P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis - PubMed (original) (raw)

P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis

Carlos Matute et al. J Neurosci. 2007.

Abstract

Oligodendrocyte death and demyelination are hallmarks of multiple sclerosis (MS). Here we show that ATP signaling can trigger oligodendrocyte excitotoxicity via activation of calcium-permeable P2X(7) purinergic receptors expressed by these cells. Sustained activation of P2X(7) receptors in vivo causes lesions that are reminiscent of the major features of MS plaques, i.e., demyelination, oligodendrocyte death, and axonal damage. In addition, treatment with P2X(7) antagonists of chronic experimental autoimmune encephalomyelitis (EAE), a model of MS, reduces demyelination and ameliorates the associated neurological symptoms. Together, these results indicate that ATP can kill oligodendrocytes via P2X(7) activation and that this cell death process contributes to EAE. Importantly, P2X(7) expression is elevated in normal-appearing axon tracts in MS patients, suggesting that signaling through this receptor in oligodendrocytes may be enhanced in this disease. Thus, P2X(7) receptor antagonists may be beneficial for the treatment of MS.

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Figures

Figure 1.

Oligodendrocytes express functional P2X receptors with high Ca2+permeability. A, ATP and BzATP, but not α,β-meATP, evoke inward, nondesensitizing currents, which are potentiated in the absence of Ca2+and Mg2+. ATP currents are blocked by PPADS (100 μ

), a nonselective P2X antagonist. B, C, The P2X7 antagonists oATP (1 m

) and BBG block ATP and BzATP currents. **p < 0.01. D, E, ATP and BzATP induce a rapid increase in [Ca2+]i, an effect that is blocked by PPADS (50 μ

) and in Ca2+-free incubation buffer, and greatly reduced by the P2X7 antagonists oATP (1 m

) and BBG (100 n

). **p < 0.01 and ***p < 0.001.

Figure 2.

P2X7 receptors are expressed in oligodendrocytes and myelin. A, Western blot illustrating the presence of P2X7 receptor in rat optic nerve (rON) and rat oligodendrocytes in culture (rOL). B, P2X7 receptors revealed by double-immunofluorescence labeling in rat MBP+ oligodendrocytes in culture (left column) and in optic nerve APC+ oligodendrocytes (right column). Scale bar, 20 μm. C, Electron microscopic images of longitudinal (top) and transversal (bottom) sections showing immunogold staining of P2X7 receptors in myelin sheaths (inset and arrowheads). Scale bars, 300 nm.

Figure 3.

P2X receptor activation triggers oligodendrocyte death in dissociated cultures and in isolated optic nerve in vitro, as well as MS-like lesions in vivo. A, Toxicity of ATP (left) and BzATP (right) in optic nerve oligodendrocytes in culture, as measured 24 h later with the calcein viability assay. Oligodendrocyte death is observed after exposure to ATP and BzATP at 1 m

and 10 μ

, respectively (*p < 0.05 for comparison between agonist and control). This is prevented by coapplication of the broad-spectrum P2X antagonist PPADS, by P2X7 receptor antagonists oATP and BBG, and by Ca2+ removal from the culture medium (# p < 0.05 for each comparison between agonist and the various conditions). B, C, Perfusion of rat optic nerves with aCSF-containing ATP results in chromatin condensation and caspase-3 activation in oligodendrocytes labeled with APC, an effect that is prevented by coapplication with P2X7 antagonists. Scale bar, 10 μm. *p < 0.05, ***p < 0.001 for comparison between control and treatment with agonist; # p < 0.05, ## p < 0.01 for comparison between agonist and agonist plus antagonist treatment. D, BzATP slowly infused onto the rabbit optic nerve induces defined lesions with microgliosis [isolectin B4 (IB4)] as well as demyelination and axonal damage, as shown by staining with antibodies to MBP and dephosphorylated NFH, respectively. Left and right panels correspond to low and high magnification of damaged nerves, respectively (scale bars, 500 and 50 μm). E, BzATP-induced lesions are prevented by coapplication of P2X7 antagonists BBG or oATP. Scale bar, 500 μm. The histogram shows the extent of damaged and demyelinated area attributable to BzATP application and its reduction with BBG and oATP. **p < 0.01.

Figure 4.

P2X7 antagonists ameliorate chronic EAE-associated neurological symptoms and restore normal axon conduction velocity. A, B, Established chronic EAE induced in C57BL/6 mice by immunization with MOG improves after treatment with oATP (p < 0.001) and BBG (p < 0.01) starting at 21 d after injection (arrows). C, The increase in axon conduction latency in the corticospinal tract of mice with chronic EAE (***p < 0.001 vs control, non-immunized animals) is greatly attenuated after treatment with oATP and BBG (## p < 0.01 and ### p < 0.001, respectively, compared with MOG immunized mice). D, Immunoblotting with tissue samples from lumbar spinal cord of mice with chronic EAE (left) and corresponding histogram (middle; OD, optic density) show a decrease in the levels of MBP and P2X7 receptor that is inhibited by BBG. Sample loading was normalized to actin levels. Symbols in histogram denote a statistically significant reduction of protein levels in vehicle-treated mice versus control, non-immunized mice (*p < 0.05 and **p < 0.01) and an increase in BBG-treated mice versus the vehicle group (# p < 0.05). P2X7 levels (right) correlate with those of MBP (r = 0.44 and p < 0.05, Pearson's correlation). E, Nissl and MBP immunostaining of consecutive longitudinal sections from the lumbar spinal cord of mice with chronic EAE. Arrows point to same blood vessels. Demyelination is evident in mice treated with vehicle but not in mice treated with P2X7 antagonist BBG. Nissl staining reveals that demyelination in the former mice may also occur in the absence of signs of inflammation. Scale bar, 100 μm. Data shown in C–E were obtained at 40 dpi.

Figure 5.

P2X7 receptor levels are increased in MS. A, Hoechst (top) and immunofluorescence staining of oligodendrocytes (APC+ cells) with antibodies to P2X7 receptors in the human optic nerve from controls and normally appearing MS samples. Merge images show that P2X7 receptors are located in oligodendrocytes in both control and MS samples. In turn, cells of the microglial lineage (CD68+) also show P2X7 immunolabeling. Scale bar, 20 μm. B, Western blot of optic nerve homogenates from sex- and age-matched controls (C) and MS cases. Sample loading was normalized to the intensity of bands corresponding to actin.

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P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis - PubMed (original) (raw)