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. 2011 Oct 7;286(40):34722-32.
doi: 10.1074/jbc.M111.280826. Epub 2011 Aug 11.

Long Term Potentiation Is Impaired in Membrane Glycoprotein CD200-deficient Mice: A Role for Toll-like Receptor Activation

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Free PMC article

Long Term Potentiation Is Impaired in Membrane Glycoprotein CD200-deficient Mice: A Role for Toll-like Receptor Activation

Derek A Costello et al. J Biol Chem. .
Free PMC article

Abstract

The membrane glycoprotein CD200 is expressed on several cell types, including neurons, whereas expression of its receptor, CD200R, is restricted principally to cells of the myeloid lineage, including microglia. The interaction between CD200 and CD200R maintains microglia and macrophages in a quiescent state; therefore, CD200-deficient mice express an inflammatory phenotype exhibiting increased macrophage or microglial activation in models of arthritis, encephalitis, and uveoretinitis. Here, we report that lipopolysaccharide (LPS) and Pam(3)CysSerLys(4) exerted more profound effects on release of the proinflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNFα), in glia prepared from CD200(-/-) mice compared with wild type mice. This effect is explained by the loss of CD200 on astrocytes, which modulates microglial activation. Expression of Toll-like receptors 4 and 2 (TLR4 and -2) was increased in glia prepared from CD200(-/-) mice, and the evidence indicates that microglial activation, assessed by the increased numbers of CD11b(+) cells that stained positively for both MHCII and CD40, was enhanced in CD200(-/-) mice compared with wild type mice. These neuroinflammatory changes were associated with impaired long term potentiation (LTP) in CA1 of hippocampal slices prepared from CD200(-/-) mice. One possible explanation for this is the increase in TNFα in hippocampal tissue prepared from CD200(-/-) mice because TNFα application inhibited LTP in CA1. Significantly, LPS and Pam(3)CysSerLys(4), at concentrations that did not affect LTP in wild type mice, inhibited LTP in slices prepared from CD200(-/-) mice, probably due to the accompanying increase in TLR2 and TLR4. Thus, the neuroinflammatory changes that result from CD200 deficiency have a negative impact on synaptic plasticity.

Figures

FIGURE 1.
FIGURE 1.
TLR2- and TLR4-induced increases in inflammatory cytokines are enhanced in glia prepared from CD200−/− mice. Incubation of mixed glia prepared from wild type mice in the presence of LPS (100 ng/ml; a–c) or Pam3Csk4 (Pam; 100 ng/ml; d–f) increased supernatant concentrations of IL-1β, IL-6, and TNFα (**, p < 0.01; ***, p < 0.001; ANOVA; n = 4–8), and the effect of LPS and Pam3Csk4 was significantly greater in cells prepared from CD200−/− mice (+++, p < 0.001; ANOVA; n = 4–8). Error bars, S.E.
FIGURE 2.
FIGURE 2.
MHCII+ CD11b+ and CD40+ CD11b+ cells are increased in glia prepared from CD200−/− mice. Shown is the mean percentage of CD11b+ cells that also stained positively for MHCII+ (top panels) and CD40 (bottom panels). Data are presented as target proteins versus side scatter (SSC). The right-hand panels illustrate representative overlays.
FIGURE 3.
FIGURE 3.
CD200 is expressed on astrocytes. CD200 expression was observed on GLAST+ cells from purified astrocytic cultures obtained from wild type (a) but not CD200−/− (b) mice. CD200 was observed in membrane, but not cytosolic, fractions prepared from purified astrocytes obtained from wild type mice. GFAP expression was observed in the cytosolic fraction (c).
FIGURE 4.
FIGURE 4.
Expression of TLR2 and TLR4 is increased in glia prepared from CD200−/− mice. TLR4 mRNA (a) and TLR2 mRNA (d) and the number of CD11b+ cells that stained positively for TLR4 (b and c) and TLR2 (e and f) were increased in glia prepared from CD200−/− compared with wild type mice (*, p < 0.05; Student's t test for independent means; n = 5). Mean data from densitometric analysis (g) and a sample immunoblot (h) reveal that phosphorylated IκBα is increased in cells prepared from CD200−/− compared with wild type mice (*, p < 0.05; Student's t test for independent means; n = 4–6). Error bars, S.E.
FIGURE 5.
FIGURE 5.
Markers of microglial activation are increased in cells prepared from CD200−/− mice. a and b, expression of CD40 mRNA, but not CD11b mRNA, was significantly greater in mixed glia prepared from CD200−/− compared with wild type mice (*, p < 0.05; Student's t test for independent means; n = 4–5). c–f, flow cytometric analysis revealed that the percentage of CD11b+ cells was similar in wild type and CD200−/− (c), but the percentage of CD11b+ cells that also stained positively for CD40 (d and e) and MHCII (f and g) was significantly greater in mixed glia obtained from CD200−/− compared with wild type mice (*, p < 0.05; ***, p < 0.001; Student's t test for independent means; n = 4–8). Error bars, S.E.
FIGURE 6.
FIGURE 6.
The increase in hippocampal expression of TLR2 and TLR4 in CD200−/− mice is coupled with a deficit in LTP. a, greater numbers of CD11b+CD45low cells were found in the hippocampus of CD200−/− compared with wild type mice (***, p < 0.001; Student's t test for independent means), and expression of CD200R expression (b) and CD40 (c) was greater in tissue prepared from CD200−/− compared with wild type mice. TLR2 (d) and TLR4 (f) expression on CD11b+CD45low cells was greater in tissue prepared from CD200−/− compared with wild type mice, whereas TLR2 mRNA (e) and TLR4 mRNA (g) expression were significantly increased in hippocampal tissue prepared from CD200−/− compared with wild type mice (*, p < 0.05; **, p < 0.01; Student's t test for independent means; n = 5). h, TBS (arrow) induced LTP in CA1 synapses of hippocampal slices prepared from wild type mice (15 slices from 11 mice). LTP, measured as mean percentage EPSP slope in the last 5 min of the experiment, was significantly reduced in slices prepared from CD200−/− mice relative to wild type mice (p < 0.001; 12 slices from seven mice). Sample recordings immediately before and 60 min following TBS are shown for wild type and CD200−/− mice (scale bars, 1 mV/20 ms). Error bars, S.E.
FIGURE 7.
FIGURE 7.
Increased hippocampal expression of TNFα in CD200−/− mice may underlie the associated deficit in LTP. IL-1α and IL-1β were similar in tissue prepared from wild type and CD200−/− mice (a and b), but TNFα was significantly increased (p < 0.05; Student's t test for independent means; c), as revealed by sample immunoblots and analysis of densitometric data. Application of TNFα (3 ng/ml) to hippocampal slices significantly impaired LTP relative to vehicle controls (p < 0.05; unpaired Student's t test; three slices from two mice; d). Sample EPSP traces immediately prior to and 60 min following TBS are presented (scale bars, 1 mV/20 ms). Error bars, S.E.
FIGURE 8.
FIGURE 8.
LTP is attenuated by LPS in CD200−/− mice. a, perfusion of LPS (20 μg/ml) for 60 min prior to TBS (arrow) decreased LTP in slices prepared from wild type mice (five slices from five mice), and the mean percentage EPSP slope in the last 5 min of the experiment was significantly decreased compared with control slices (p < 0.0001; 15 slices from 11 mice). b and c, LTP in slices prepared from wild type mice was unaffected by perfusion of 10 μg/ml LPS for 20 min prior to TBS (b; seven slices from six mice relative to control 15 slices from 11 mice), but LTP was attenuated in slices from CD200−/− mice (c; p < 0.05; 13 slices from nine mice relative to control 12 slices from seven mice). Sample EPSP traces immediately prior to and 60 min following TBS are presented (scale bars, 1 mV/20 ms). Error bars, S.E.
FIGURE 9.
FIGURE 9.
LTP is attenuated by Pam3Csk4 in CD200−/− mice. a, perfusion of Pam3Csk4 (20 μg/ml) for 60 min prior to TBS (arrow) decreased LTP in slices prepared from wild type mice (three slices from three mice), and the mean percentage EPSP slope in the last 5 min of the experiment was significantly decreased compared with control slices (15 slices from 11 mice; p < 0.001). b and c, LTP in slices prepared from wild type mice was unaffected by perfusion of 10 μg/ml Pam3Csk4 for 20 min prior to TBS (b; four slices from three mice relative to control 15 slices from 11 mice). However, LTP was attenuated in slices from CD200−/− mice following treatment with 10 μg/ml Pam3Csk4 (c; six slices from five mice relative to control 12 slices from seven mice; p < 0.05). Sample EPSP traces immediately prior to and 60 min following TBS are presented (scale bars, 1 mV/20 ms). Error bars, S.E.

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