A role for ErbB signaling in the induction of reactive astrogliosis

Reactive astrogliosis is a hallmark of many neurological disorders, yet its functions and molecular mechanisms remain elusive. Particularly, the upstream signaling that regulates pathological responses of astrocytes is largely undetermined. We used a mouse traumatic brain injury model to induce astrogliosis and revealed activation of ErbB receptors in reactive astrocytes. Moreover, cell-autonomous inhibition of ErbB receptor activity in reactive astrocytes by a genetic approach suppressed hypertrophic remodeling possibly through the regulation of actin dynamics. However, inhibiting ErbB signaling in reactive astrocytes did not affect astrocyte proliferation after brain injury, although it aggravated local inflammation. In contrast, active ErbB signaling in mature astrocytes of various brain regions in mice was sufficient to initiate reactive responses, reproducing characterized molecular and cellular features of astrogliosis observed in injured or diseased brains. Further, prevalent astrogliosis in the brain induced by astrocytic ErbB activation caused anorexia in animals. Therefore, our findings defined an unrecognized role of ErbB signaling in inducing reactive astrogliosis. Mechanistically, inhibiting ErbB signaling in reactive astrocytes prominently reduced Src and focal adhesion kinase (FAK) activity that is important for actin remodeling, although ErbB signaling activated multiple downstream signaling proteins. The discrepancies between the results from loss- and gain-of-function studies indicated that ErbB signaling regulated hypertrophy and proliferation of reactive astrocytes by different downstream signaling pathways. Our work demonstrated an essential mechanism in the pathological regulation of astrocytes and provided novel insights into potential therapeutic targets for astrogliosis-implicated diseases.


Figure S3
Effects of dnEGFR expression on ErbB receptor activation and molecular hallmarks in reactive astrocytes. (a) Astrogliosis was induced by stab wound injury in the cortices of both Mlc1-dnEGFR and littermate control mice. Shown are representative GFAP-immunostaining results of injured cortices 3 days or 7 days post injury (dpi). Note the similar increase in GFAP immunoreactivity in the area adjacent to the injury sites in both groups. (b) Nestin expressed in reactive cortical astrocyte 3 days post injury. Cortical slices from Mlc1-dnEGFR and littermate control mice with or without injury were immunostained for GFAP and nestin. Note there were neither GFAP nor nestin expressing in intact cortices. (c) Representative real-time RT-PCR results of EGFR/dnEGFR transcripts in injured cortical tissues of Mlc1-dnEGFR and littermate control mice 3 days post injury. Gel image showed the PCR products of four repeats after full run (40 cycles) of real-time RT-PCR. Note the apparent increase of EGFR/dnEGFR PCR products in Mlc1-dnEGFR group. (d) Quantitative analysis of the transcript increase of dnEGFR in the injured cortical tissues of a Mlc1-dnEGFR mouse in comparison with that of littermate control 3 days post injury. **, P = 0.0012, n = 3 replicates, unpaired t test. (e) Activities of ErbB receptors in primary astrocytes of Mlc1-dnEGFR and control mice induced by ErbB receptor ligands. Primary astrocytes were treated with saline (Ctrl), rhEGF (1 µg/ml), or rhNRG1 (100 ng/ml) for 15 min, respectively, and ErbB receptors and their phosphorylation levels in cell lysates were examined by Western blotting using specific antibodies. (f) Quantitative analyses of ErbB phosphorylation levels revealed by Western blotting in astrocytes stimulated by ErbB ligands. Phosphorylation levels of indicated proteins were normalized by its total protein levels. ##, P < 0.01; #, P < 0.05; as compared with the treatment with saline. ***, P < 0.001; **, P < 0.01; *, P < 0.05; as compared with control astrocytes with the same treatment. n = 3 for each protein, paired t test.

Figure S4
AAV-TRE-YFP targeted normal astrocytes in the uninjured cortices and reactive astrocytes in the injured cortices. AAV-TRE-YFP labeled cells were immunopositive for astrocyte marker glutamine synthetase (GS) in either the injured or the uninjured cortices (a), but only positive for nestin in the injured cortices (b), of Mlc1-dnEGFR and Mlc1-tTA mice. Images were taken under the 40X oil-immersion objective of a Zeiss710 confocal microscope. Note the reactive astrocytes labeled by YFP in the injured cortices of both mice on 3 days post injury (dpi) exhibited bigger sizes than those in the uninjured cortices. Moreover, the reactive astrocytes labeled by YFP in the injured cortices of Mlc1-dnEGFR mice were smaller than those in control mice.

Figure S5
Morphological exhibition of cortical astrocytes by TRE-YFP expression. (a) Comparison of the labeling of cortical reactive astrocytes by TRE-YFP expression and fluorescence dye filling. AAV-TRE-YFP labeled astrocytes in injured cortices 3 days post injury were identified under a fluorescence microscope and filled with fluorescence dye Alexa Fluor 568 by glass micropipettes. Shown are projected images of a labeled astrocyte that were captured at Z-stack series by a Zesiss710 confocal microscope with a 40X oil-immersion objective. Note that YFP labeled cell bodies and all fine processes and exhibited a more complete morphology of the cell than the fluorescence dye did. (b) Shown is the gallery of Z-stack serial images of the same astrocyte in a. Note at every optical slice, YFP exhibited better coverage of the cell than Alexa Fluor 568 did due to labeling of a live cell. Moreover, it was noticeable that the biggest fluorescence area of the astrocyte taken by the confocal microscope was within 6.3-µm Z-axial range through its cell body (from 18.09 µm to 24.12 µm).

Figure S6
Colocalization of Olig2 + and Ki67 + nuclei induced by stab wound injury. Uninjured or injured cortices from wild type mice were sectioned and immunostained for Olig2 and Ki67. Note that injury induced the number of Olig2 + nuclei as well as Ki67 + nuclei increased in cortical regions adjacent to the injury sites. Arrows indicate the colocalization of Olig2 and Ki67 in the same nuclei. Arrowheads indicate the nuclei positive for Olig2 only.

Figure S7
ErbB2 V664E was expressed in various brain regions in Mlc1-ErbB2 V664E mice. Brain slices from Mlc1-ErbB2 V664E and littermate control mice 20 days after Dox withdrawal were immunostained for ErbB2. Note ErbB2 + cells increased dramatically in different brain regions of Mlc1-ErbB2 V664E mice as compared with that of littermate controls.

Figure S8
Cell-autonomous activation of ErbB signaling in astrocytes promoted cell proliferation. (a) Primarily cultured astrocytes from Mlc1-ErbB2 V664E brain exhibited different morphology from that of control mice. Shown are representative images of cultured astrocytes in bright field, or immunostained for GFAP, Acsbg1, or RC2. Note normal astrocytes cultured in vitro were flat and adhered well to the dish bottom, while astrocytes isolated from Mlc1-ErbB2 V664E brain were plump and small. (b) Cell growth rate of cultured astrocytes from Mlc1-ErbB2 V664E mice was much faster than that from control mice. Primary cultured astrocytes were plated into 24-well plate at 5×10 3 cells per well, and cells in each well were trypsinized and counted at different time points. **, P < 0.01; ***, P < 0.001; n = 3 replicates for each time point, one-way ANOVA. (c) Increased Olig2 + nuclei in the brain of Mlc1-ErbB2 V664E mice. Brain slices from Mlc1-ErbB2 V664E and littermate control mice 20 days after Dox withdrawal were immunostained for Olig2. (d) Colocalization of Olig2 with Ki67 in some nuclei in the cortices of Mlc1-ErbB2 V664E mice. White arrows, Ki67 + Olig2 + nuclei. (e) Localization of Olig2 + nuclei in nestin + cells in the brain of Mlc1-ErbB2 V664E mice. White arrows, nestin + Olig2 + cells. A nucleus (DAPI + ) for a nestin + cell was identified by the association with its main cell body. (f) Reactive astrocytes in Mlc1-ErbB2 V664E mice exhibited gene expression characteristics of both A1 and A2 subtypes. Total RNA extracted from the cortices of Mlc1-ErbB2 V664E and control mice were subjected to real-time RT-PCR with specific primers to examine the characteristic genes of A1 or A2 subtypes. Results shown were normalized by the mRNA levels of internal control gapdh and the same genes in control mice. n = 3 replicates for each gene.

Figure S9
Spontaneous astrogliosis induced by ErbB activation in astrocytes was accompanied by inflammation throughout the brain. (a) Reactive microglia was induced in the brain of Mlc1-ErbB2 V664E mice. Shown are representative immunostaining results of GFAP and microglia marker Iba1 in brain slices of Mlc1-ErbB2 V664E and littermate control mice 20 days after Dox withdrawal. (b) Quantitative analyses of Iba1 + cell densities in the cortices and corpora callosa in Mlc1-ErbB2 V664E mice and littermate controls. *, P = 0.0287; **, P = 0.0007; n = 3 for each group, paired t test. (c) No expression of ErbB2 V664E in reactive microglia or leukocytes of Mlc1-ErbB2 V664E mice. Shown are representative immunostaining results of ErbB2 and Iba1, or ErbB2 and CD45, in the cortices of Mlc1-ErbB2 V664E mice after Dox withdrawal.

Figure S10
Inflammation in the brain of Mlc1-ErbB2 V664E mice was induced by reactive astrocytes. (a) Iba1 + cells were not reactive in the brain of Mlc1-ErbB2 V664E mice with early astrogliosis. Shown are representative immunostaining results of GFAP and Iba1 in cortical slices of Mlc1-ErbB2 V664E and littermate control mice 3 days after Dox withdrawal. Note that cortical astrocytes already started to express GFAP as this stage. (b) Quantitative analyses of Iba1 + and GFAP + cell densities in the cortices of Mlc1-ErbB2 V664E mice and littermate controls 3 days after Dox withdrawal. **, P = 0.001, n = 3 for each group, paired t test. (c) Only GFAP + cells in the brain of Mlc1-ErbB2 V664E mice with early astrogliosis had proliferation marker Ki67. Shown are representative immunostaining results of GFAP and Ki67 in cortical slices of Figure S11 Mlc1-tTA did not target enteric glia in the gastrointestinal tract. (a) ErbB2 was not overexpressed in gastrointestinal tract of Mlc1-ErbB2 V664E mice. Stomach sections from Mlc1-ErbB2 V664E or control mice 20 days after Dox withdrawal were immunostained for GFAP and ErbB2. Enteric glia were labeled by GFAP immunostaining. Arrowheads indicate the cells positive for GFAP but not ErbB2. (b) AAV-TRE-YFP did not label cells in gastrointestinal tract of Mlc1-tTA mice. Small intestine of Mlc1-tTA mice were exposed and 2 µl AAV-TRE-YFP was injected into the intestine wall. One day later the injected part was isolated and sectioned for immunostaining of GFAP. Enteric glia in the submucosa of plicae circulares were positive for GFAP (arrowheads). No YFP signal was detected in the virus-injected intestine sections. There were some spots in the sections exhibited higher green autofluorescence due to tissue folding or overlapping at the edge.

Figure S12
No detectable Src and FAK activities in the uninjured cortices by immunostaining. Cortical slices from uninjured brains of Mlc1-dnEGFR and littermate control mice were co-immunostained by mouse antibodies against Aldh1L1 or Acsbg1, both are previously reported markers for cortical astrocytes, and rabbit/goat antibodies against the active forms of FAK or Src, respectively. Although the antibody against Aldh1L1 failed to label cortical astrocytes, the immunostaining results showed there were neither FAK nor Src activities detected in normal astrocytes of either Mlc1-dnEGFR or control mice.