Elsevier

Neuroscience

Volume 319, 5 April 2016, Pages 35-45
Neuroscience

Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons

https://doi.org/10.1016/j.neuroscience.2016.01.048Get rights and content

Highlights

  • ATP administration to cultured neurons killed them via mitochondrial dysfunction.

  • Levels of ATP-induced neuronal death depended on expression levels of P2X7 receptor.

  • ATP-induced neuronal death was exacerbated by co-cultured astrocytes.

Abstract

Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5 mM ATP for 2 h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.

Introduction

ATP plays critical roles in the neuron-glia network as a neuro- and glio-transmitter in the brain (Pankratov et al., 2006, Higashi et al., 2011, Segawa et al., 2014, Rodrigues et al., 2015), and P2 receptors (P2Rs) are involved in ATP-mediated signaling in the brain neuronal cells. Of P2Rs, P2X7Rs have unique characteristics and are activated by high concentrations (ca. mM) of ATP under pathophysiological conditions such as ischemia, resulting in the formation of a non-selective cationic channel/pore, through which large molecules of up to 900 Da can pass (North, 2002, Skaper et al., 2009). There is controversy on functional expression of P2X7R in brain neuronal cells, especially neurons and astrocytes (Pedata et al., 2015 and references therein). However, recent findings have demonstrated that functional expression of P2X7Rs is found in neurons and astrocytes not only in vitro, but also in situ and in vivo conditions (Arbeloa et al., 2012, Kamatsuka et al., 2014, Hirayama et al., 2015).

Among neuronal cells, P2X7Rs are known to exhibit different functionality (Duan and Neary, 2004, Franke and Illes, 2006). Exposure of neurons to high concentrations of ATP activates P2X7Rs, resulting in their death (Nishida et al., 2012). In microglia, a typical immune cell in the brain, activation of P2X7Rs leads to generation of reactive oxygen species (ROS) and pro-inflammatory cytokines, and contributes to exacerbation of brain injury (North, 2002, Sim et al., 2004, Anderson and Nedergaard, 2006, Duan and Neary, 2004, Jabs et al., 2007, Surprenant and North, 2009). In addition, microglial P2X7Rs play a role in the regulation of their migration (Higashi et al., 2011). On the other hand, P2X7Rs expressed by astrocytes are constitutively activated by ATP released by themselves in an autocrine/paracrine manner under exogenous ligand-free resting conditions (Nagasawa et al., 2009, Kamatsuka et al., 2014). As one of the roles of their constitutive activation in astrocytes, we recently revealed that P2X7Rs regulate their engulfing activity (Yamamoto et al., 2013). In two strains of mice, which have the identical nucleotide sequence of cDNA of P2X7R, P2X7R channel/pore activity was greater in the astrocytes obtained from SJL-strain mice, their engulfing activity also being greater, than in astrocytes from ddY-strain mice (Kido et al., 2014). Furthermore, we demonstrated that the difference in channel/pore activity of P2X7Rs between the two mouse strain-derived astrocytes was due, at least in part, to the different expression profiles of their splice variants, but not the total cellular expression level of P2X7Rs (Kido et al., 2014).

Recent accumulating evidence indicates that functional alteration of P2X7Rs induced by SNPs is associated with mood disorders such as bipolar disorders, major depressive disorders, etc. (Bartlett et al., 2014, Caseley et al., 2014, Sperlagh and Illes, 2014). In addition, Cao et al. demonstrated that a decrease in ATP release from astrocytes induced depressive-like behavior in mice (Cao et al., 2013). Together, it is suggested that functional alteration of ATP/P2X7R-mediated signaling in the neuron-glia network might be a determinant of the development and/or clinical outcome of mood disorders.

Neurons play a central role in brain neuronal activity, and their dysfunction leads not only to cognitive impairment but also mental disorders (Moylan et al., 2014, Sperlagh and Illes, 2014). Since ATP is a neuro-transmitter, fine-tuned regulation of its signaling is critical for neuronal function. Nevertheless, to our knowledge, it is unknown whether different expression levels of P2X7Rs in neurons result in difference in the level of ATP-induced neuronal death, and whether the co-existence of astrocytes with different P2X7R activity has a different effect on the neuronal response to ATP.

Here, we examined the ATP-sensitivity of neurons in pure cultures and co-cultures with astrocytes obtained from SJL- and ddY-strain mice, and mechanism underlying the difference.

Section snippets

Reagents

The chemicals and reagents for experiments were purchased from Wako Pure Chemical Ind. (Osaka, Japan) except where otherwise noted.

Neuron cultures

Primary cultured cortical neurons as a pure neuron culture were prepared by the method reported previously with slight modification (Nagasawa et al., 2004, Nishida et al., 2012). All experiments were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University and were performed according to the Guidelines for Animal Experimentation of

ATP-induced neuronal death

First, we examined whether or not treatment of neurons in pure cultures at 11–13 DIV with ATP induced their death. As shown in Fig. 1a, the treatment decreased the number of MAP2-immuno-reactive neurons for both SJL- and ddY-neurons. On comparing neuronal viability between the two types of neurons, the viability was found to be significantly less in SJL-neurons than in ddY-neurons (Fig. 1b). This decreased viability of both types of neurons was almost completely restored to the control level on

Discussion

In this study, we found that (1) treatment of cultured neurons with ATP induced their death via mitochondrial dysfunction and PARP activation, (2) the ATP-induced neuronal death depended on the expression level of P2X7Rs, and (3) co-culture of neurons with astrocytes increased the ATP-induced their death. Overall, it is suggested that functional expression of P2X7Rs in neurons is one of the critical factors that determine their vulnerability to ATP, and the co-existence of astrocytes

Conclusion

We have demonstrated that treatment of neurons with a high concentration of ATP induces their death, the expression level of P2X7Rs is a critical determinant for their death, and the co-existence of astrocytes increases the vulnerability of neurons to ATP.

Conflicts of interest

The authors declare that there are no conflicts of interest, financial or otherwise.

Acknowledgement

A part of this study was financially supported by a Grant-in-Aid for Scientific Research (C) (24590128) from the Japan Society for the Promotion of Science (JSPS).

References (36)

  • X. Cao et al.

    Astrocyte-derived ATP modulates depressive-like behaviors

    Nat Med

    (2013)
  • E.A. Caseley et al.

    Non-synonymous single nucleotide polymorphisms in the P2X receptor genes: association with diseases, impact on receptor functions and potential use as diagnosis biomarkers

    Int J Mol Sci

    (2014)
  • R.A. Cunha

    Regulation of the ecto-nucleotidase pathway in rat hippocampal nerve terminals

    Neurochem Res

    (2001)
  • S. Duan et al.

    P2X(7) receptors: properties and relevance to CNS function

    Glia

    (2004)
  • H. Franke et al.

    P2X7 receptor expression after ischemia in the cerebral cortex of rats

    J Neuropathol Exp Neurol

    (2004)
  • Y. Higashi et al.

    Microglial zinc uptake via zinc transporters induces ATP release and the activation of microglia

    Glia

    (2011)
  • Y. Hirayama et al.

    Astrocyte-mediated ischemic tolerance

    J Neurosci

    (2015)
  • R. Jabs et al.

    Lack of P2X receptor mediated currents in astrocytes and GluR type glial cells of the hippocampal CA1 region

    Glia

    (2007)
  • Cited by (0)

    View full text