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. 2008 Nov 12;28(46):11980-8.
doi: 10.1523/JNEUROSCI.2920-08.2008.

G-protein-coupled receptor screen reveals a role for chemokine receptor CCR5 in suppressing microglial neurotoxicity

Kazushige Gamo  1 , Sumiko Kiryu-Seo, Hiroyuki Konishi, Shunsuke Aoki, Kouji Matsushima, Keiji Wada, Hiroshi Kiyama
Affiliations

G-protein-coupled receptor screen reveals a role for chemokine receptor CCR5 in suppressing microglial neurotoxicity

Kazushige Gamo et al. J Neurosci. .

Abstract

G-protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins, and several GPCRs have been implicated in signaling between neurons and glia to protect neurons from pathological stresses. Here, we have used a screening strategy to investigate GPCRs that are involved in neuronal protection. The real-time PCR was performed using 274 primers targeting nonsensory GPCR mRNAs, which were listed on the database. The cDNAs from control and nerve-injured hypoglossal nuclei of mouse brain were used, and the alterations of PCR products were compared. This screen and the subsequent in situ hybridization screen exhibited six GPCR mRNAs which were prominently and convincingly induced in nerve-injured hypoglossal nuclei. Among these candidates, the chemokine receptor CCR5 was selected, based on the marked induction in CCR5 mRNA in microglia after nerve injury. The mRNA expression of ligands for CCR5, such as regulated on activation normal T-cell expressed and secreted (RANTES/CCL5), MIP-1alpha, and MIP-1beta, were induced in injured motor neurons, indicating that CCR5 and its ligands were expressed in microglia and neurons, respectively, in response to nerve injury. In vitro, lipopolysaccharide (LPS)-induced expression of mRNAs for inflammatory cytokines (IL-1beta, IL-6, and tumor necrosis factor-alpha) and inducible nitric oxide synthase (iNOS) in microglia were all suppressed by RANTES. Those suppressions were not observed in microglia from CCR5 null mice. In addition, nerve injury-induced motor neuron death seen in wild type C56BL/6J mice was accelerated in CCR5 knock-out C57BL/6J. These results may suggest that CCR5-mediated neuron-glia signaling functions to protect neurons by suppressing microglia toxicity.

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Figures

Figure 1.
Figure 1.
In situ hybridization screening. ISH screening demonstrated six upregulated (A) and three downregulated (B) gene expressions 5 d after nerve injury. The left columns (a–i) show emulsion autoradiography under dark-field illumination, and the right columns (j–r) show high-power bright-field photograph counterstained with thionine. The mRNA-positive signal (silver grain) for F2RL1 (a, j), MC4R (b, k), AGTR2 (g, p), TRHR (h, q), and VIPR2 (i, r) were observed on neurons. Those for CCR5 (c, l), CX3CR1 (d, m), GPR84 (e, n), and P2RY12 (f, o) are seen on microglia, which locate adjacent to the large-sized neurons and heavily stained with thionine. Scale bars: left, 50 μm; right, 25 μm.
Figure 2.
Figure 2.
mRNAs for MIP-1α, MIP-1β, and RANTES are upregulated in nerve-injured hypoglossal motor neurons. A, Expression of mRNAs for CCR5 ligands 5 d after axotomy. The left columns show film autoradiography demonstrating that mRNA expressions are observed in injured side of hypoglossal nucleus (right side), and the right columns show emulsion autoradiography, indicating that those hybridization signals (silver grain) are mainly observed on neurons. Scale bars: left, 1 mm; right, 25 μm. B, RT-PCR analysis of mRNA expression for MIP-1α, MIP-1β, and RANTES in control and injured hypoglossal nuclei 5 d after axotomy.
Figure 3.
Figure 3.
Expression profiles of mRNAs for CCR5 and its ligands (MIP-1α, MIP-1β, RANTES) in injured hypoglossal nucleus after axotomy. Semiquantification of mRNA expression levels from film autoradiography of hybridized tissue sections. Data represent the average intensity of the positive signals from film autoradiograms. Error bars indicate SE (statistically significant differences by paired t test, *p < 0.01).
Figure 4.
Figure 4.
RANTES suppressed LPS-induced mRNA expression of inflammatory cytokines in microglia. Microglia from wild-type (WILD) and CCR5−/− (CCR5 KO) mice were cultured and treated for 6 h with 1 μg/ml LPS with and without 0.5 μg/ml RANTES. mRNA expression for IL-1β, IL-6, iNOS, and TNF-α was examined by RT-PCR. Error bars indicate SD (statistically significant differences by paired t test, *p < 0.001). Each bar represents results of five separate experiments.
Figure 5.
Figure 5.
Motor neuron death is accelerated in CCR5-deficient mice. The representative thionine-stained sections of hypoglossal nucleus from wild-type (WILD) and CCR−/− (CCR5 KO) mice. The hypoglossal nucleus is shown by the dashed line. The right side hypoglossal nerve was transected. Note that the number of motor neurons survived in the nerve-injured side of CCR5 null mouse is significantly less than those in wild type. cc, Central canal; XII, hypoglossal nucleus. Scale bar, 50 μm.
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