Astroglial P2X7 receptor current density increased following long-term exposure to rotenone

Abstract

The role of the interaction between neurons and glial cells in the pathogenesis of neurodegenerative diseases is gaining more attention. Neuroinflammation participates in the progressive nature of diverse neurologic diseases including Parkinson's disease, Alzheimer's disease and multiple sclerosis. Activated microglia release neurotoxic molecules, which take part in the neuroinflammatory responses. Astrocytes are also key players in these responses. Reactive astrocytes secrete inflammatory factors, including tumor necrosis factor-α (TNF-α). This secretion can be regulated by extracellular ATP mediated through P2X7 receptors. However, whether the activity of astrocytic P2X7 receptors changes in Parkinson's disease and whether these changes would influence the secretion of inflammatory factors in astrocytes are still unclear. In our study, through immunocytochemistry, whole-cell patch clamp and ELISA assay, we found that P2X7 receptors were expressed in midbrain astrocytes, and that, rotenone, a Parkinson's disease model used at a low concentration (2-20 nM) for 48 h increased the P2X7 receptor current density and thereby inhibited the secretion of TNF-α. Our research suggests that rotenone can regulate cytokine secretion of astrocytes through elevated P2X7 channel current density and, in turn, take part in the neuroinflammatory process in the rotenone Parkinson's disease model.

Fig. 1a, b

Expression of P2X7 receptors in cultured astroglia. a Representative pictures of astrocytes which were stained by GFAP (green, A1), P2X7 receptor (red, A2) antibody and Hoechst dye (blue, A3) after 72 h in culture. A4 Merged picture. Immunofluorescence signal intensity of GFAP was stronger in the cells showing hypertrophy with long processes (thin arrows) than in the polygonal type cells (thick arrows). P2X7 receptors were preferentially expressed in the hypertrophic cells, while the signal intensity was relatively low, or sometimes could not be detected in the polygonal type cells. Bar = 100 μm. b Representative pictures of astrocytes which were treated by various concentrations of rotenone for various time periods. B1 0 nM rotenone (control) for 24 h, B2 2 nM for 24 h, B3 20 nM for 24 h, B4 200 nM for 24 h, B5 0 nM rotenone (control) for 48 h, B6 2 nM for 48 h, B7 20 nM for 48 h, B8 200 nM for 48 h. Cells clustered into islands after 72 h in culture (B5–8). The signal intensity of P2X7 receptors in astrocytes that remained in the treatment group (B2–4, 6–8) was not significantly different from that of the control group (B1, 5). Bar = 50 μm

Fig. 2a, b, c

Rotenone influences the P2X7 receptor current densities in a time- and concentration-dependent way. a Sample traces of inward currents induced by 300 μM BzATP in astrocytes after treatment with 0, 2, 20 or 200 nM rotenone for 24 or 48 h. Numbers in parentheses represent the membrane capacitance of astrocytes investigated. b Histogram of P2X7 receptor current densities after different treatments. *P_{200 vs. 0 nM, 24 h} < 0.05, n = 18. ^†P_{2 vs. 0 nM, 48 h} < 0.05, n = 16. ^‡P_{20 vs. 0 nM, 48 h} < 0.01, n = 16. ^§P_{200 vs. 0 nM, 48 h} < 0.05, n = 15. ^¶P_{20 vs. 2 nM, 48 h} < 0.05, n = 16. ^‖P_{0 nM, 48 vs. 24 h} < 0.05, n = 17. c The inward current elicited by 100 μM BzATP can be blocked by 10 μM BBG

Fig. 3a, b

Rotenone influences the P2X7 receptor current densities, which are related to cell age or LPS pretreatment. a After 24 h recovery, astrocytes were treated with rotenone for 24 or 48 h (groups 24hours and 48hours) before the currents were tested by 100 μM BzATP. After 48 h recovery, astrocytes were treated with 2 and 20 nM rotenone for 24 h (group 24hours′), and the current densities did not increase. b After 24 h recovery, astrocytes were treated with rotenone and 10 μg/ml LPS for 24 or 48 h (groups 24hours and 48hours) before the currents were tested by 100 μM BzATP

Fig. 4

Rotenone inhibits secretion of TNF-α from astrocytes. Except group blank, the cells of all groups were treated with 10 μg/ml LPS for 48 h along with respective treatments. The cells of group blank were cultured for 48 h without any treatment. Control LPS alone, rotenone 20 nM, apyrase 5 U/ml, BzATP 100 μM. *P_{rotenone vs. control} < 0.05. ^†P_{rotenone+BBG vs. rotenone} < 0.05. ^‡P_{BzATP vs. control} < 0.01. ^§P_{rotenone+BzATP vs. rotenone} < 0.05