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Lentiviral-Mediated Overexpression of the 18 kDa Translocator Protein (TSPO) in the Hippocampal Dentate Gyrus Ameliorates LPS-Induced Cognitive Impairment in Mice

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Lentiviral-Mediated Overexpression of the 18 kDa Translocator Protein (TSPO) in the Hippocampal Dentate Gyrus Ameliorates LPS-Induced Cognitive Impairment in Mice

Wei Wang et al. Front Pharmacol.

Abstract

The 18 kDa translocator protein (TSPO) is involved in the immune/inflammatory response. However, the exact role that TSPO plays in neuroinflammation-induced cognitive impairment is still elusive. The purpose of our present study was to investigate the effects of lentiviral-mediated hippocampal overexpression of the TSPO in a mouse model of LPS-induced cognitive impairment. We established a mouse cognitive impairment model using systematic daily administration of lipopolysaccharide (LPS) (0.5 mg/kg). Microinjection of the dentate gyrus of the mouse with lentiviral vectors, which contained a cDNA targeting TSPO (Lv-TSPO), resulted in a significant increase in TSPO expression and allopregnanolone production. Mice treated with LPS showed cognitive deficits in the novel object recognition test and the Morris water maze test that could be ameliorated by TSPO overexpression. In addition, TSPO overexpression reversed LPS-induced microglial activation and accumulation of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Moreover, TSPO overexpression attenuated the LPS-induced impairment of hippocampal neurogenesis. Our results suggest that local overexpression of TSPO in the hippocampal dentate gyrus alleviated LPS-induced cognitive deficits, and its effects might be mediated by the attenuation of inflammatory cytokines, inhibition of microglial activation, and promotion of neurogenesis.

Keywords: TSPO; allopregnanolone; cognitive impairment; hippocampus; neurogenesis; neuroinflammation.

Figures

FIGURE 1
FIGURE 1
Experimental design. (A) Treatment schedule and test order for the optimized dose of lipopolysaccharide (LPS). LPS or saline was administered consecutively for 6 days following a 1-week acclimatization. The open field test (OFT) was performed 12, 16, 20, and 24 h after LPS/saline administration over the subsequent 6 days. (B) Treatment schedule and test order for evaluating the effects of TSPO overexpression and LPS. Brdu was injected three times at 3-h intervals. The next day, lentiviral vectors containing TSPO-cDNA (Lv-TSPO) or the negative control (NC) sequence were infused into the bilateral dentate gyrus (2 × 108 tu, 1 μl/side) of the mouse hippocampus. Behavioral tests were carried out from day 15 to day 26. LPS (0.5 mg/kg) or saline was injected (i.p.) once daily from day 14 to day 26, and the animals were sacrificed on day 27. MWM, Morris water maze; OFT, open field test; NOR, novel object recognition; LV, lentiviral vectors; SAC, sacrifice.
FIGURE 2
FIGURE 2
Locomotor activity after administration of different doses of LPS. The open field test was conducted at different times after the first LPS injection. (A) Line crossing times in the OFT. (B) Rears in the OFT. LPS (0.2, 0.5, and 1 mg/kg) decreased locomotor activity 12 and 16 h after injection, but it recovered after 20 h, except in the 1 mg/kg mice (n = 6). P < 0.05, ∗∗∗P < 0.001 vs. saline group.
FIGURE 3
FIGURE 3
Lv-TSPO increased TSPO expression and allopregnanolone levels in the hippocampus. (A) Microinjection of Lv-TSPO induced high, specific expression of EGFP (green) in the dentate gyrus, as observed under fluorescence microscopy. Scale bar = 200 μm. (B) Lv-TSPO increased hippocampal TSPO expression significantly. Protein bands on the gel, and the expression level was normalized to β-actin as an internal control. Values are expressed as fold-change over the mean values of the negative control mice. (C) Lv-TSPO significantly increased allopregnanolone expression. The data are expressed as the means ± SEM (n = 6). ∗∗∗P < 0.001 vs. NC.
FIGURE 4
FIGURE 4
Lv-TSPO ameliorates cognitive impairment in LPS-exposed mice. All behavioral tests were performed as described in the Methods section. Both LPS and Lv-TSPO have no effect on spontaneous locomotor activity, as tested by the open field test. (A) Line crossing times in the OFT. (B) Rears in the OFT. (C) Swimming speed showed no difference among groups during the MWM test. (D) Escape latency during the acquisition training process showed no difference at each time point among all the groups. (E,F) LPS decreased the time for platform crossing and the percentage of target quadrant occupancy during time in the probe trial. The reduction was reversed by Lv-TSPO. (G) LPS decreased the index of recognition in the NOR test, while Lv-TSPO could reverse this reduction. The data are expressed as the means ± SEM (n = 10). P < 0.05, ∗∗P < 0.01 vs. NC + saline, #P < 0.05 vs. NC + LPS. MWM, Morris water maze; OFT, open field test; NOR, novel object recognition; Lv, lentiviral vectors.
FIGURE 5
FIGURE 5
Lv-TSPO suppresses the LPS-induced accumulation of inflammatory cytokines and microglial activation in the hippocampus. (A) Representative images of Iba1-labeled activated microglia in the hippocampal dentate gyrus. Activated microglia are shown in green. Scale bar = 150 μm. (B) Quantification of the Iba1-labeled cells. LPS increased Iba1-labeled cells, while Lv-TSPO attenuated this increase. (C) LPS increased the integral optical density (IOD) of Iba1 staining, which was attenuated by Lv-TSPO. The data are expressed as the means ± SEM (n = 4). LPS increased the accumulation of IL-1β (D), IL-6 (E) and TNF-α (F), all of which were reversed by Lv-TSPO. The data are expressed as the means ± SEM (n = 6). P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. NC + saline, #P < 0.05, ###P < 0.001 vs. NC + LPS, &P < 0.05 vs. Lv-TSPO + saline.
FIGURE 6
FIGURE 6
Lv-TSPO ameliorates the reduction of neurogenesis caused by LPS. (A) Micrographs and quantification of Brdu-labeled cells (red) in the dentate gyrus. Scale bar = 200 μm. (B) Confocal micrographs and quantification of NeuN (green) and Brdu (red) co-labeled cells in the dentate gyrus. Scale bar = 50 μm (C) Brdu-labeled cells were decreased by LPS, which was reversed by Lv-TSPO. (D) LPS decreased the number of NeuN- and Brdu- co-labeled cells, which was reversed by Lv-TSPO. (E) The percentage of new neurons among the Brdu-labeled cells was decreased by LPS, while this was attenuated by Lv-TSPO. The data are expressed as the means ± SEM (n = 4). ∗∗P < 0.01 vs. NC + saline, #P < 0.05 vs. NC + LPS.

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