Calcium-permeable AMPA͞kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors Wenbin Deng, Paul A. Rosenberg, Joseph J. Volpe, and Frances E. Jensen* Department of Neurology and Program in Neuroscience, Children’s Hospital and Harvard Medical School, Boston, MA 02115 Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved March 14, 2003 (received for review October 31, 2002) Hypoxic–ischemic brain injury in premature infants results in OLs (11), and OL cell lines (12–14) in vitro, and AMPA͞kainate cerebral white matter lesions with prominent oligodendroglial receptor blockade is protective against hypoxic–ischemic white injury and loss, a disorder termed periventricular leukomalacia matter injury in the developing rat brain (15). (PVL). We have previously shown that glutamate receptors The occurrence of hypoxic–ischemic insults in human prema- mediate hypoxic–ischemic injury to oligodendroglial precursor ture infants is usually unpredictable, often making pretreatment cells (OPCs) in a model of PVL in the developing rodent brain. We less feasible. The efficacy of specific AMPA͞kainate antagonists used primary OPC cultures to examine the mechanism of cellular as a posttreatment strategy for PVL, however, remains unclear. toxicity induced by oxygen–glucose deprivation (OGD) to sim- Previous studies have focused only on a monophasic insult to ulate brain ischemia. OPCs were more sensitive to OGD-induced OLs, yet repeated episodes of hypoxic–ischemic injury com- toxicity than mature oligodendrocytes, and OPC toxicity was monly occur clinically in human premature infants (1). OLs that attenuated by nonselective [2,3-dihydroxy-6-nitro-7-sulfamoyl- have survived one ischemic episode may be exposed to a benzo[f]quinoxaline (NBQX), 6-cyano-7-nitroquinoxaline-2,3-di- subsequent insult. In neurons, hypoxic–ischemic precondition- one], ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ing is known to afford protection against subsequent, more (AMPA)-preferring (GYKI 52466), kainate-preferring (␥-D- severe insults (16, 17). However, the consequences and impli- glutamylaminomethanesulfonic acid), or Ca2؉-permeable AMPA͞ cations of sublethal insults to OLs are unknown, as is the kainate receptor antagonists (joro spider toxin, JSTx) administered involvement of AMPA͞kainate receptors. either during or after OGD. Furthermore, NBQX or JSTx blocked OGD-induced Ca2؉ influx. Relevant to recurrent hypoxic–ischemic Materials and Methods insults in developing white matter, we examined the effects of Cell Culture, Pharmacological Treatment, Oxygen-Glucose Depriva- sublethal OGD preconditioning. A prior exposure of OPCs to tion, and Cell Viability͞Differentiation͞Proliferation Assays. Primary sublethal OGD resulted in enhanced vulnerability to subsequent OPCs were isolated from mixed glial cultures of newborn excitotoxic or OGD-induced injury associated with an increased Sprague–Dawley rat forebrains by using a selective detachment ,(Ca2؉ influx. AMPA͞kainate receptor blockade with NBQX or JSTx procedure (18, 19), further purified by differential adhesion (20 either during or after sublethal OGD prevented its priming effect. and maintained in a chemically defined medium (19, 21). Unless Furthermore, OGD preconditioning resulted in a down-regulation otherwise indicated, pharmacological agents were applied 10 of the AMPA receptor subunit GluR2 on cell surface that increased min before exposure of the cells to kainate or OGD. Cell death Ca2؉ permeability of the receptors. Overall, these data suggest that was quantitatively assessed by using a trypan blue exclusion aberrantly enhanced activation of Ca2؉-permeable AMPA͞kainate method (22) or by measuring the extent of lactate dehydrogenase receptors may be a major mechanism in acute and repeated release (13, 14). Cell lineage progression was monitored immu- hypoxic–ischemic injury to OPCs in disorders of developing cerebral nocytochemically with stage-specific OL markers: A2B5, O4, white matter, such as PVL. O1, and myelin basic protein (MBP) (20). Cell proliferation was measured by determining the incorporation of BrdUrd into ypoxic-ischemic insults to the premature infant, particularly replicating DNA of cells. See Supporting Materials and Methods, Hduring the period from 24 to 32 weeks postconception, which is published as supporting information on the PNAS web result in prominent cerebral white matter injury and permanent site, www.pnas.org. areas of hypomyelination, a disorder termed periventricular ͞ 2؉ 45 ␮ 45 leukomalacia (PVL). PVL is a predominant age-specific form of Ca Uptake. Cultures were incubated with CaCl2 (8 Ci ml; NEUROSCIENCE brain injury in the premature infant and is the leading cause of 1Ciϭ 37 GBq) at room temperature for 10 min, then washed subsequent neurological disability, such as spastic motor deficits with Hanks’ balanced salts solution (HBSS) and lysed with 1% (cerebral palsy) and cognitive deficits (1). The incidence of low Triton X-100 in HBSS. Radioactivity in the whole lysate was birth weight infants is increasing because of improved survival counted by liquid scintillation. rates of premature babies, yet currently no specific treatment exists for this disorder. The neuropathological hallmarks of PVL Biochemical Measurements of Surface Expressed Receptors. Biotiny- are focal and diffuse white matter lesions with prominent lation of cell-surface protein in combination with immunoblot- oligodendrocyte (OL) injury and loss (1). ting of total versus surface receptors were performed as de- During the developmental period of greatest risk for PVL, scribed in ref. 23 and in Supporting Materials and Methods. human cerebral white matter is populated primarily by OL precursor cells (OPCs) (2). OPCs have been well characterized both in vitro and in vivo (3–6). These cells express non-N-methyl- This paper was submitted directly (Track II) to the PNAS office. ␣ D-aspartate (NMDA) glutamate receptors, including ␣-amino- Abbreviations: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; OL, oligo- dendrocyte; JSTx, joro spider toxin; OGD, oxygen–glucose deprivation; OPC, oligodendro- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kai- glial precursor cell; PVL, periventricular leukomalacia; NBQX, 2,3-dihydroxy-6-nitro-7- nate receptors, but not functional NMDA receptors (7). sulfamoylbenzo[f]quinoxaline; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; NMDA, Glutamate agonists or oxygen–glucose deprivation (OGD) in- N-methyl-D-aspartate; GAMS, ␥-D-glutamylaminomethanesulfonic acid. duce excitotoxic injury to differentiated OLs (8–10), immature *To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.1136624100 PNAS ͉ May 27, 2003 ͉ vol. 100 ͉ no. 11 ͉ 6801–6806 Downloaded by guest on September 30, 2021 Fig. 1. Immunochemical characterization and response to OGD-induced toxicity of OL lineage cells. (A) Stage-specific cultures. Cultures were allowed to differentiate for 0, 2, 6, or 10 days and stained for the OL stage-specific markers A2B5, O4, O1, and MBP, as well as the astrocytic marker GFAP. Percent of immunolabeled cells in the total population and characteristic morphology for each stage are shown. (Scale bar ϭ 50 ␮m.) (B) Effect of OGD duration on OPC death. Day 0 cultures were subjected to OGD for 0, 0.5, 1, 2, or 6 h, and cell survival was determined at 24 h. (C) Differential sensitivity of OL lineage cells at various maturational stages to OGD-induced death. Stage-specific cultures were exposed to OGD for 2 h, and cell survival was assayed at 24 h. Data Analysis. All data represent the mean Ϯ SEM of at least cultures. NBQX dramatically attenuated OPC death induced by three separate experiments, and all assays were performed in OGD (2 h) or kainate (300 ␮M), an excitotoxin that activates triplicate. Statistical differences were assessed by one-way anal- both AMPA and kainate receptors (24), in a dose-dependent ϭ Ϯ Ϯ ␮ ysis of variance with Tukey post hoc analysis for multiple manner (EC50 9.26 1.6 and 12.3 1.8 M, respectively). comparisons. Student’s t test was used when only two indepen- NBQX (100 ␮M) achieved 80 Ϯ 7% cell survival in OGD- dent groups were compared. induced death and completely blocked cell death induced by kainate (300 ␮M) that caused 90 Ϯ 6% OPC death during 24 h Results (data not shown). We further examined the effect of the Oligodendroglial Sensitivity to OGD-induced Toxicity in Vitro Is De- nonselective AMPA͞kainate [6-cyano-7-nitroquinoxaline-2,3- velopmentally Regulated. Immunocytochemical characterization dione (CNQX) 100 ␮M], AMPA-preferring (GYKI 52466, 100 and representative cell images of stage-specific OL cultures are ␮M), kainate-preferring [␥-D-glutamylaminomethanesulfonic shown in Fig. 1A. We first examined OGD-induced toxicity to acid (GAMS), 500 ␮M], and Ca2ϩ-permeable AMPA͞kainate these cultures. The time course of vulnerability of OPCs (un- [joro spider toxin (JSTx) 1 ␮M] receptor antagonists on OGD- differentiated cultures) showed that 2-h OGD exposure killed induced OPC death (Fig. 2). The NMDA receptor blocker Ϸ60% of the cells by 24 h (Fig. 1B), and thus this duration of MK-801 (10 ␮M) was used as a negative control. The receptor OGD was used for further studies. Stage-specific cultures were antagonists were applied either 10 min before or immediately obtained by allowing the OPC cultures to differentiate for 0, 2, after a 2-h OGD exposure and remained in