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. 2010 Jun 9:7:33.
doi: 10.1186/1742-2094-7-33.

Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

Mandi Gandelman  1 , Hugo Peluffo, Joseph S Beckman, Patricia Cassina, Luis Barbeito
Affiliations

Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

Mandi Gandelman et al. J Neuroinflammation. .

Abstract

Background: During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X(7) receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X(7) receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.

Methods: We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X(7) receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.

Results: Repeated stimulation by ATP or the P2X(7)-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X(7) receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X(7) receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.

Conclusions: Here we found that P2X(7) receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X(7) receptor. Thus, pharmacological inhibition of P2X(7) receptor might reduce neuroinflammation in ALS through astrocytes.

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Figures

Figure 1
Figure 1
ATP induced a neurotoxic phenotype in non-transgenic astrocytes. (A) Motor neuron stained for p75NTR cultured on the top of an astrocyte monolayer (B) Motor neuron survival in coculture with astrocytes pretreated with ATP (100 μM, top graph) as described in the diagram (bottom). Astrocytes treated for 5, 3 or 1 day(s) received 3, 2 or 1 ATP addition(s) correspondingly. (C) Survival of motor neurons in pure cultures exposed to conditioned media from control or ATP-pretreated astrocytes (100 μM, 5 days, 3 additions). (D) GFAP immunofluorescence of control and ATP-treated astrocytes (100 μM, 5 days, 3 additions) (E) Motor neuron survival in co-culture with astrocytes pretreated with ATP and apyrase (5 U/ml), ADP, AMP or Adenosine (ADO, 0.1 μM, 5 days) on motor neuron survival. Data are expressed as percentage of control, mean ± SEM from at least three independent experiments. *p < 0.05, significantly different from untreated control.
Figure 2
Figure 2
P2X7r activation triggered astrocyte-mediated neurotoxicity by inducing oxidative stress. (A) Motor neuron survival in co-culture with astrocytes pre-treated with ATP (100 μM, 5 days) or BzATP (10 μM, 48 hours) and the P2X7r inhibitor BBG (1 μM). (B) Motor neuron survival in co-culture with astrocytes pre-treated with NAME (1 mM), MnTBAP (0.1 mM) or urate (0.2 mM) and BzATP before co-culture. Data are expressed as the percentage of control, mean ± SEM from at least three independent experiments. *p < 0.05, significantly different from untreated control.
Figure 3
Figure 3
SOD1G93A astrocytes exhibit ATP-dependent neurotoxicity, proliferation, and increased ATP degradation. (A) Motor neuron survival in co-culture with SOD1G93A astrocytes pre-treated for 48 hours with the P2X7r inhibitor BBG (1 μM) or the ATP-hydrolyzing enzyme apyrase (5 U/ml) (B) Effect of apyrase treatment on SOD1G93A astrocyte proliferation in culture. (C) Degradation of exogenously added ATP by SOD1G93A or non-transgenic astrocytes astrocytes. Data are expressed as percentage of non-transgenic control, mean ± SEM from at least three independent experiments. Data are expressed as percentage of non-transgenic control, mean ± SEM from at least three independent experiments. *p < 0.05, significantly different from non-transgenic control.
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