Chondroitin sulfate proteoglycan tenascin-R regulates glutamate uptake by adult brain astrocytes

Abstract

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.

Keywords: Astrocytes; Chondroitin Sulfate; Extracellular Matrix; Glutamate; Tenascin.

FIGURE 1.

CS-56 immunoreactivity localizes to astrocytes but not oligodendrocytes in the adult mouse cerebral cortex. A, representative images of immunostaining with CS-56 antibody in the adult mouse cerebral cortex. Schematic diagram of a coronal section of the mouse brain (A1) is shown, and the fluorescence image of the enclosed region appears in A2. Cx, cerebral cortex; cc, corpus callosum. Scale bar, 300 μm. B–F, representative double immunostaining with CS-56 antibody (B1–G1; green) and cell marker antibodies (B2–G2; red) are shown. Each pair of images was merged and is indicated in the 3rd column (B3–G3). The markers included S100β (B2), GLAST (C2), and GS (D2) mature astrocyte markers; GSTπ (E2) and myelin basic protein (MBP) (F2), mature oligodendrocyte markers; and NG2 (G2), an immature oligodendrocyte marker. For control staining, normal mouse IgM (H), normal mouse IgG (I), or normal rabbit IgG (J) was used instead of the primary antibodies. The inset in C3 is an enlargement of the enclosed region. Scale bar, 100 μm. Note that the CS-56-immunoreactive patches closely encased S100β-positive astrocytic nuclei (arrows in B1–B3) and partially co-localized with GLAST-positive astrocytes (enlarged region in C3), demonstrating their astrocytic origin. However, two other astrocytic markers, GS (D) and GFAP (data not shown), barely overlapped with CS-56 immunoreactivity. As shown in E1–E3 and F1–F3, mature oligodendrocyte markers did not co-localize with CS-56 immunoreactivity; likewise, the immature oligodendrocyte marker NG2 did not (G1–G3) overlap with CS-56-positive patches.

FIGURE 2.

Representative electron micrographs of CS-56 immunoreactivity in the adult mouse cerebral cortex. A, CS-56 immunoreactivity was detected in a cell with narrow cytoplasm and an irregularly shaped nucleus with rich chromatin, all of which are morphological characteristics of the astrocyte (As). B, fine cellular process with CS-56-immunoreactive membrane (arrowheads) is juxtaposed with an axonal terminal (T) and a postsynaptic spine (S). C, CS-56-immunoreactive processes are attached to a capillary (Cap) with an endothelial nucleus (End) in the form of end feet (arrows). Scale bars, A and C are1 μm; B is 500 nm.

FIGURE 3.

Purification and identification of mouse brain CSPGs. A, purification steps for glycoproteins from the adult mouse cerebral cortex are indicated. B, representative silver-stained polyacrylamide gel of fractionated purified glycoproteins. Lane M, protein size markers (kDa). The protein bands in the right lane were cut out, in-gel-digested, and subjected to MALDI-TOF mass spectrometry. Mass fingerprints for phosphacan, versican, brevican, neurocan, and tenascin-R were obtained, and protein-specific peptides were identified using Mascot software. Band identities are indicated on the right side.

FIGURE 4.

TNR mRNA is localized in CS-56-positive astrocytes in the adult mouse cerebral cortex. In situ hybridization for TNR mRNA (arrows in A and B) and immunostaining with CS-56 antibody (A, red) or WFA (B, red) was performed in single sections of the adult mouse cerebral cortex. Note that halo-like CS-56 immunoreactivity surrounded the TNR mRNA-containing cell bodies, although WFA-positive perineuronal nets did not. To identify TNR mRNA-positive cells, in situ hybridization for TNR mRNA (arrows in C1 and D1, red), immunostaining with the astrocyte marker S100β (C2, green), and the mature oligodendrocyte marker GSTπ (D2, green) were simultaneously performed in the adult mouse cerebral cortex. The TNR mRNA-positive cells were a subpopulation of astrocytes with nuclear S100β immunoreactivity (arrows in C). GSTπ-positive oligodendrocytes were distinct from TNR mRNA-positive cells (arrow in D). Scale bars in A and B are 100 μm; C and D are 50 μm.

FIGURE 5.

TNR immunoreactivity in cultured astrocytes. A, representative confocal images of anti-S100β (A1, red) and anti-GFAP (A2, green) double-immunostaining in cultured astrocytes (A3, merged image of A1 and A2). We determined the number of S100β and GFAP immunoreactive cells and then calculated their ratios to total cells (DAPI-positive cells) (A4). Values are shown as the mean ± S.E. of three different experiments. B, representative confocal images of double-immunostaining with anti-S100β (B1, red) and CS-56 (B2, green) in cultured astrocytes. We measured the ratio of CS-56-positive cells to S100β-positive astrocytes (B4). Values are shown as the mean ± S.E. of three different experiments. C, representative confocal images of double labeling with anti-S100β (C1, red) and anti-TNR (C2, green) antibodies in cultured astrocytes. We determined the ratio of TNR-immunoreactive cells to S100β-positive cells (C4). Values are shown as the mean ± S.E. of three different experiments. Scale bar, 50 μm. Note that TNR-positive cells composed a subpopulation of cultured astrocytes expressing S100β (arrows in C).

FIGURE 6.

Knockdown of TNR expression decreases glutamate uptake in cultured astrocytes. Glutamate (A), d-serine (B), and ATP (C) release assays were performed with cultured astrocytes transfected with scrambled or two different TNR siRNAs (si-1 and si-2) for 72 h. Gliotransmitters were measured after treating cells for 10 min with BzATP (500 μm), DHPG (50 μm), or tACPD (500 μm) in KRS. D, glutamate uptake was determined after incubating with 20 nm l-[³H]glutamate in KRS for 10 min. In a separate set of glutamate uptake experiments, cells were preincubated with the pan-glutamate transporter antagonist TBOA (300 μm) for 15 min, and glutamate uptake was then examined as noted above. Values are shown as the mean ± S.E. of nine cell samples from three different experiments. *, p < 0.05; **, p < 0.01, significantly different from value obtained for cells transfected with scrambled siRNA. Note that glutamate uptake activity was decreased by TNR knockdown.

FIGURE 7.

Knockdown of TNR expression decreases GLAST expression in cultured astrocytes. A, real time PCR analysis of mRNA expression of astrocyte-specific genes in cultured astrocytes transfected with scrambled or TNR-specific siRNAs (si-1 and si-2) for 72 h. B, representative Western blot analysis of GLAST, GLT-1, and GAPDH proteins. GAPDH was used as a loading control. Lane M, protein size markers (in kDa). The positions of monomer (arrow) and dimer (arrowhead) bands of GLAST are indicated. Numbers indicate independent cell samples. C and D, semi-quantitative densitometric data for GLAST (C) and GLT-1 (D) expression are shown. Values are shown as the mean ± S.E. of nine cell samples from three different experiments. *, p < 0.05; **, p < 0.01, significantly different from value obtained for cells transfected with scrambled (scra) siRNA. Note that TNR siRNA expression resulted in a marked decrease in GLAST expression but not GLT-1 expression. E, representative double immunostaining of cultured astrocytes with TNR and GLAST antibodies. Scale bar, 50 μm. TNR and GLAST were co-localized in cultured astrocytes (arrow). F, real time PCR analysis of mRNA expression of proteoglycan and GLAST genes in cultured astrocytes transfected with scrambled siRNA or proteoglycan-specific siRNAs (two, si-1 and si-2, for each proteoglycan) for 72 h. Note that conventional lecticans were not involved in the regulation of GLAST expression.