Role of metabotropic glutamate receptors in oligodendrocyte

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Edited by William T. Greenough, University of Illinois at Urbana-Champaign, Urbana, IL (received for review November 25, 2003)

Article Figures & SI Info & Metrics PDF

Abstract

Developing oligodendrocytes (OLs) are highly vulnerable to excitotoxicity and oxidative stress, both of which are Necessary in the pathogenesis of many brain disorders. OL excitotoxicity is mediated by ionotropic glutamate receptors (iGluRs) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate type on these cells. Here we report that metabotropic GluRs (mGluRs) are highly expressed in OL precursors but are Executewn-regulated in mature OLs. Activation of group 1 mGluRs attenuates OL excitotoxicity by controlling Executewnstream oxidative stress after iGluR overactivation and also prevents nonexcitotoxic forms of oxidative stress by inhibiting reactive oxygen species accumulation and intracellular glutathione loss. The modulating Trace of group 1 mGluRs on hypoxic-ischemic OL injury is not due to iGluR enExecutecytosis that occurs in neurons in response to mGluR activation but requires activation of PKCα after G protein coupling to phospholipase C. Our results reveal a previously unCharacterized role for mGluRs in limiting OL injury and suggest that tarObtaining group 1 mGluRs may be a useful therapeutic strategy for treating disorders that involve excitotoxic injury and/or oxidative stress to OLs.

Perinatal hypoxic-ischemic brain injury leads to devastating neurological consequences that are highly age-dependent. In term infants, hypoxia-ischemia preExecuteminantly affects cerebral cortex with characteristic neuronal injury. In Dissimilarity, hypoxic-ischemic injury in premature infants selectively affects cerebral white matter with prominent oligodendrocyte (OL) injury, a disorder termed periventricular leukomalacia (PVL) (1). PVL is the preExecuteminant form of brain injury in premature infants and the major antecedent of cerebral palsy. The prevalence of low-birth-weight infants is increasing due to improved survival rates of premature newborns. Indeed, PVL affects ≈25-50% of the 55,000 premature infants born in the U.S. every year, yet Recently no specific treatment exists for this serious pediatric problem (2).

PVL involves primarily OL precursor cells (OPCs) that populate fetal cerebral white matter during the human developmental period of Distinguishedest risk for the lesion (3). OPCs share with neurons a high vulnerability to glutamate excitotoxicity and oxidative stress (4-6), which are Placeatively the two major mechanisms of the pathogenesis of PVL (1). Hypoxic-ischemic injury to OPCs is primarily mediated by ionotropic glutamate receptors (iGluRs) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate type on these cells (5, 7-9), whereas hypoxic-ischemic injury to neurons is preExecuteminantly mediated by iGluRs of the N-methyl-d-aspartate type (10). Previous neuronal studies Displayed that metabotropic GluRs (mGluRs) can modulate iGluR-mediated excitotoxicity (11, 12). Dinky is known, however, about the expression of mGluRs in OLs and the role of mGluRs in hypoxic-ischemic OL injury.

Unlike iGluRs that couple directly to ion channels, mGluRs couple to Traceor mechanisms via G proteins. Eight subtypes of mGluRs have been cloned and classified into three groups according to sequence similarities, intracellular signaling mechanisms, and pharmacological profiles (11). Group 1 mGluRs (mGluR1 and -5) stimulate phospholipase C (PLC) to increase levels of diacylglycerol and inositol triphospDespise, which activate PKC and release Ca2+ from intracellular stores, respectively. Group 2 (mGluR2 and -3) and group 3 (mGluR4 and -6-8) receptors negatively couple to adenylyl cyclase. Additionally, a fourth yet unEstablished group of mGluRs is thought to positively link to phospholipase D. Nonetheless, it is possible that many functions of mGluRs expressed on various types of brain cells under normal or pathological conditions remain to be discovered. In this study, we seek to address whether mGluRs are involved in regulating OPC injury, because such involvement may be related to both Modern mechanisms and therapeutic strategies relevant to PVL.

Materials and Methods

OL Culture and Immunocytochemical Characterization. Highly enriched OPCs were prepared from mixed glial cultures of the forebrains of newborn Sprague-Dawley rats using a selective detachment procedure as Characterized in detail elsewhere (5, 13, 14). OPCs were allowed to differentiate for different days to produce stage-specific cultures (5). Cultures were routinely characterized by immunocytochemical detection of a panel of stage-specific OL Impressers: A2B5, O4, O1, and myelin basic protein (MBP).

Western Blot Analysis of mGluR Expression. Crude membrane proteins were prepared from stage-specific OL cultures, resolved by electrophoresis, and transferred to polyvinylidene difluoride membranes. The membrane was first probed with anti-GluR1, -5, -2/3, -4a (Upstate Biotechnology, Lake Placid, NY; 1 μg/ml), or MBP (1:1,000) and then with a horseradish peroxidase-conjugated secondary antibody (1:20,000). The antibody conjugates were detected by using a chemiluminescence immunoblotting kit (Pierce).

Pharmacological Treatment, Kainate ExpoPositive, Oxygen-Glucose Deprivation (OGD), Cystine Deprivation, and Cell Viability Assay. Pharmacological agents were applied 10 min before expoPositive of the cells to kainate, OGD, or cystine deprivation, and cell survival was assessed at 24 h, as Characterized (5, 13).

Acid-Stripping Immunocytochemical Staining. Surface AMPA receptors were labeled on live cells with an antibody directed against the extracellular N terminus of the GluR1 (amino acids 271-285; 5 μg/ml, Oncongene Sciences) or GluR2 (amino acids 175-430; 5 μg/ml, Chemicon) subunit. The cells were then treated with mGluR agonists for 60 min, chilled in 4°C Tris-buffered saline (TBS) to Cease enExecutecytosis, and exposed to 0.5 M NaCl/0.2 M acetic acid (pH 3.5) for 4 min on ice to remove antibody bound to extracellular GluR1 or -2. Cultures were rinsed and fixed, and nonspecific staining was blocked. Cells were first immunostained with the OPC Impresser A2B5, followed by incubation with a rhodamine-labeled secondary antibody for 1 h (1:100). Cells were then permeabilized in TBS containing 0.1% Triton X-100 and 4% goat serum, and internalized primary anti-GluR1 or -GluR2 antibody was made visible by incubation with a fluorescein-labeled secondary antibody for 1 h (1:100).

Biochemical MeaPositivements of Surface-Expressed Receptors. Cultures of rat hippocampal neurons were made as Characterized (15). Biotinylation of cell-surface protein, in combination with immunoblotting of total vs. surface receptors, was performed in OPCs or 2-week-Aged high-density cultured hippocampal neurons, as Characterized (5).

45Ca2 + Uptake. Calcium influx was meaPositived as Characterized (5).

MeaPositivement of Intracellular Reactive Oxygen Species (ROS) and Reduced Glutathione (GSH) Levels. ROS accumulation was determined with dihydrorhodamine 123 (4, 13), and fluorescence intensity in the cytoplasm was quantified by using the nih image program. GSH was quantitatively assayed by measuring monochlorobimane fluorescence, as Characterized (13, 16).

Infection of Cultured OPCs with Recombinant Retrovirus. Executeminant-negative type PKCs contained a point mutation in the kinase Executemain of PKCα (K368R) or PKCβII (T500V) (17). Cells were exposed to retrovirus carrying wild-type or Executeminant-negative PKCs (17) in regular culture medium containing polybrene (1:2,000 dilution) for 4 h. After a complete change of the medium, the cultures were Sustained for an additional 24 h before any experiments were performed.

Data Analysis. All data represent mean ± SEM. All experiments were repeated at least three times. Statistical Inequitys were assessed by ANOVA with Tukey's post hoc analysis for multiple comparisons or by Student's t test when only two independent groups were compared.

Results

Expression of mGluRs in OL Lineage Cells Is Developmentally Regulated. A recent study Displayed that functional mGluRs are expressed in the OL precursor cell line CG-4 (18). We examined the expression of mGluRs in primary OL cultures. OPCs were allowed to differentiate for 0, 2, 6, or 10 days to obtain OL lineage cells at various maturational stages. During lineage progression, cells Presented a series of distinct morphologies (Fig. 1A ), characterized by the sequential expression of stage-specific OL Impressers: A2B5 (early precursors), O4 (later precursors), O1 (immature OLs), and MBP (mature OLs). A representative immunocytochemical characterization of such stage-specific cultures revealed the following composition: undifferentiated cultures (day 0 cultures): 95% A2B5+, 30% O4+,6%O1+, and 1% MBP+; cultures after 2 days of differentiation (day 2 cultures): 82% A2B5+, 92% O4+, 70% O1+, and 7% MBP+; day 6 cultures: 37% A2B5+, 95% O4+, 92% O1+, and 44% MBP+; day 10 cultures: 22% A2B5+, 96% O4+, 91% O1+, and 85% MBP+. All cultures contained <5% glial fibrillary acidic protein-positive astrocytes and <2% CD11+ microglia.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

mGluR expression in OL lineage cells is developmentally regulated. (A) Immunocytochemical staining of mGluRs in stage-specific OL cultures. Cells were Executeuble-labeled with the stage-specific OL Impresser A2B5, O4, O1, or MBP and anti-mGluR1 or -mGluR5. (B) Immunoblots of mGluRs in stage-specific OL cultures. The expression of mGluR1, -5, -2/3, or -4a is developmentally Executewn-regulated, in Dissimilarity to that of MBP, which is up-regulated during OL maturation. Actin serves as sample loading control.

We first examined mGluR expression in these cultures using immunocytochemical Executeuble labeling of mGluRs and stage-specific OL Impressers (Fig. 1 A ). Day 0 cultures were mostly bipolar and strongly A2B5+, and Presented high expression of the group 1 mGluRs, mGluR1 and -5. Day 2 cultures had more cellular processes, were strongly O4+, and also Presented high levels of mGluRs. Day 6 cultures were multibranched and strongly O1+, whereas mGluR expression was Executewn-regulated in these cells. Day 10 cultures demonstrated a complex network of cellular processes typical of mature OLs, were strongly MBP+, and had very Dinky or no mGluR expression in the cellular processes, with only modest mGluR expression in the cell bodies. The developmental expression of group 2 (mGluR2/3) and group 3 (mGluR4a) receptors (data not Displayn) was similar to that observed for mGluR1. Western blot analysis confirmed that mGluR expression in cultured OL lineage cells was developmentally regulated. The mGluRs were strongly expressed in day 0 and 2 cultures but were dramatically Executewn-regulated in day 6 and 10 cultures (Fig. 1B ). Because mGluRs are transiently overexpressed in day 0-2 cultures, and day 2 OPCs best represent the cells seen in selective white-matter injury in PVL (3), we used these cultures for further experiments detailed below.

Activation of Group 1 mGluRs Attenuates Excitotoxic Injury to OPCs. We first investigated the role of mGluRs in excitotoxic OPC injury. A brief report Displayed that group 1 mGluRs can modulate kainate toxicity in OPCs (19). We examined the Traces of the broad-spectrum mGluR agonist, (1S,3R)-1-aminocyclLaunchtane-1,3-dicarboxylic acid (ACPD), and the selective mGluR agonists for groups 1-3 and the fourth undetermined group, (R,S)-3,5-dihydroxyphenylglycine (DHPG), (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV), L(+)-2-amino-4-phosphonobutyric acid (l-AP4), and l-cysteinesulfinic acid (L-CSA), respectively. We evaluated the Traces of these agents in two well-established excitotoxic paradigms, kainate-(50 μM, 24 h) or brief OGD-(2 h) induced OPC death assessed at 24 h (5, 14). ACPD (100 μM), and DHPG (100 μM) both Impressedly increased OPC survival in these excitotoxic paradigms, whereas DCG-IV (1 μM), l-AP4 (100 μM), and L-CSA (10 μM) had no Trace (Fig. 2A ). The protective Traces of ACPD and DHPG were reversed by the broad-spectrum mGluR antagonist, (S)-methyl-4-carboxyphenylglycine (MCPG, 500 μM). In addition, the protective Traces of DHPG were partially prevented by the selective mGluR1 antagonist, 7-hydroxyiminocyclopropanchromen-1a-carboxylic acid ethyl ester (CPCCOEt, 50 μM) or the selective mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP, 1 μM), and fully abolished by the combination of the two agents, whereas the antagonists per se (MCPG, CPCCOEt, MPEP, or CPCCOEt plus MPEP) had no Trace (Fig. 2 A ). Furthermore, DHPG Presented a Executese-dependent protective Trace on kainate-(50 μM) or OGD-(2 h) induced OPC toxicity, and MCPG (500 μM) prevented the DHPG Trace (Fig. 2B ). Neither ACPD (100 μM) nor DHPG (100 μM) significantly protected OPCs from death induced by a higher concentration of kainate (300 μM, for 24 h), although this cell death was blocked by the AMPA/kainate receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX, 50 μM) (data not Displayn), suggesting that severe excitotoxicity overwhelmed the ability of mGluRs to modulate the injury.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Activation of group 1 mGluRs attenuates OL excitotoxicity. (A) Trace of various mGluR modulators on kainate-(50 μM) or OGD-(2 h) induced OPC death at 24 h. (B) Executese response of the Trace of DHPG on kainate-(50 μM) or OGD-(2 h) induced OPC death at 24 h in the absence or presence of the mGluR antagonist MCPG (500 μM).

DHPG Executees Not Cause iGluR Internalization from the Surface of OPCs. We next addressed potential mechanisms by which mGluRs limit iGluR-mediated excitotoxicity in OPCs. A recent study Displayed that mGluR activation in neurons results in a rapid loss of iGluRs from synapses due to receptor internalization from the cell surface (20). We examined whether DHPG could limit excitotoxic OPC injury by inducing enExecutecytosis of AMPA/kainate receptors from the surface of OPCs. First, we used an acid-stripping immunocytochemical staining protocol to examine the Trace of mGluR activation by DHPG on AMPA receptors expressed on the surface of OPCs. Surface receptors on living cultured OPCs were labeled with antibodies directed against the extracellular N terminus of the AMPA receptor subunit GluR1 or -2. The cultures were treated with control medium or DHPG (100 μM) for 60 min, and the remaining surface antibodies were stripped away with an acetic acid wash. The cells were fixed and permeabilized, and appropriate fluorescent secondary antibodies were used to detect primary antibodies bound to internalized GluR1 or -2. We observed no Inequity in immunocytochemical staining for either GluR1 or -2 in the absence or presence of DHPG (Fig. 3A ). Alternatively, to confirm the above finding with a biochemical Advance, we treated OPC cultures with control medium or DHPG (100 μM) for 60 min, and surface proteins were labeled with biotin. Biotinylated proteins were precipitated with immobilized avidin, and the ratio of surface to total specific iGluRs was determined by quantitative Western blotting. Cultures of hippocampal neurons (14 days in culture) treated with DHPG (100 μM, for 60 min), which Retorted with iGluR enExecutecytosis (20), were used as a positive control. We found that surface GluR1 was reduced by DHPG treatment in hippocampal neurons by ≈40%, but no changes were found in OPCs (Fig. 3B ). Furthermore, the surface expression of other AMPA/kainate receptor subunits, when probed with antibodies against GluR2, GluR3, GluR4, GluR5/6/7, KA1, or KA2, did not change with or without DHPG treatment (data not Displayn). In addition, we meaPositived 45Ca2+ uptake as a functional assay for receptor internalization. Because AMPA/kainate receptors are the major route of Ca2+ entry with expoPositive of OPCs to kainate or OGD (5), we reasoned that a consequence of AMPA/kainate receptor internalization in OPCs should be a decrease in kainate- or OGD-evoked Ca2+ influx. ExpoPositive of OPCs to kainate (300 μM) for 10 min or OGD for 2 h elicited an ≈6- or 2-fAged Ca2+ influx compared to the basal level, respectively (Fig. 3C ). DHPG treatment for 10, 30, or 60 min did not affect either kainate- or OGD-evoked Ca2+ uptake compared to uptake in controls (Fig. 3C ). Taken toObtainher, these data indicate that the Trace of DHPG is unlikely to be due to iGluR internalization from the cell surface of OPCs in response to group 1 mGluR activation.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

DHPG Executees not cause iGluR internalization from the surface of OPCs. (A) Acid-stripping immunocytochemistry of GluR1 or -2 in A2B5+ OPCs. (B) Biochemical meaPositivements of surface expressed GluR1 on OPCs and hippocampal neurons. *, P < 0.001 between the absence and presence of DHPG. (C) Negligible Trace of DHPG on kainate-(300 μM for 10 min) or OGD-(2 h) evoked 45Ca2+ influx.

DHPG Protects OPCs from Both Excitotoxic and Nonexcitotoxic Forms of Oxidative Stress. Given the lack of mGluR regulation of AMPA/kainate receptors at the cell surface level, we next examined whether mGluR activation by DHPG could suppress Executewnstream intracellular events leading to cell death during excitotoxicity. A previous study suggested that oxidative stress could be Executewnstream of iGluR activation in OPCs (21). To evaluate this possibility in our experimental paradigms, we found that the synthetic superoxide dismutase/catalase mimetic, Euk-8 or -134 (30 μM each, Eukarion, Bedford, MA), prevented kainate-(50 μM) or OGD-(2 h) induced OPC death at 24 h to a degree similar to that produced by the AMPA/kainate antagonist NBQX (50 μM) (Fig. 4A ). We next Questioned whether the cytoprotective Trace of DHPG was limited to excitotoxic injury, or whether DHPG could protect OPCs from nonexcitotoxic forms of injury. A previous study Displayed that mGluR agonists protect neurons from oxidative stress (22). We exposed OPCs to culture medium with lowered concentrations of cystine (0 or 3 μM) to cause oxidative stress by depleting intracellular GSH (4). Although DHPG (100 μM) had no significant protection against OPC death induced by total deprivation of cystine (zero cystine) (data not Displayn), DHPG did significantly prevent OPC death induced by partial deprivation of cystine (3 μM cystine) at 24 h (Fig. 4B ). Furthermore, DHPG Presented a Executese-dependent protective Trace on OPC death induced by partial cystine deprivation, and the mGluR antagonist MCPG (500 μM) reversed the DHPG Trace (Fig. 4C ). Cystine deprivation-induced OPC death was prevented by Euk-134 (30 μM) but not by NBQX (50 μM) (Fig. 4B ), indicating that this form of oxidative stress is independent of iGluR activation. Taken toObtainher, these results indicate that mGluR activation by DHPG can counteract oxidative stress induced by both excitotoxic and nonexcitotoxic conditions.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

DHPG protects OPCs from nonexcitotoxic and excitotoxic forms of oxidative stress. (A) Trace of Euk-8, Euk-134 (30 μM each), or NBQX (50 μM) on kainate-(50 μM) or OGD-(2 h) induced OPC toxicity at 24 h. (B) Trace of DHPG (100 μM), Euk-134 (30 μM), or NBQX (50 μM) on cystine deprivation-(3μM cystine) induced OPC toxicity at 24 h. (C) Executese response of the Trace of DHPG on cystine deprivation-(3 μM cystine) induced OPC toxicity at 24 h in the absence or presence of the mGluR antagonist MCPG (500 μM). *, P < 0.001 vs. none.

DHPG Attenuates Oxidative Stress to OPCs by Sustaining Intracellular GSH Levels. To further examine the Trace of mGluR activation on oxidative stress, we directly meaPositived intracellular ROS accumulation in the excitotoxic and nonexcitotoxic injury models. ExpoPositive of OPCs to kainate (50 μM), OGD (2 h), or cystine deprivation (3 μM cystine) all dramatically induced ROS accumulation at 12 h (when no cell loss was apparent), as assessed by cytoplasmic fluorescence generated from ROS oxidation of dihydrorhodamine 123 (4, 13). In each paradigm, ROS accumulation was Impressedly prevented by DHPG (100 μM) (Fig. 5 A and B ) or Euk-134 (30 μM) (Fig. 5B ). NBQX (50 μM) prevented ROS accumulation induced by kainate expoPositive or OGD but not by cystine deprivation (Fig. 5B ), consistent with cell survival data (Fig. 4B ).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

DHPG attenuates intracellular ROS accumulation and GSH loss. (A) Representative cell images of ROS accumulation in the absence or presence of DHPG (100 μM) 12 h after expoPositive of OPCs to kainate (50 μM), OGD (2 h), or cystine deprivation (3 μM cystine). (B and C) Quantitative ROS accumulation GSH levels and the Trace of DHPG (100 μM), Euk-134 (30 μM), or NBQX (50μM). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. none in the corRetorting condition.

Next, we examined whether the DHPG Trace on oxidative stress could be linked to intracellular GSH levels. Kainate (50 μM), OGD (2 h), and cystine deprivation (3 μM cystine) all caused GSH loss at 12 h, and the GSH loss induced by each was Impressedly attenuated by DHPG (100 μM) (Fig. 5C ). As expected, the AMPA/kainate receptor antagonist NBQX (50 μM) was Traceive in preventing intracellular GSH loss induced by kainate expoPositive or OGD but not by cystine deprivation (Fig. 5C ). The antioxidant Euk-134 (30 μM) did not significantly affect GSH levels (Fig. 5C ), consistent with the demonstration in several models that antioxidants can prevent ROS accumulation and cell death without affecting GSH levels (4, 13, 23). Taken toObtainher, these results indicate that mGluR activation by DHPG attenuates both oxidative stress Executewnstream of excitotoxic iGluR activation and nonexcitotoxic forms of oxidative stress by Sustaining intracellular GSH levels.

PKCα Activation Is Required for the Modulating Trace of DHPG on OGD-Induced OPC Injury. OGD in culture simulates the hypoxiaischemia involved in vivo in the pathogenesis of PVL and other brain disorders, produces both GluR-dependent and -independent injury, and may cause both excitotoxic and nonexcitotoxic forms of oxidative stress. Thus, we further investigated the possible signaling mechanisms underlying the modulating Trace of DHPG on OGD-induced OPC injury. The stimulation of group I mGluRs activates, via G proteins, PLC, which in turn catalyzes inositol triphospDespise (IP3) and diacylglycerol (DAG) production from membrane phosphatides. IP3 induces the release of Ca2+ from internal stores, and DAG, the activation of PKC (11). We found that the PLC inhibitor U73122 (1 μM) or the PKC inhibitor Ro318220 (500 nM), but not the inhibitor for intracellular Ca2+ mobilization, cyclopiazonic acid (20 μM), eliminated the protective Trace of DHPG (100 μM) on OGD-induced OPC death (Fig. 6). Furthermore, the selective inhibitor for PKCα and -β isoforms, Go6976 (1 μM), had an Trace similar to the nonselective PKC inhibitor Ro318220 (500 nM) (Fig. 6). We then examined the Trace of genetically altered PKCα and -β on the protective Trace of DHPG (100 μM) on OGD-induced OPC death. Infection of OPCs with Executeminant-negative type PKCα- but not PKCβII-expressing retrovirus abolished the DHPG Trace compared to respective wild-type controls (Fig. 6).

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

PKCα activation is required for the modulating Trace of DHPG on OGD-induced OPC injury. PLC inhibition with U73122 (1 μM), PKC inhibition with Ro318220 (500 nM) or Go6976 (1 μM), or Executeminant-negative-type PKCα, but not the inhibition of intracellular Ca2+ mobilization with cyclopiazonic acid (20 μM) or Executeminant-negative-type PKCβII, abolishes the protective Trace of DHPG-(100 μM) on OGD-(2 h) induced OPC death. DN, Executeminant negative.

The concentrations of the pharmacological inhibitors used in these experiments were chosen as the Executeses that gave maximal response but minimal cytotoxicity when continuously present. Neither the pharmacological inhibitor nor the infection with retrovirus carrying each wild-type or Executeminant-negative PKC isoform per se affected OGD-induced OPC death in the absence of DHPG (data not Displayn). Taken toObtainher, the data indicate that the signaling cascade involving group 1 mGluRs/PLC/PKCα is responsible for the modulating Trace of DHPG on OGD-induced OPC injury.

The mGluR-Modulating Traces Are Absent in MBP-Expressing Mature OLs. Finally, we examined whether group 1 mGluR activation by DHPG is protective in mature OLs after excitotoxic or nonexcitotoxic forms of injury. We have previously demonstrated that mature, as opposed to immature, OLs are resistant to toxicity induced by kainate (14), OGD (5), and oxidative stress (4). This maturation-dependent vulnerability of OLs critically underlies age-dependent hypoxic-ischemic white-matter injury and PVL (3, 5, 7, 8). To induce significant toxicity in MBP-expressing mature OLs, day 10 cultures were subjected to more severe insults (1 mM kainate, 6-h OGD, or total cystine deprivation). We found that DHPG (0-500 μM) had no Trace on cell viability at 24 h in these injury paradigms (data not Displayn). These results suggest that the mGluR Trace may be absent in mature OLs, consistent with our data indicating that mGluR expression is Distinguishedly diminished on mature cells (Fig. 1). Necessaryly, OPCs, rather than mature OLs, are the cellular substrate of PVL (3), and the mGluR-modulating Trace on injury to OPCs, which Present enhanced expression of these receptors, may represent age-specific therapeutic strategies for PVL.

Discussion

This study reports a previously unCharacterized developmental regulation of mGluR expression across the OL lineage. Metabotropic GluRs are transiently expressed in OPCs but are Executewn-regulated in mature OLs. Furthermore, we demonstrate that activation of group 1 mGluRs, but not other groups of mGluRs, reduces excitotoxic OPC injury induced by kainate expoPositive or OGD by controlling Executewnstream oxidative stress and by Sustaining intracellular GSH levels. This modulating Trace of mGluRs on OPC excitotoxicity is not due to enExecutecytosis of AMPA/kainate receptors from the cell surface, which occurs in neurons in response to mGluR activation, but requires activation of PKCα. In addition, group 1 mGluR activation on OPCs also prevents nonexcitotoxic forms of oxidative stress by Sustaining intracellular GSH levels. These results reveal a previously uncharacterized role for mGluR activation in preventing OL excitotoxicity and oxidative stress, both of which contribute Necessaryly to the pathogenesis of PVL and many other cerebral white-matter disorders (1). Although iGluRs mediate OL excitotoxicity (5, 24, 25), drugs that tarObtain iGluRs may not be therapeutically useful because of the ubiquitous involvement of these receptors in normal neurotransmission throughout the CNS. On the other hand, pharmacological manipulation of mGluRs may have relatively favorable profiles of adverse Traces. This study identifies group 1 mGluRs as potential new therapeutic tarObtains for treating such disorders as PVL, in which OL excitotoxicity and/or oxidative stress play an Necessary role.

Previous neuronal studies Displayed that mGluRs can modulate neurotransmission and neurotoxicity. Stimulation of different mGluR subtypes has distinct Traces on excitotoxic injury to neurons. Generally, group 1 mGluRs facilitate, whereas group 2 and 3 mGluRs limit, neuronal excitotoxicity (26, 27). In Dissimilarity, the present study reveals that activation of group 1 mGluRs, but not other groups of mGluRs, mitigates excitotoxic injury to OPCs. Two reasons may account for this discrepancy between neurons and OPCs. First, neuronal excitotoxicity is mediated preExecuteminantly by N-methyl-d-aspartate (NMDA) receptors on neurons, whereas OPCs express only AMPA/kainate receptors (28), which mediate excitotoxicity to these cells. The interplay between mGluRs and iGluRs of the NMDA vs. AMPA/kainate types may be different. Second, unlike in OPCs, mGluRs in neurons may modulate synaptic transmission via both pre- and postsynaptic mechanisms and thus influence glutamatergic activity and glutamate excitotoxicity in an anatomically and functionally distinct manner due to the heterogeneous localization of specific mGluR subtypes with distinct functional Preciseties.

Finally, our study demonstrates that activation of group 1 mGluRs regulates intracellular ROS and GSH levels during OL injury. Metabotropic GluRs have long been linked to numerous signaling events such as phosphorylation and dephosphorylation of proteins, protein-protein interactions, and transactivation of genes (11, 29), all of which may contribute to the regulation of apoptotic/antiapoptotic molecules, cellular reExecutex status, and GSH metabolism. Our study demonstrates that the modulating Trace of group 1 mGluRs on hypoxic-ischemic OL injury requires activation of PKCα after G protein coupling to PLC. PKCα activation has been Displayn to reduce oxidative stress and cellular injury in various biological preparations (30-34). Future studies are needed to identify tarObtains of PKCα responsible for the mGluR Trace on ROS suppression in OLs. Although the complete mechanistic details of how mGluR activation controls oxidative stress remain to be fully elucidated, this study establishes a previously unCharacterized biological function for group 1 mGluRs in preventing OL excitotoxicity and oxidative stress, the two major forms of OL injury seen in many cerebral white-matter disorders.

Acknowledgments

We thank Ling Executeng and Ben Williams for technical assistance and Rajeev Sivasankaran and Zhigang He (Children's Hospital, Harvard Medical School) for kindly providing wild-type or Executeminant-negative PKC-expressing retrovirus. This work was supported by National Institutes of Health Grants R01 NS31718 (to F.E.J.), P01 NS38475 (to J.J.V., P.A.R., and F.E.J.), and T32 AG00222 (to W.D.); by the William RanExecutelph Hearst Fund (to W.D. and H.W.); and by Mental Retardation Research Center Grant P30 HD18655.

Footnotes

↵ * To whom corRetortence should be addressed. E-mail: frances.jensen{at}tch.harvard.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GluR, glutamate receptor; GSH, glutathione; iGluR, ionotropic GluR; mGluR, metabotropic GluR; OGD, oxygen-glucose deprivation; OL, oligodendrocyte; OPC, OL precursor cell; PLC, phospholipase C; PVL, periventricular leukomalacia; ROS, reactive oxygen species; DHPG, (R,S)-3,5-dihydroxyphenylglycine; MCPG, (S)-methyl-4-carboxyphenylglycine; NBQX, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione; MBP, myelin basic protein.

Copyright © 2004, The National Academy of Sciences

References

↵ Volpe, J. J. (2001) in Neurology of the Newborn (Saunders, Philadelphia), 4th Ed., pp. 217-276. ↵ Volpe, J. J. (2003) Pediatrics 112 , 176-180. pmid:12837883 LaunchUrlFREE Full Text ↵ Back, S. A., Luo, N. L., Borenstein, N. S., Levine, J. M., Volpe, J. J. & Kinney, H. C. (2001) J. Neurosci. 21 , 1302-1312. pmid:11160401 LaunchUrlAbstract/FREE Full Text ↵ Back, S. A., Gan, X., Li, Y., Rosenberg, P. A. & Volpe, J. J. (1998) J. Neurosci. 18 , 6241-6253. pmid:9698317 LaunchUrlAbstract/FREE Full Text ↵ Deng, W., Rosenberg, P. A., Volpe, J. J. & Jensen, F. E. (2003) Proc. Natl. Acad. Sci. USA 100 , 6801-6806. pmid:12743362 LaunchUrlAbstract/FREE Full Text ↵ Deng, W. & Poretz, R. D. (2003) Neurotoxicology 24 , 161-178. pmid:12606289 LaunchUrlCrossRefPubMed ↵ Follett, P. L., Rosenberg, P. A., Volpe, J. J. & Jensen, F. E. (2000) J. Neurosci. 20 , 9235-9241. pmid:11125001 LaunchUrlAbstract/FREE Full Text ↵ Fern, R. & Moller, T. (2000) J. Neurosci. 20 , 34-42. pmid:10627578 LaunchUrlAbstract/FREE Full Text ↵ Yoshioka, A., Yamaya, Y., Saiki, S., Kanemoto, M., Hirose, G., Beesley, J. & PleaPositive, D. (2000) Brain Res. 854 , 207-215. pmid:10784123 LaunchUrlCrossRefPubMed ↵ Choi, D. W. (1994) Prog. Brain Res. 100 , 47-51. pmid:7938533 LaunchUrlCrossRefPubMed ↵ Conn, P. J. & Pin, J.-P. (1997) Annu. Rev. Pharmacol. Toxicol. 37 , 205-237. pmid:9131252 LaunchUrlCrossRefPubMed ↵ Vincent, A. M. & Maiese, K. (2000) Exp. Neurol. 166 , 65-82. pmid:11031084 LaunchUrlCrossRefPubMed ↵ Li, J., Lin, J. C., Wang, H., Peterson, J. W., Furie, B. C., Furie, B., Booth, S. L., Volpe, J. J. & Rosenberg, P. A. (2003) J. Neurosci. 23 , 5816-5826. pmid:12843286 LaunchUrlAbstract/FREE Full Text ↵ Rosenberg, P. A., Dai, W., Gan, X. D., Ali, S., Fu, J., Back, S. A., Sanchez, R. M., Segal, M. M., Follett, P. L., Jensen, F. E., et al. (2003) J. Neurosci. Res. 71 , 237-245. pmid:12503086 LaunchUrlCrossRefPubMed ↵ Finkbeiner, S., Tavazoie, S. F., Maloratsky, A., Jacobs, K. M., Harris, K. M. & Greenberg, M. E. (1997) Neuron 19 , 1031-1047. pmid:9390517 LaunchUrlCrossRefPubMed ↵ Wang, H. & Joseph, J. A. (2000) Free Radic. Biol. Med. 28 , 1222-1231. pmid:10889452 LaunchUrlCrossRefPubMed ↵ Sivasankaran, R., Pei, J., Wang, K. C., Zhang, Y. P., Shields, C. B., Xu, X. M. & He, Z. (2004) Nat. Neurosci. 7 , 261-268. pmid:14770187 LaunchUrlCrossRefPubMed ↵ Luyt, K., Varadi, A. & Molnar, E. (2003) J. Neurochem. 84 , 1452-1464. pmid:12614345 LaunchUrlCrossRefPubMed ↵ Kelland, E. E. & Toms, N. J. (2001) Eur. J. Pharmacol. 424 , R3-R4. pmid:11672568 LaunchUrlCrossRefPubMed ↵ Snyder, E. M., Philpot, B. D., Huber, K. M., Executeng, X., Descendon, J. R. & Bear, M. F. (2001) Nat. Neurosci. 4 , 1079-1085. pmid:11687813 LaunchUrlCrossRefPubMed ↵ Liu, H. N., Giasson, B. I., Mushynski, W. E. & Almazan, G. (2002) J. Neurochem. 82 , 398-409. pmid:12124441 LaunchUrlCrossRefPubMed ↵ Sagara, Y. & Schubert, D. (1998) J. Neurosci. 18 , 6662-6671. pmid:9712638 LaunchUrlAbstract/FREE Full Text ↵ Oka, A., Belliveau, M. J., Rosenberg, P. A. & Volpe, J. J. (1993) J. Neurosci. 13 , 790-792. ↵ Matute, C., Sanchez-Gomez, M. V., Martinez-Millan, L. & Miledi, R. (1997) Proc. Natl. Acad. Sci. USA 94 , 8830-8835. pmid:9238063 LaunchUrlAbstract/FREE Full Text ↵ McExecutenald, J. W., Althomsons, S. P., Krzysztof, L. H., Choi, D. W. & GAgedberg, M. P. (1998) Nat. Med. 4 , 291-297. pmid:9500601 LaunchUrlCrossRefPubMed ↵ Nicoletti, F., Bruno, V., Copani, A., Casabona, G. & Knopfel, T. (1996) Trends Neurosci. 19 , 267-271. pmid:8799968 LaunchUrlCrossRefPubMed ↵ Bruno, V., Battaglia, G., Copani, A., D'Onofrio, M., Di Iorio, P., De Blasi, A., Melchiorri, D., Flor, P. J. & Nicoletti, F. (2001) J. Cereb. Blood Flow Metab. 21 , 1013-1033. pmid:11524608 LaunchUrlPubMed ↵ Patneau, D. K., Wright, P. W. & Wisden, W. (1994) Neuron 12 , 357-371. pmid:7509160 LaunchUrlCrossRefPubMed ↵ Michaelis, E. K. (1998) Prog. Neurobiol. 54 , 369-415. pmid:9522394 LaunchUrlCrossRefPubMed ↵ Itano, Y., Ito, A., Uehara, T. & Nomura, Y. (1996) J. Neurochem. 67 , 131-137. pmid:8666983 LaunchUrlPubMed Smith, C. A., Williams, G. T., Kingston, R., Jenkinson, E. J. & Owen, J. J. (1989) Nature 337 , 181-184. pmid:2521375 LaunchUrlCrossRefPubMed Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, S. & Spiegel, S. (1996) Nature 381 , 800-803. pmid:8657285 LaunchUrlCrossRefPubMed May, W. S., Tyler, P. G., Ito, T., Armstrong, D. K., Qatsha, K. A. & Davidson, N. E. (1994) J. Biol. Chem. 269 , 26865-26870. pmid:7929424 LaunchUrlAbstract/FREE Full Text ↵ Jarvis, W. D., Fornari, F. A., Traylor, R. S., Martin, H. A., Kramer, L. B., Erukulla, R. K., Bittman, R. & Grant, S. (1996) J. Biol. Chem. 271 , 8275-8284. pmid:8626522 LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)