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. 2017 Apr 28:11:116.
doi: 10.3389/fncel.2017.00116. eCollection 2017.

Aquaporin 4-Mediated Glutamate-Induced Astrocyte Swelling Is Partially Mediated through Metabotropic Glutamate Receptor 5 Activation

Zhongfang Shi  1   2   3   4   5 , Wei Zhang  1   2   3   4   5 , Yang Lu  1   2   3   4   5 , Yi Lu  1   2   3   4   5 , Lixin Xu  1   2   3   4   5 , Qing Fang  1   2   3   4   5 , Min Wu  1   2   3   4   5 , Mei Jia  1   2   3   4   5 , Yujiao Wang  1   2   3   4   5 , Liping Dong  1   2   3   4   5 , Xu Yan  1   2   3   4   5 , Shaohua Yang  1   2   3   4   5   6 , Fang Yuan  1   2   3   4   5
Affiliations

Aquaporin 4-Mediated Glutamate-Induced Astrocyte Swelling Is Partially Mediated through Metabotropic Glutamate Receptor 5 Activation

Zhongfang Shi et al. Front Cell Neurosci. .

Abstract

Astrocytes are one of the most abundant cell types in the mammalian central nervous system (CNS), and astrocyte swelling is the primary event associated with brain edema. Glutamate, the principal excitatory amino acid neurotransmitter in the CNS, is released at high levels after brain injury including cerebral ischemia. This leads to astrocyte swelling, which we previously demonstrated is related to metabotropic glutamate receptor (mGluR) activation. Aquaporin 4 (AQP4), the predominant water channel in the brain, is expressed in astrocyte endfeet and plays an important role in brain edema following ischemia. Studies recently showed that mGluR5 is also expressed on astrocytes. Therefore, it is worth investigating whether AQP4 mediates the glutamate-induced swelling of astrocytes via mGluR5. In the present study, we found that 1 mM glutamate induced astrocyte swelling, quantified by the cell perimeter, but it had no effect on astrocyte viability measured by the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays. Quantitative reverse transcription polymerase chain reaction analyses revealed that AQP4, among AQP1, 4, 5, 9 and 11, was the main molecular expressed in cultured astrocytes. Glutamate-induced cell swelling was accompanied by a concentration-dependent change in AQP4 expression. Furthermore, RNAi technology revealed that AQP4 gene silencing inhibited glutamate-induced astrocyte swelling. Moreover, we found that mGluR5 expression was greatest among the mGluRs in cultured astrocytes and was co-expressed with AQP4. Activation of mGluR5 in cultured astrocytes using (S)-3,5-dihydroxyphenylglycine (DHPG), an mGluR5 agonist, mimicked the effect of glutamate. This effect was abolished by co-incubation with the mGluR5 antagonist fenobam but was not influenced by DL-threo-β-benzyloxyaspartic acid (DL-TBOA), a glutamate transporter inhibitor. Finally, experiments in a rat model of transient middle cerebral artery occlusion (tMCAO) revealed that co-expression of mGluR5 and AQP4 was increased in astrocyte endfeet around capillaries in the penumbra, and this was accompanied by brain edema. Collectively, these results suggest that glutamate induces cell swelling and alters AQP4 expression in astrocytes via mGluR5 activation, which may provide a novel approach for the treatment of edema following brain injury.

Keywords: aquaporin 4; astrocyte; glutamate; metabotropic glutamate receptor 5; swelling.

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Figures

Figure 1
Figure 1
Glutamate-induced cell swelling in cultured astrocytes. (A) Measurement of astrocyte survival after treatment with 0, 1, or 10 mM glutamate for 48 h by cell counting kit-8 (CCK-8) assay showed that 1 mM glutamate had no effect on astrocytes viability, whereas 10 mM glutamate decreased astrocyte viability. (B) Lactate dehydrogenase (LDH) release rates measured using an LDH kit showed that 1 and 10 mM glutamate had no effect on astrocyte viability. (C,E) Immunofluorescence staining for GFAP (shown in green). Representative images showing the effects of exposure of astrocytes to 0, 1, or 10 mM glutamate for 48 h (C), and 1 mM glutamate for 0, 1, 3, 6, 12, 24, or 48 h (E). Scale bar shows 200 μm. (D) Quantitative analysis of image data from panels in (C) showed that astrocyte cell perimeter significantly increased after exposure to 1 or 10 mM glutamate for 48 h. (F) Quantitative analysis of image data from panels in (E) showed that astrocytes swelled 1 h after treatment with 1 mM glutamate and maintained a similar level of swelling at 3 and 6 h. Astrocytes swelling then decreased at 12 and 24 h but again increased at 48 h compared to the control group (the dotted line). Control group corresponds to “0” in (A), (B), (D), (F). *P < 0.05, compared with control; #P < 0.05, compared with 1 h; n = 9; graphed data show mean ± SD.
Figure 2
Figure 2
Glutamate-induced changes in aquaporin 4 (AQP4) expression in cultured astrocytes. (A,B) Change in expression AQP family subtype mRNAs after incubation with 1 mM glutamate for 48 h. Results showed that AQP4 expression was the highest, followed by AQP11. Besides AQP4 and AQP11, the expression of AQP1, 5, and 9 mRNAs were also found in cultured astrocytes. After incubation in 1 mM glutamate for 48 h, the expression of AQP1, 4, 5, 9 and 11 were all significantly increased approximately two-fold compared to levels before incubation. (C,G) Immunofluorescence staining for AQP4 (shown in green). Representative images showing effects of 0, 1, or 10 mM glutamate for 48 h (C), and exposure to 1 mM glutamate for 1, 3, 6, 12, 24, or 48 h (G). Scale bar shows 200 μm. (D–H) Quantitative analysis of data from (C) and (G). (E,F) Levels of AQP4 protein and mRNA after 48-h glutamate incubation were by detected Western blot and quantitative reversetranscription-PCR (qRT-PCR), respectively. Compared with the control group, 1 or 10 mM glutamate both induced an increase in AQP4 expression. (I) AQP4 mRNA expression was measured by qRT-PCR. Results showed that AQP4 mRNA significantly increased at 1 h after treatment with 1 mM glutamate, and this level was maintained at 3 h. AQP4 expression was reduced to a normal level at 6 and 12 h, before falling significantly below the normal level at 24 h. However, expression of AQP4 mRNA again increased at 48 h. Control group corresponds to “0” in (D), (E), (F), (H), (I). *P < 0.05, compared with control; #P < 0.05, compared with 1 h; n = 9; graphed data show mean ± SD.
Figure 3
Figure 3
Glutamate-induced swelling of astrocytes was mediated by AQP4. (A,C) Levels of AQP4 protein in cultured astrocytes were measured by immunofluorescence and western blot, respectively, after incubation with glutamate and AQP4 siRNAs. In the images, AQP4 is shown in green (A). Scale bar shows 200 μm. (B) Quantitative analysis of data from (A). The results showed that 1 mM glutamate increased the level of AQP4 protein. This was inhibited by AQP4 siRNA transfection, but not the mismatch siRNA transfection. (D) Expression of AQP4 mRNA was detected by qRT-PCR. The result was consistent with that of AQP4 protein expression. (E) Astrocytes were transfected with siRNA for 24 h, before exposure to 1 mM glutamate for 48 h. Representative photos taken with an inverted fluorescence microscope show GFAP immunofluorescence in green. Scale bar shows 200 μm. (F) Quantitative analysis of cell perimeter from images in (E). One millimolar glutamate caused an increase in astrocyte perimeter which was blocked when AQP4 siRNA was transfected into astrocytes to silence the gene. In contrast, mismatch siRNA transfection failed to inhibit the increase of astrocyte perimeter induced by glutamate. *P < 0.05, compared with AQP4 siRNA (−), 0 mM glutamate; #P < 0.05, compared with mismatch; n = 9; graphed data show mean ± SD.
Figure 4
Figure 4
mGlu5 mediated glutamate-induced astrocyte swelling and changes in AQP4 expression. (A) Expression of metabotropic glutamate receptor (mGluR) in cultured astrocytes, as measured by RT-PCR. (B) Quantitative analysis of data from (A). Results showed that mGluR1, 3, 5, 7 and 8 were expressed in astrocytes, and mGluR5 expression was the highest. (C) Co-expression of AQP4 and mGluR5 in cultured astrocytes, as measured by double-immunofluorescence staining. (D) Quantitative analysis of cell perimeters after GFAP immunofluorescence staining. Compared to the control, the cell perimeters of astrocytes in the Glu and DHPG groups were larger, whereas the DMSO group showed no difference compared to the control. Cell perimeters in the Glu + Fenobam group were smaller than in the Glu group, and likewise smaller in the DHPG + Fenobam group vs. the DHPG group. However, there were no cell perimeter differences between the Glu + DL-TBOA and Glu groups. (E) Quantitative analysis of qRT-PCR results showed that AQP4 mRNA expression in the Glu and DHPG group was higher than in the control group, whereas the DMSO group did not differ from control. Compared to the Glu group, expression was lower in the Glu + Fenobam group, but the level in the Glu + DL-TBOA group did not differ. The level of AQP4 mRNA was lower in the DHPG + Fenobam group compared to the DHPG group. *P < 0.05, compared to the control group; #P < 0.05, compared to the Glu group; &P < 0.05, compared to the DHPG group; n = 9; graphed data show mean ± SD. DHPG, (S)-3,5-dihydroxyphenylglycine, an mGluR5 agonist; DL-TBOA, DL-threo-β-benzyloxyaspartic acid, a glutamate transporter-1 (GLT-1) inhibitor.
Figure 5
Figure 5
Increased co-expression of mGluR5 and AQP4 in a rat model of transient middle cerebral artery occlusion (tMCAO). (A) Hematoxylin and eosin (HE) staining showed that neuronal cells in the sham group were arranged regularly, and glial cells and capillary morphogenesis were normal. After tMCAO, most cell arrangement was disordered, with pyknotic or severely shrunken nuclei; in certain areas, hypervacuolization and cellular edema were found. (B) Double immunofluorescence staining showed the expression of GFAP (red) and AQP4 (green) in astrocytes, which increased after tMCAO. (C) Double immunofluorescence staining showed that mGluR5 (red) and AQP4 (green) were rarely co-expressed in pericapillary areas in the normal brain, but co-expression was increased in the pericapillary area and cell membranes of astrocytes after tMCAO. Scale bar shows 20 μm. (D) Statistical analysis of chart (B). (E) Colocalization of AQP4 and mGluR5 was determined using ImageJ software. Three independent fields containing more than three randomly selected images per condition in three different experiments were assessed and analyzed, and the average Mander’s overlap coefficient was calculated. *P < 0.05, compared to the sham group, graphed data show mean ± SD.
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