Temporal expression of P2X7 purinergic receptor during the course of experimental autoimmune encephalomyelitis

doi:10.1016/j.neuint.2010.08.021

Neurochemistry International

Volume 57, Issue 7, December 2010, Pages 823-829

https://doi.org/10.1016/j.neuint.2010.08.021 Get rights and content

Abstract

Purinergic P2X₇ receptors are nucleotide-gated ion channels widely distributed in brain. Strong evidence suggests that they are involved in cross-talk between glial and neuronal cells. These receptors activated under pathological conditions may participate in regulation of inflammatory response and cell death.

In this study we show the expression of P2X₇ protein and mRNA during the course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), in different stages of the disease (4, 6, 8, 10 post-immunization). The enhanced expression of the receptor at the level of both mRNA and protein was observed in the peak of neurological symptoms and was connected mostly with neurons. However, early overexpression of receptor’ protein was observed also in an asymptomatic phase of EAE and was tightly related to astrocytic pool of cells. This suggests the early involvement of this kind of receptor into pathological mechanisms leading for symptoms characteristic for EAE.

Research highlights

▶ The expression of P2X₇ purinergic receptor is enhanced during the course of EAE. ▶ Early overexpression of P2X₇R in an asymptomatic phase of EAE is tightly related to astrocytes. ▶ The overexpression of P2X₇R in a symptomatic phase is connected with neurons.

Introduction

Purines, mainly ATP, are important signaling molecules and potent mediators of neuron–glia communication in the central nervous system (CNS) under physiological conditions (Fields and Burnstock, 2006, Inoue et al., 2007). In healthy brain neurons and astrocytes release low concentrations of purines to the extracellular space. In pathological conditions, however, enhanced release of these nucleotides from cellular compartments is noted. They derive either from cells dying at the site of injury or are released from dysfunctional astrocytes (James and Butt, 2002, Neary et al., 1999) and activate purinergic receptors localized on different cells. It was suggested that in a variety of insults such as trauma, ischemia and inflammation, ATP may be involved in mechanisms of cytotoxicity through the purinergic receptors (Di Virgilio, 1995, Le Feuvre et al., 2003).

Family of purinergic receptors is divided into classes: metabotropic P2Y and ionotropic P2X receptors (Abbracchio and Burnstock, 1994). Activation of ionotropic group (P2XR) leads to the influx of Na⁺ and Ca²⁺ and efflux of K⁺ through non-selective cationic channels and to the cellular depolarization. Subtype of ionotropic receptors – P2X₇R – expresses the ability to form a non-selective pore in plasma membrane at high concentrations of agonist (ATP) released into the extracellular space by activated or dying cells (Adinolfi et al., 2005). Activation of the P2X₇R by high levels of nucleotide leads to a release of inflammatory mediators such interleukin-1( (Ferrari et al., 1997, Gabel, 2007, Mingam et al., 2008) and TNF-α (Kucher and Neary, 2005) from lipopolysaccharide (LPS)-stimulated microglia, and to the cytotoxicity. Forming of plasma membrane pores is associated with the loss of membrane potential connecting with the permeabilization and results in cell death via necrotic or apoptotic way (Di Virgillio et al., 1998, Morelli et al., 2001).

Due to these properties, P2X₇R evokes much interest, since it is expressed in many types of cells. Recently, a great deal of attention is focusing on the relation between ATP-activated receptors and inflammatory/neurodegenerative changes observed in many pathological states in CNS (Le Feuvre et al., 2002, Wang et al., 2004, Takenouchi et al., 2010), including multiple sclerosis (MS) (Matute, 2008). MS is a chronic, degenerative disease of the CNS that is characterized by inflamed lesions infiltrated by immune cells, demyelination, oligodendroglial cell death and damage of axons (Prineas et al., 2002). It was shown that activation of P2X₇R on oligodendrocytes may be involved in the MS pathology contributing to tissue damage (Matute et al., 2007).

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune inflammatory state of the CNS and is the commonly used rodent model of human demyelinating disease MS.

The expression of P2X₇R has not been assigned in the course of EAE as yet, although it was shown to be enhanced in oligodendrocytes during the severe neurological symptoms (Matute et al., 2007). Therefore, the present study was undertaken to analyze temporary changes in receptor's protein and mRNA levels starting from the very early stage of EAE. Knowing the fact that in the central nervous system P2X₇R is expressed not only in microglia and oligodendroglia but also in other brain cell populations like neurons (Anderson and Nedergaard, 2006) and astrocytes (Duan et al., 2003), analysis was directed towards these types of cells.

Section snippets

Animals and immunization procedure

All procedures involving animals were carried out under a protocol approved by the local Experimental Animal Care and Use Committee in accordance with international ethical guidelines for care and use of laboratory animals.

Female Lewis rats weighing 190–210 g were used throughout the study. Rats were bred in the animal house at the Mossakowski Medical Research Centre, Polish Academy of Sciences (Warsaw, Poland) and housed on a 12-h dark/light cycle in temperature and humidity controlled

Animals

Neurological deficits in animals, determined daily, were scored in the 5 steps scale (see Section 2). Neurological symptoms characteristic for EAE started to develop at day 7–8, peaked at 10–11 day, and then recovered, as shown in Fig. 1. Progressive development of paralysis of tail and hind limbs leading to paraplegia and loss of reflex in immunized animals were observed. Full recovery of neurological deficits was reached at day 15 when EAE rats did not reflect neurological features of

Discussion

The role of purinergic receptors in neurodegenerative/neuroinflammatory disorders is recently highlighted, indicating a crucial involvement of P2X₇R pathway in the progression of the diseases due to the secretion of inflammatory mediators and apoptotic process (Franke et al., 2006, Takenouchi et al., 2010).

It has been already shown that some of purinergic receptors are connected with pathology of autoimmune-lesioned CNS. Overexpression of P2X₄R was observed in macrophages accumulating in brain

Acknowledgments

This study was supported by funds from the Polish Ministry of Science and Higher Education to the Mossakowski Medical Research Centre, Polish Academy of Sciences.

References (49)

M.P. Abbracchio et al.
Purinoceptors: are there families of P2X and P2Y purinoceptors?
Pharmacol. Ther.
(1994)
C.M. Anderson et al.
Emerging challenges of assigning P2X7 receptor function and immunoreactivity in neurons
Trends Neurosci.
(2006)
M.M. Ayers et al.
Early glial responses in murine models of multiple sclerosis
Neurochem. Int.
(2004)
P. Chomczyński et al.
Single-step method of RNA isolation by acid guanidinum thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987)
F. Di Virgilio
The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death
Immunol. Today
(1995)
D. Ferrari et al.
ATP-mediated cytotoxicity in microglial cells
Neuropharmacology
(1997)
L.H. Guo et al.
Lesional accumulation of P2X(4) receptor(+) macrophages in rat CNS during experimental autoimmune encephalomyelitis
Neuroscience
(2005)
G. James et al.
P2Y and P2X purinoceptor mediated Ca²⁺ signaling in glial cell pathology in the central nervous system
Eur. J. Pharmacol.
(2002)
M. Kerschensteiner et al.
Animal model – targeting experimental autoimmune encephalomyelitis lesions to a predetermined axonal tract system allows for refined behavioral testing in an animal model of multiple sclerosis
Am. J. Pathol.
(2004)
R.A. Le Feuvre et al.
Extracellular ATP and P2X7 receptors in neurodegeneration
Eur. J. Pharmacol.
(2002)

O.W. Lowry et al.

Protein measurement with Folin phenol reagent

J. Biol. Chem.

(1951)

R. Mingam et al.

In vitro and in vivo evidence for a role of the P2X₇ receptor in the release of IL-1β in the murine brain

Brain Behav. Immun.

(2008)

K. Mitosek-Szewczyk et al.

Expression of glutamate transporter GLT-1 in different regions of rat brain during the course of experimental autoimmune encephalomyelitis

Neuroscience

(2008)

J.T. Neary et al.

Trophic signaling pathways activated by purinergic receptors in rat and human astroglia

Prog. Brain Res.

(1999)

A. Nicke et al.

A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 knock-out mice

J. Biol. Chem.

(2009)

B. Sperlagh et al.

P2X7 receptors in the nervous system

Prog. Neurobiol.

(2006)

L. Strużyńska et al.

Astroglial reaction during the early phases of acute lead toxicity in the adult rat brain

Toxicology

(2001)

E. Adinolfi et al.

P2X7 receptor: death or life?

Purin. Signal.

(2005)

R.F. Booth et al.

A rapid method for the preparation of relatively pure, metabolically competent synaptosomes from rat brain

Biochem. J.

(1978)

L. Chen et al.

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R^−/− mice: evidence for loss of apoptotic activity in lymphocytes

J. Immunol.

(2006)

K.K. Daniels et al.

Simultaneous isolation of glial and neuronal fractions from rat brain homogenates: comparison of high-affinity l-glutamate transport properties

Neurochem. Res.

(1998)

F. Di Virgillio et al.

Cytolytic P2X purinoceptors

Cell Death Differ.

(1998)

S. Duan et al.

P2X7 receptor-mediated release of excitatory amino acids from astrocytes

J. Neurosci.

(2003)

S. Duan et al.

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

Glia

(2006)

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The increase in P2X7 receptor occurs during the disease in a temporal manner. The early increase of P2X7R expression before occurring of neurological symptoms, is connected with astrocytic activation whereas the peak of neurological symptoms is connected mostly with neurons (Grygorowicz et al., 2010). P2X7 receptors activated under pathological conditions may participate in regulation of inflammatory response and neuronal death.
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Temporal expression of P2X7 purinergic receptor during the course of experimental autoimmune encephalomyelitis

Abstract

Research highlights

Introduction

Section snippets

Animals and immunization procedure

Animals

Discussion

Acknowledgments

Pharmacol. Ther.

Trends Neurosci.

Neurochem. Int.

Anal. Biochem.

Immunol. Today

Neuropharmacology

Neuroscience

Eur. J. Pharmacol.

Am. J. Pathol.

Eur. J. Pharmacol.

J. Biol. Chem.

Brain Behav. Immun.

Neuroscience

Prog. Brain Res.

J. Biol. Chem.

Prog. Neurobiol.

Toxicology

P2X7 receptor: death or life?

Purin. Signal.

A rapid method for the preparation of relatively pure, metabolically competent synaptosomes from rat brain

Biochem. J.

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R−/− mice: evidence for loss of apoptotic activity in lymphocytes

J. Immunol.

Simultaneous isolation of glial and neuronal fractions from rat brain homogenates: comparison of high-affinity l-glutamate transport properties

Neurochem. Res.

Cytolytic P2X purinoceptors

Cell Death Differ.

P2X7 receptor-mediated release of excitatory amino acids from astrocytes

J. Neurosci.

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

Glia

Temporal expression of P2X₇ purinergic receptor during the course of experimental autoimmune encephalomyelitis

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R^−/− mice: evidence for loss of apoptotic activity in lymphocytes