Neural progenitor cells (NPCs) constitute a heterogeneous cell population capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes. In the adult rodent, these cells are restricted to two regions of the CNS: the subventricular zone of the lateral ventricles (Alvarez-Buylla and Garcia-Verdugo 2002) and the dentate gyrus of the hippocampus (Gage et al. 1998). In embryonic mice, the majority of neuroepithelial cells have progenitor cell properties (Kilpatrick and Bartlett 1993). NPCs can be isolated and propagated as neurospheres, in the presence of epidermal growth factor (EGF) and fibroblast growth factor (Reynolds and Weiss 1996).
Neural progenitor cell-based therapy has been successfully developed for animal models of CNS disorders such as Parkinson’s disease, Huntington’s disease, stroke injury and multiple sclerosis (Martino and Pluchino 2006). Grafting NPCs during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, improves remyelination and induces a decrease in T-cell infiltrates (Martino and Pluchino 2006). Furthermore, endogenous NPCs are recruited during EAE and migrate into areas of demyelination, where they differentiate into glial cells (Picard-Riera et al. 2002).
However, during neurodegenerative and demyelinating diseases and stroke, tissue damage and inflammation lead to the release of various cytokines and mediators as well as high levels of extracellular pro-inflammatory nucleotides such as ATP (Fields and Burnstock 2006; Khakh and North 2006). This increase in local concentrations of ATP could enhance inflammation mediated by purinergic receptors and/or lead to cell death.
P2X7 receptor (P2X7R) is a peculiar purinergic receptor as it acts both as a classical ATP-gated ion channel and can also induce cell death. This receptor is a transmembrane protein whose ligation of ATP4− leads to opening of cation channels, in particular an increase in intracellular Ca2+ concentrations (Virginio et al. 1999; North 2002). Prolonged exposure to ATP induces the formation of a non-selective pore allowing the entry of solutes up to 900 Da, which eventually leads to cell death (Virginio et al. 1999; North 2002).
Cell death has been observed in different cells of haematopoietic origin such as lymphocytes (Ferrari et al. 1999), thymocytes (Zheng et al. 1991; Auger et al. 2005), macrophages and dendritic cells (Coutinho-Silva et al. 1999) as well as in neuronal cells such as the dopaminergic neuronal immortalized cell line SN4741 (Jun et al. 2007) and retinal cholinergic neurons (Resta et al. 2005). In addition to its role in cell death, the P2X7R is involved in release of inflammatory cytokines, such as IL-1β, IL-6 and tumour necrosis factor-α (Labasi et al. 2002; Ferrari et al. 2006; Fields and Burnstock 2006). Studies on the functional role of P2X7R in the nervous system are actually an extensive area of research. P2X7R-mediates neuromediator release, like glutamate, endocannabinoids and ATP, from astrocytes (Duan et al. 2003; Walter et al. 2004; Suadicani et al. 2006), as well as from neurons in the hippocampus, cortex and spinal cord and motor neurons (Deuchars et al. 2001; Sperlagh et al. 2006; Marcoli et al. 2008).
These findings raise the possibility that the activation of P2X7R may contribute to cell death in CNS disorders. Effectively, administration of P2X7R antagonists in rat acute spinal cord injuries improves functional recovery and decrease cell death in the periphery of traumatic lesions (Wang et al. 2004). Matute et al. (2007) have also demonstrated that during chronic EAE, ATP can induce oligodendrocyte cell death via P2X7R activation and contribute to the demyelination process.
Therefore, the therapeutic potential of NPCs could also be affected negatively by P2X7R stimulation in CNS disorders. In this study, we have investigated the functional properties of P2X7R in NPCs and in particular, the sensitivity to P2X7R-mediated NPC death as these cells represent a potential cell source for cell replacement therapy.
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- Materials and methods
In this work, we show by RT-PCR and western blot that P2X7R is expressed in NPCs. Furthermore, as observed in several cell types, we find that P2X7R expressed at the plasma membrane of NPCs is present in similar amounts in DRMs and non-DRMs (Garcia-Marcos et al. 2006; Barth et al. 2007; Gonnord et al. 2009). Patch-clamp recordings showed that stimulation of P2X7R by Bz-ATP leads to the opening of this ionotropic purinergic receptor in NPCs. Prolonged activation of P2X7R with extracellular ATP or Bz-ATP leads to lysis/necrosis of NPCs accompanied by a loss of mitochondrial membrane potential. Surprisingly, NPC death mediated by P2X7R stimulation is not associated with the opening of a non-selective pore, which is assumed to be responsible for ionic imbalances leading to cell death.
The following points strongly support the conclusion that the observed biological effects are mediated by P2X7R activation: (i) high concentrations of ATP are needed to induce cell death, (ii) Bz-ATP induces NPC cell death with a greater potency than ATP (iii) the pharmacological inhibitor o-ATP which preferentially acts on P2X7R abolishes ATP-induced lytic death of NPC and (iv) ATP and Bz-ATP do not trigger cell lysis of NPC from P2X7R-deficient mice.
Activation of P2X7R can lead to cell death by apoptosis and/or lysis/necrosis, depending on the cell type (Zheng et al. 1991; Ferrari et al. 1999). P2X7R-mediated apoptosis may be linked to a signalling pathway implicating death receptors (DR). Denlinger et al. (2001) have reported that residues 436–531 of the C-terminal region of P2X7R exhibit sequence homologies with the death domain region of TNFR1, suggesting that P2X7R could interact with adaptator or effector proteins involved in caspase activation, a salient feature of apoptotic cell death. However, NPC express functionally inactive DR, whose binding by specific ligands does not induce apoptosis, because of the absence of caspase 8. In addition, in cytokine-treated NPCs, caspase 8 recruitment and activation are prevented by the high level of expression of death effector domain-containing protein PED/PEA15, which competes with caspase 8 for Fas-associated protein with death domain binding (Ricci-Vitiani et al. 2004). Moreover, NPCs express a high level of inhibitor of apoptosis protein, which inhibits tumor necrosis-related apoptosis-induced ligand-induced apoptosis in these cells (Peng et al. 2005). Activation of Fas receptors on NPCs induces proliferation rather than apoptosis (Ceccatelli et al. 2004). These findings suggest that proteins involved in DR-mediated cell death are not functional in NPCs and may explain why P2X7R mediates lysis/necrosis rather than apoptosis in NPCs.
Actually, necrosis was described more on morphological than biochemical criteria because contrary to apoptosis, its biochemical pathways are not as well defined. In thymocytes, P2X7R-induced necrosis/lysis has been shown to require the sequential activation of Src family tyrosine kinase(s), PI3 kinase, Erk1/2 and the proteasome (Auger et al. 2005). In NPCs, we found no evidence for the implication of this pathway, as none of the inhibitors of enzymes from this pathway affected NPC death (Fig. 5 and data not shown).
Mitochondrial membrane depolarization triggered by P2X7R stimulation in cells from submandibular glands was described by Garcia-Marcos et al. (2005). In agreement with their observations, we found that P2X7R activation induces a rapid mitochondrial membrane depolarization in NPCs (Fig. 7b). Mitochondria seem to play a central role in the induction of necrosis and can activate multiple death effectors. We have tested several inhibitors of biochemical pathways potentially triggered by mitochondrial dysfunction: antioxidant (butylated hydroxyanisol), protease inhibitors (Pepstatin, Tosyl phenylalanine chloromethyl ketone, N-Acetyl-Leu-Leu-Nle-CHO), inhibitor of necroptosis (Necrostatin), inhibitors of cytosolic phospholipase A2 [Arachidonyl trifluoromethyl ketone (AACOCF3), E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one haloenol lactone suicide substrate] (Syntichaki and Tavernarakis 2003). None of these inhibitors affects Bz-ATP induced NPC cell death (data not shown). These results suggest that several redundant pathways are activated simultaneously in the death process (Syntichaki and Tavernarakis 2003).
In thymocytes, P2X7R-mediated opening of the non-selective pore is required for cell death (Auger et al. 2005). However, in NPCs, P2X7R-stimulation results in lysis/necrosis of the cells without opening of the non-selective pore. Similarly, P2X7R stimulation does not seem to trigger pore formation in cells of neuronal origin. Indeed, in rat inner retina, ATP induces permeabilization only in microglia, which are cells from haematopoietic origin but not in rat retinal ganglion cells (Innocenti et al. 2004). In the latter work, the neuronal fate was not studied and it thus remains to be determined whether some of the cells undergo cell death, as NPCs. One may argue that our observations stem from the use of NPCs from C57BL/6 mice in which the P2X7R bears a Pro-451 to Leu mutation associated with a decrease in pore formation and cell death in mature T lymphocytes and thymocytes (Adriouch et al. 2002; Auger et al. 2005). Importantly, we found no evidence for a Bz-ATP-mediated dye-uptake pore in BALB/c mouse NPCs (data not shown) which indicates that the absence of pore formation is not a peculiarity of C57BL/6 NPCs.
Pannexin-1 has recently been shown to be involved in non-selective pore formation and IL-1β release triggered by P2X7R (Pelegrin and Surprenant 2006). This was established by silencing pannexin-1 with small-interfering RNA and pannexin-1 inhibitory peptide. We found that pannexin-1 is expressed in NPCs (Fig. 5b), therefore the absence of non-selective pore formation in NPCs does not appear to be due to a deficiency in pannexin-1 expression. Different studies suggest that the ability to form pores correlates with the density of cell surface P2X7R (Hickman et al. 1994; Narcisse et al. 2005). P2X7R expression increases during the differentiation of monocytes into macrophages (Hickman et al. 1994), as does the ability to form pores. Similarly, primary foetal human astrocytes treated with IL-1β showed increased surface P2X7R expression, which was associated with an increase in pore formation (Narcisse et al. 2005). We could hypothesize that immature neural cells do not express sufficient P2X7R sub-units to recruit proteins (such as pannexin-1) involved in non-selective pore formation or to generate critical amounts of a second messenger needed to open the pore (Faria et al. 2005). Thus, NPCs die by an original pathway that has not been described yet as it is independent of the non-selective pore and involves an undefined biochemical pathway.
During acute spinal cord injury in rats, high levels of ATP are released in the peritraumatic zone, and more cells die in areas of high ATP release than in regions with low ATP concentrations at the same distance from the lesion (Wang et al. 2004). In addition, it has been shown in EAE that pathogenic T lymphocytes of wild-type mice are eliminated in the CNS more efficiently than T cells of P2X7Rko animals. These experiments show that the amount of ATP needed to induce P2X7R-mediated cell death can be reached in the CNS during EAE (Chen and Brosnan 2006). Previous studies did not investigate NPC impairment because of ATP, but these cells are also likely to be targeted. Thus, endogenous ATP release from damaged cells could lead to cell death in vivo, thereby limiting endogenous repair and diminishing the efficiency of cell therapy by NPCs in neurodegenerative and demyelinating diseases as well as stroke.
Administration of P2X7R antagonists may represent a useful therapeutic approach to treat neurodegenerative disease, as it could inhibit cell death of repairing NPCs in vivo. In fact, o-ATP and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate, antagonists of P2X7R, have been shown to improve recovery from spinal cord injury in rats (Wang et al. 2004) and o-ATP and Brilliant Blue G attenuate symptoms associated with chronic EAE in mouse (Matute et al. 2007). P2X7R antagonists could also diminish inflammation by inhibiting release of inflammatory cytokines, such as IL-1β, IL-18 and tumour necrosis factor-α, by microglia and macrophages. The recent development of stable P2X7R-selective antagonists may help in controlling inflammatory infiltrates and decrease the number of dying cells in the CNS.
Neural progenitor cells might represent a potent source of cells for cell-based therapy as they can be obtained from different tissues (embryonic, foetal and adult) and do not form teratocarcinoma, in vivo, unlike embryonic stem cells (Martino and Pluchino 2006). Transplantation of NPCs has also been shown to improve recovery from different CNS inflammatory diseases (Martino and Pluchino 2006). However, the therapeutic use of NPCs requires a profound understanding of the biological behaviour of NPCs. The present study indicates that delivery of NPCs to replace damaged cells or to improve repair could be restrained because of the high level of ATP released in the lesion.