Extracellular ATP has become increasingly recognized to have a major role in neurodegenerative processes, but its role in astrocyte-mediated neuronal death has not been explored. Here, we found that spinal cord astrocytes assume a neurotoxic phenotype in response to extracellular ATP, leading to the induction of motor neuron death in co-cultures. Furthermore, evidence indicates that endogenous ATP stimulates SOD1G93A astrocytes in basal conditions and contributes to the maintenance of their neurotoxic phenotype.
Non-transgenic astrocytes required multiple stimuli with ATP over several days to induce the neurotoxic phenotype, while a single stimulus with the P2X7
r-selective agonist BzATP was sufficient to activate astrocytes to induce the same extent of motor neuron death. BzATP is most potent as an agonist for P2X7
r, but it is also a weaker agonist of P2X1
r and P2X3
]. The involvement of P2X7
r was further implicated in the activation of astrocyte neurotoxicity by the antagonist BBG, as it completely inhibited the action of ATP and BzATP. BBG is a selective antagonist for both P2X7
r and P2X5
]. Thus, P2X7
r appears to be the most likely receptor responsible for inducing the neurotoxic phenotype in astrocytes.
We have previously shown that oxidative stress induced by superoxide and nitric oxide forming peroxynitrite in non-transgenic astrocytes leads to a neurotoxic phenotype [19
]. Here we found that oxidative stress induced by BzATP stimulation mediated the transition of non-transgenic astrocytes to a neurotoxic phenotype, as NOS inhibitors as well as superoxide and peroxynitrite scavengers prevented their neurotoxicity towards motor neurons. In a similar way, Skaper et al showed that P2X7
r activation in microglia stimulated peroxynitrite production and led to death of co-cultured neurons [54
]. Thus, amplification of oxidative stress by P2X7
r signaling in microglia and astrocytes could lead to the generation of an adverse environment for vulnerable neurons during neurodegenerative processes.
astrocytes in culture display a neurotoxic phenotype that is maintained by chronic oxidative stress and autocrine pro-inflammatory signaling [14
], we investigated whether they also presented alterations in extracellular ATP signaling. Indeed, our results indicate that SOD1G93A
astrocytes display basally augmented extracellular ATP signaling as evidenced by an ATP-dependent neurotoxic phenotype, increased ATP-dependent proliferation, and increased extracellular ATP metabolism. Thus, ATP emerges as an extracellular factor that could chronically maintain the SOD1G93A
astrocyte aberrant phenotype in an autocrine/paracrine manner.
We found that SOD1G93A
astrocytes degraded ATP faster than non-transgenic astrocytes, ruling out that their basal alteration in ATP signaling could be caused by a decrease in its extracellular degradation, thereby allowing ATP to accumulate near receptors. An increase in ATP degradation could also be induced in non-transgenic astrocytes exposed to BzATP or LPS. We have previously shown that LPS induces a neurotoxic phenotype in astrocytes, leading to motor neuron death [42
]. Increased ATP degradation and/or ectonucleotidase upregulation has been previously described in neural tissue after cortical stab wound and acute ischemia [55
]. This phenomenon might reflect a cellular attempt to prevent over-activation of purinergic receptors during increases in extracellular ATP, thus promoting the return of extracellular ATP signaling to homeostasis.
Degradation of ATP by ectonucleotidases cannot only terminate deleterious ATP signaling, but also initiates ADP and adenosine signaling through P2Y and P1 receptors. To our surprise, in non-transgenic astrocytes, ATP degraded with apyrase, ADP, AMP, or adenosine led to ~35% more motor neuron attachment and survival compared to untreated controls. Because survival is determined 48 hours after plating of the motor neurons freshly isolated from spinal cords, any treatment that increases attachment of motor neurons will result in an increase of motor neuron survival above the untreated control. These results illustrate how the astrocyte phenotype can be modulated from toxic to highly trophic by changing the balance between ATP, ADP and adenosine signaling through P2X, P2Y or adenosine receptors.
In animal models of ALS, proliferative activated astrocytes interact with microglia to accelerate disease progression [57
]. Remarkably, we found that modulating ATP signaling in SOD1G93A
astrocytes with apyrase or BBG blocked their neurotoxic phenotype, completely preventing astrocyte-mediated death of motor neurons. A role for ATP and P2X7
r in the SOD1G93A
model was recently proposed by D'Ambrosi et al [41
], who showed that SOD1G93A
microglia are sensitized to BzATP activation. A combination of aberrant ATP signaling in astrocytes and microglia could generate a positive feedback loop driving a sustained inflammatory response in the spinal cord. The results presented here and the findings in SOD1G93A
] suggest that P2X7
r inhibition in ALS could slow disease progression by decreasing astrocyte and microglial activation.
Taken together, the present work supports the idea that extracellular ATP acting through P2X7r causes astrocytes to develop a neurotoxic phenotype. In SOD1G93A astrocytes evidence suggests that P2X7r is basally activated and contribute to their toxicity towards motor neurons. Thus, modulation of astrocyte P2X7r during disease could lead to decreased oxidative stress and inflammatory signaling and in turn the switch to a more trophic phenotype towards neurons. A better understanding of ATP and P2X7r signaling in astrocytes could contribute to the development of novel protective therapies in ALS and other neurodegenerative diseases where astrocytes are involved.