Expression And Effect Of P2X₇R In Oligodendrocyte Precursor Cells And The Role Of P2X₇R In Hypoxic-ischemic Brain Injury

Perinatal hypoxia–ischemia (HI) is a significant primary cause of brain damage of neonates. Periventricular white matter injury (PWMI) is the predominant form of brain injury underlying this neurological morbidity. The spectrum of chronic PWMI includes focal cystic necrotic lesions (periventricular leukomalacia; PVL) and focal, multifocal, or diffuses myelination disturbances. Recent neuroimaging studies support that the incidence of diffuse myelination disturbances is emerging as the predominant lesion in premature infants. Premature delivery and improved neonatal intensive care have led to a significant increase in the number of preterm survivors with long-term neurological deficits. OPCs have been demonstrated to be intrinsically more vulnerable to injury than mature oligodendrocytes under conditions of oxidative stress, oxygen– glucose deprivation (OGD) in vitro, hypoxia-ischemia in vivo, and glutamate receptor -mediated excitotoxicity. Perinatal hypoxia–ischemia (HI) is a significant primary cause of PWMI. The fact that OPCs appear coinciding with the high-risk period for PWMI and is selectively targeted by HI suggests that OPCs are the major target cell of injury in PWMI. However, Our understanding of the fundamental mechanisms involved in selective injury of OPCs to HI in the developing brain is still limited.ATP is now regarded as one of the most ubiquitous neuromodulator and acting as an intercellular signaling molecule. The receptors for extracellular ATP are classified into P2X and P2Y subclasses. Among seven members of P2X ionotropic receptors activated by extracellular ATP, the P2X₇ subtype is unique in that it can function as a cation channel, a nonselective pore that has been linked to the release of cytokines and the induction of cell death, or even a signaling complex coupled with multiple downstream components. Recent studies have documented that the P2X₇R is involved in the regulation of diverse functions in central nervous system (CNS) cells. In addition to modulating of neurotransmitter release, P2X₇R has recently been reported to be involved in neurodegenerative processes by regulating intracellular Ca²⁺ concentration, interleukin-1β(IL-1β) processing and release, and multiple caspase activation under pathological conditions such as inflammation, mechanical injury, ischemia, and stress. This receptor has been proposed to be potential therapeutic target sites in disorders of the nervous system, such as ischemia-reperfusion injury, Alzheimer's disease, spinal cord injury and neuropathic pain.However, the functional role of P2X₇R in glia has remained relatively unexplored. A recent study showed that a large number of O4⁺ OPCs as well as neurons died after being exposed to high levels of extracellular ATP in spinal cord, and P2X₇R antagonist OxATP significantly diminished cell death in both gray and white matter, suggesting P2X₇R may be involved in response to OPCs injury. We asked whether P2X₇R may be expressed in OPCs of developing brains, and whether this receptor per se is implicated in the process of OPCs damage or defense. To address these questions, first, we examined the expression and functional responses of P2X₇R on purified primary OPCs. Second, we tested the changes of P2X₇R expression on OPCs cultures after OGD for 2 h in vitro. Third, to confirm the changes of P2X₇R expression in a more intact system, a neonatal hypoxic-ischemic injury model was employed in postnatal (P) 3 rats. We investigated temporal changes of the P2X₇ receptor in various brain areas of the P3 rats.I. Isolation and identification of OPCs from neonate rats.1. Primary OPCs were isolated from mixed glial cultures of newborn Sprague Dawley rat forebrains by using a selective detachment procedure, further purified by differential adhesion, and maintained in a chemical conditioned medium. Immunocytochemical analysis revealed thatmore than 90% of the isolated cells expressed the OPCs specific marker NG2.2. OPCs possess unique physiological properties, exhibite a moderate input resistance, delayed rectifier K⁺ currents ( IK ) that could be antagonized partly by TEA-Cl and CsCl, small tetrodotoxin (TTX) sensitive Na⁺ currents that failed to generate typical action potentials.II. Expression and function of P2X₇R in OPCs.1. We demonstrated the expression of the P2X₇R in OPCs by RT-PCR and Western blot. By immunostaining, we found that P2X₇R colocalize with NG2 on the OPCs.2. The application of ATP at 100μM induced a significant increase in [Ca²⁺]i. Intracellular calcium concentrations peaked quicklyand the magnitudes of the calcium responses to ATP were significantly blocked by incubation in calcium-free solution with EGTA This indicates that the effect of ATP was mediated by a P2X receptor. The average amplitude of the Ca²⁺ signals induced by BzATP (100nM) were significantly higher than that induced by ATP (100nM). Pretreatment with BBG (100nM), which was a selective antagonist for P2X₇R in nanomole range, inhibited the responses to ATP and BzATP, indicating the participation of P2X₇R.3. after the treatment of BzATP (300μM), an increase in intracellular Lucifer yellow fluorescence intensity in OPCs was detected. Lucifer yellow uptake induced by BzATP was greatly reduced by BBG (300 nM), further confirming the participation of P2X₇R.4. BzATP (100μM) produced significant release of cytosolic LDH into the culture medium, compared to the control OPCs. Pretreatment with the P2X₇R antagonist BBG (100 nM) attenuated the toxicity effect of BzATP. BBG per se had no effect on the viability of OPCs..III. Role of P2X₇R in hypoxic-ischemic damage to OPCs1. LDH release assays showed that 2 h OGD exposure killed ⁴0% of the cells by 2 h. BBG applied 10 min before OGD partially protected OPCs from OGD-induced cell death. Furthermore, BzATP exacerbated OGD induced OPCs injury, and the enhancement in toxicity could not be prevented by BBG. Taken together, these results reveal that BzATP is able to cause P2X₇R-mediated toxicity to OPCs under normoxic conditions. Furthermore, OGD-induced OPCs toxicity is due partly to the activiation of P2X₇R.2. RT-PCR and immunoblots revealed that P2X₇R mRNA and protein levels was significantly decreased in OPCs cultures compared with controls at 2 h after OGD.IV. P2X₇R expression in neonatal rats and its changes after hypoxic- ischemic brain damage.1. Immunoblots revealed a upward trend in the P2X₇ protein levels in cerebral cortex, subcortical white matter and hippocampus of neonatal rats from P0^P28.2. We performed a confocal microscopy study in P3 rat CNS, Coronal sections (10μm) at the hippocampal level were double labeled for NG2 (green) and P2X₇R (red). We found that anti-P2X₇ Ab labeled NG²⁺ OPCs in the cerebral cortex, hippocampus, and white matter. Moreover, strong immunoreactivity was also observed in the neuronal cell bodies at the hippocampal pyramidal cell layers and cerebral cortex.3. In comparison to the sham operation controls, a down-regulation of the mRNA and protein in cerebral cortex, subcortical white matter and hippocampus of ischemic hemisphere 2 h after HI was found. A substantial loss of ipsilateral P2X₇R immunostaining in the lateral corpus callosum, adjacent cortex, and hippocampus was observed in comparison to the controls. Immunofluorescence double-labeling studies for P2X₇R and NG2 reveal a significantly decrease in the number of NG²⁺/P2X₇R⁺ OPCs in corpus callosum of the HI group. The post-ischemic down-regulation of P2X₇R suggested a role for this receptor in the pathophysiology of hypoxic-ischemic injury of OPCs.In our study, we found that OPCs express functional P2X₇R that can mediate extracelluar Ca²⁺ influx, formation of large pores and cell death. OPC cultures exhibited sensitivity to OGD toxicity, which can be attenuated by P2X₇R-preferring antagonists, BBG. The expression of P2X₇R was down-regulated in OPCs cultures and in ischemic cerebral cortex, subcortical white matter and hippocampus following HI. These findings indicate that P2X₇R involves in the process of hypoxic-ischemic white matter injury.