (NeuN⁺, red), and no recombined microglia (Iba⁺, red) or Olig2⁺ precursors (red) can be detected. Colabeling was determined by using confocal microscopy, and colabeled cells were manually counted. (B) FGF signaling is active in cortical astrocytes. Quantitative real-time PCR of RNA from FACS-purified GFP⁺ astrocytes and GFP⁻ cells reveals expression of Fgfr1, Fgfr2, and FGF-responsive genes (Sprouty1, Erm, Er81). The neuronal markers Fox3 and Tbr1 confirm the purity of the astrocytes. (C–E) FGF signaling is down-regulated in the FGFR mutants 3 wk after TM treatment. (C) Quantitative real-time PCR analysis for FACS purified GFP⁺ astrocytes from control and mutant cortices indicates a decrease in FGFR expression and signaling in the LOF mutant (mean ± SEM, *P < 0.05 and **P < 0.001). (D and E) Colabeling by in situ hybridization for Fgfr1 or Erm mRNA and immunostaining for s100 reveals that fewer astrocytes express Fgfr1 and Erm in the mutant cortex. A cell was considered positive for Fgfr1 or Erm if more than five bright spots, a threshold set as being above background, were found in contact or overlapping with the Hoechst-stained nucleus (blue). Arrow, colabeled cells; arrowhead, astrocytes not expressing Erm or Fgfr1. (Scale bars: 20 μm.)

Fig. 2.

Loss of FGF signaling results in the activation of astrocytes in the cortex. (A) The intermediate neurofilament protein GFAP (green) is increased in the neocortical gray matter (ctx) of the LOF mutant 4 wk after TM treatment. (Lower) Higher magnifications of boxed areas. Hoechst (blue) is used as a counterstain. (Scale bars: Upper, 150 μm; Lower, 50 μm.). (B) Vimentin and nestin (red) are also increased in the LOF-mutant neocortex and colabel with GFAP (green). (Scale bar: 20 μm.)

Fig. 3.

Loss of FGF signaling in astrocytes induces microglia activation. (A) Cell proliferation, determined by immunostaining for the cell proliferation marker Ki-67 (red), is increased at 2 wk after TM treatment in the neocortex of the mutant, but then resolves back to control levels at 4 wk after TM treatment (mean ± SEM, **P < 0.001). Ki-67⁺ cells do not colabel with GFAP in the mutant neocortex. (B) The proliferating cells in the mutant are not astrocytes (s100⁺, red), but are oligodendrocyte precursors (Olig2⁺, red) or microglia (Iba1⁺, red); colabeling is yellow (arrowheads). (Scale bars: 20 μm.)

Fig. 4.

Loss of FGF signaling in cortical pyramidal neurons does not induce astrocyte activation. (A) CamK2a-CreER confers recombination of a universal Rosa26^{lox-stop-lox-EGFP} Cre-reporter allele in pyramidal neurons in the cortex. GFP⁺ cells colabel with the neuronal marker NeuN (arrows) in the neocortex of the CamK2a-CreER;Rosa26^{lox-stop-lox-EGFP} mice 3 wk after TM treatment. (Scale bars: Left, 100 μm; Right, 20 μm.) (B) No GFAP⁺ reactive astrocytes (red) are detected in the gray matter of CamK2a-CreER Fgfr triple mutants 3 wk after TM treatment. cc, corpus callosum; ctx, neocortex; hip, hippocampus. (Scale bar: 100 μm.)

Fig. 5.

Astrocytes that lack FGF signaling become reactive. Colabeling by immunofluorescence for GFAP⁺ protein and in situ hybridization for Fgfr1 or Erm RNA reveals that reactive astrocytes do not express Fgfr1 or Erm mRNA in the LOF mutant (n = 0 of 100 cells examined by confocal imaging). (Scale bar: 20 μm.)

Fig. 6.

FGF signaling inhibits injury-induced astrocyte activation. Controls, LOF mutants, and GOF mutants were subjected to a cortical stab wound injury 3 wk after TM treatment. Representative images of coronal sections of GFAP immunostaining (red; Hoechst, blue) were taken at 1, 3, and 20 d post injury (dpi). Integrated intensity of GFAP staining was quantified in boxed areas parallel to the plane of the neocortex (ctx) immediately adjacent to the edge of the lesion (dotted line) for 1 and 3 d post injury, 130 μm from the lesion for 20 d post injury (to avoid the scar), and, in all cases, 100 μm from the corpus callosum (cc). Intensities were normalized to the measurement in the matching position of the contralateral hemisphere. The graphs represent the relative change of the integrated intensity to the mean measurement in controls, which was set as 1 (mean ± SEM; *P < 0.05 and **P < 0.001). hip, hippocampus. (Scale bars: 100 μm.)

Fig. 7.

Aberrant FGF signaling reduces glial scar formation. (A) Immunostaining for GFAP in the neocortex (ctx) 20 d after a stab wound injury reveals significantly smaller scars not only in the GOF mutants, as expected, but also, surprisingly, in the LOF mutants. The area covered by scars and the integrated intensity of GFAP staining in the scar were measured in the boxed areas as described in Materials and Methods. For analysis, coronal sections were positionally matched between brains along the stab wound, which extends perpendicularly to the section along the anterior–posterior axis and which, because of the triangular shape of the blade, is deeper in more posterior areas. Changes are relative to the mean value obtained for controls (set as 1; mean ± SEM; *P < 0.05). hip, hippocampus. (Scale bars: Upper, 200 μm; Lower, 50 μm.) (B) Proliferation (Ki-67 staining) of reactive astrocytes (GFAP staining) around the lesion 3 d after injury is reduced in LOF mutants, but increased in GOF mutants, providing a potential explanation for why both mutants exhibit smaller scars. Colabeling for GFAP and Ki-67 (arrows) was quantified by confocal microscopy of 20-μm sections (mean ± SEM; **P < 0.001). (Scale bar: 25 μm.)