Toll-like receptor 2 is required for opioids-induced neuronal apoptosis

Abstract

Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, is widely expressed in various systems, including the immune and nervous systems and plays a critical role in controlling innate and adaptive immune responses. We previously reported that opioids inhibit cell growth and trigger apoptosis. However, the underlying mechanism by which TLR2 mediates apoptosis in response to opioids is not yet known. Here we show that chronic morphine treatment in primary neurons dramatically increased the expression of TLR2 at both the messenger RNA and protein levels. In addition, TLR2 deficiency significantly inhibited chronic morphine-induced apoptosis in primary neurons. Activation of caspase-3 after morphine treatment is impaired in TLR2 deficient primary neurons. Moreover, morphine treatment failed to induce an increased level of phosphorylated glycogen synthase kinase 3 beta (GSK3beta) in TLR2 deficient primary neurons, suggesting an involvement of GSK3beta in morphine-mediated TLR2 signaling. These results thus demonstrate that opioids prime neurons to undergo apoptosis by inducing TLR2 expression. Our data suggest that inhibition of TLR2 is capable of preventing opioids-induced damage to neurons.

Figure 1

Morphine induces the expression of TLR2 in wild type neurons. A, Quantitative real time RT-PCR analysis of TLR2 expression showed a significant increase in wild type primary cortical neurons treated with morphine for 4 days. Neurons were treated with 15 µM morphine for 16 h, 2 d and 4 d, respectively. * p < 0.05 vs. untreated neurons. Results represent mean ± s.e.m. of three independent experiments. B, Immunostaining in wild type neurons showed a dramatic increase of TLR2-immunoreactive neurons after 15 µM morphine treatment for 6 days. Scale bars, 75 µm.

Figure 2

A deficiency of TLR2 is resistant to morphine-induced neuronal apoptosis. A, Bar graph showed the percentage of neuronal cell death in wild type (WT) and TLR2 knockout (TLR2 KO) neurons identified with PI staining. WT and TLR2 KO neurons were treated with 5 µM, 15 µM, or 50 µM morphine for 6 days, respectively. Results represent mean ± s.e.m. of three independent experiments. * p < 0.05 vs. untreated WT neurons. ** p < 0.05 vs. WT neurons treated with 15 µM morphine. *** p < 0.05 vs. WT neurons treated with 50 µM morphine. B, Representative light microscopic images showed TUNEL-positive neurons. WT and TLR2 KO neurons were treated with or without 15 µM morphine for 6 days. The dark brown color showed TUNEL-positive neuronal apoptosis (Red arrow head). Magnification 200 ×.

Figure 3

Morphine causes caspase-3 activation in wild type neurons but not in TLR2 knockout neurons. A, Total and active cleaved caspase-3 in protein levels were determined by western blot. WT and TLR2 KO neurons were treated with morphine at 5 µM or 15 µM for 6 days. Representative results of the level of cleaved caspase-3 and total caspase-3 are shown at the left of each pane. Data are representative of three independent experiments. * p < 0.05 vs. untreated WT neurons. ** p < 0.05 vs. WT neurons treated with 15 µM morphine. B, WT and TLR2 KO neurons were treated with or without 15 µM morphine for 6 days and immunostained with antibodies to cleaved caspase-3 (Red) and NeuN (neuronal marker) (Green). Merged images displayed co-localizations of cleaved caspase-3 and NeuN (Yellow) (White arrow head). Scale bars, 18.75 µm. Data are representative of three separate experiments.

Figure 4

TLR2 deficiency in neurons blocks morphine-enhanced GSK3β serine-9 phosphorylation. A, Phosphorylated serine-9 GSK3β (p-Ser9-GSK3β) and total GSK3β in protein levels were examined by western blot. WT and TLR2 KO neurons were treated with 5 µM or 15 µM morphine for 6 days, respectively. Data are representative of three independent experiments. B, WT and TLR2 KO neurons were treated with or without 15 µM morphine for 6 days and immunostained with p-Ser9-GSK3β antibody. C, Phosphorylated Akt (p-Akt) was determined by western blot. We treated WT and TLR2 KO primary neurons with or without 15 µM morphine for 6 days and examined for the level of phosphorylated Akt (p-Akt). Data are representative of three independent experiments.