Role of metabotropic glutamate receptor 5 signaling and homer in oxygen glucose deprivation-mediated astrocyte apoptosis
© Paquet et al.; licensee BioMed Central Ltd. 2013
Received: 8 February 2013
Accepted: 11 February 2013
Published: 14 February 2013
Group I metabotropic glutamate receptors (mGluR) are coupled via Gαq/11 to the activation of phospholipase Cβ, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. In addition to functioning as neurotransmitter receptors to modulate synaptic activity, pathological mGluR5 signaling has been implicated in a number of disease processes including Fragile X, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, epilepsy, and drug addiction. The expression of mGluR5 in astrocytes has been shown to be increased in several acute and chronic neurodegenerative conditions, but little is known about the functional relevance of mGluR5 up-regulation in astrocytes following injury.
In the current study, we investigated primary mouse cortical astrocyte cell death in response to oxygen glucose deprivation (OGD) and found that OGD induced both necrotic and apoptotic cell death of astrocytes. OGD resulted in an increase in astrocytic mGluR5 protein expression, inositol phosphate formation and extracellular regulated kinase (ERK1/2) phosphorylation, but only inositol phosphate formation was blocked with the mGluR5 selective antagonist MPEP. Cortical astrocytes derived from mGluR5 knockout mice exhibited resistance to OGD-stimulated apoptosis, but a lack of mGluR5 expression did not confer protection against necrotic cell death. The antagonism of the inositol 1,4,5 trisphosphate receptor also reduced apoptotic cell death in wild-type astrocytes, but did not provide any additional protection to astrocytes derived from mGluR5 null mice. Moreover, the disruption of Homer protein interactions with mGluR5 also reduced astrocyte apoptosis.
Taken together these observations indicated that mGluR5 up-regulation contributed selectively to the apoptosis of astrocytes via the activation of phospholipase C and the release of calcium from intracellular stores as well as via the association with Homer proteins.
KeywordsAstrocyte Metabotropic glutamate receptor Cell death Inositol phosphate Oxygen glucose deprivation Homer
Extracellular regulated kinase
Metabotropic glutamate receptor
2-Methyl-6-(phenylethynyl) pyridine hydrochloride
Oxygen glucose deprivation
Several neurotransmitter receptors are expressed in astrocytes including the G protein-coupled receptors (GPCRs) for the excitatory neurotransmitter glutamate. The metabotropic glutamate receptors (mGluRs) comprise the family C of GPCRs, which are characterized by sequence similarities in their seven transmembrane spanning helical domains and large extracellular amino-terminal venus fly trap domain [1–3]. Eight distinct mGluRs have been identified and are divided into three subgroups based on sequence homology and G protein coupling-specificity. Group I mGluRs (mGluR1 and mGluR5) are predominantly coupled to the activation of phospholipase C (PLC) via Gαq/11, whereas Group II (mGluR2 and mGluR3) and Group III (mGluR4, mGluR6, mGluR7 and mGluR8) mGluRs negatively regulate adenylyl cyclase via Gαi. Group I mGluR stimulation leads to Gαq/11-mediated intracellular activation of second messenger pathways, βγ activation of ion channels, as well as stimulation of G protein-independent pathways [2–4]. Pharmacological and biochemical experiments have shown that mGluR5 is the predominant group I mGluR subtype expressed in cultures of brain astrocytes [5, 6] although mGluR1 is also expressed in astrocytes in the spinal cord .
Astrocytic mGluR5 has been shown to play key roles in glia-neuron interactions, regulation of glutamate reuptake, and the coupling of the neurovasculature to neuronal activity [8–10]. However, the roles of mGluR5 in both astrocytic and neuronal survival following injury are poorly understood. In neurons, Group I mGluRs have been reported to both protect against and exacerbate cell death, depending on the model of toxicity employed, the neuronal subtype involved, and prior activation state of the receptors themselves [11–17]. The variety of signaling pathways activated by mGluR1/5 may explain these observations. Group I mGluRs coupled to Gαq/11 proteins stimulate the activation of PLC resulting in diacylglycerol and inositol-1,4,5-triphosphate (InsP3) formation. By interacting with InsP3 receptors, InsP3 induces calcium release from intracellular stores potentially leading to inappropriate cell death . On the other hand, mGluR1/5 stimulation also leads to activation of other signaling pathways important for cell survival/proliferation, such as extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT [19, 20]. Consistent with these opposing effects on cell survival, the mGluR1/5 scaffold protein Homer has been shown to promote the activation of both pro- and anti-survival signaling pathways [19, 21].
Group I mGluR expression is up-regulated in models of epilepsy, brain injury, amyotrophic lateral sclerosis, and multiple sclerosis [22–25]. Alterations in glutamate signaling are also known to play a prominent role in ischemic brain injury and both apoptotic and necrotic astrocytes have been observed following in vivo ischemic insults [26–30]. However, very little is known about the functional consequences of elevated mGluR5 expression on astrocyte survival following injury. Therefore, in the present study, we utilized an in vitro model of ischemia/reperfusion, oxygen glucose deprivation (OGD), to evaluate the potential role of alterations in mGluR5 protein expression following the injury of astrocytes. We find that OGD induces astrocytic cell death by both apoptotic and necrotic mechanisms and results in a rapid increase in mGluR5a expression that is independent of gene transcription. The observed OGD-induced apoptotic cell death is associated with increased mGluR5-dependent inositol phosphate formation and is reduced in astrocytes isolated from mGluR5 knockout mice. Moreover, OGD-induced apoptotic death of cortical astrocytes was reduced following IP3 receptor antagonism and Tat-Homer peptide-mediated interference of Homer interactions with mGluR5. Taken together, these observations indicate that upregulation of mGluR5 expression contributes to OGD-induced cell death via a Gαq/11 and Homer mediated mechanism.
OGD induces both necrotic and apoptotic cell death of cortical asrtocytes
OGD-induced increase in mGluR5 protein expression correlates with an increase in mGluR5-dependent inositol phosphate signaling
OGD-induced increase in mGluR5 protein expression correlates with an increase in mGluR5-dependent inositol phosphate signaling
mGluR5 promotes apoptotic, but not necrotic astrocytic cell death
OGD induced apoptosis requires mGluR5-dependent InsP3 receptor activation
Role for Homer in OGD mediated astrocytic cell death
In the present study, we show that OGD induces both apoptotic and necrotic astrocytic cell death. We find that similar to what is observed for both acute and chronic neurodegenerative conditions, such as pain, amyotrophic lateral sclerosis, multiple sclerosis, and epilepsy [22–25, 31–33] OGD also results in an increase in mGluR5a expression. We find InsP formation in cortical astrocytes is increased following OGD and that the treatment of cultures with the mGluR selective antagonist MPEP, not only attenuates OGD-mediated InsP formation, but the genetic deletion of mGluR5 selectively protects against OGD-induced apoptosis. The effects of mGluR5 are independent of the activation of the ERK1/2 signaling pathway, but appear to be associated with increased signaling via the PLC-InsP3-calcium pathway and Homer protein interactions.
We show that the mGluR5a expressed in response to injury are functional and activated after the ischemic insult. Group I mGluRs are known for their high constitutive and agonist-independent activity when they are overexpressed in heterologous systems . However, in this case, because OGD-induced mGluR5 expression in astrocytes is not as high as those found in neuronal cultures or in transfected cells , increased mGluR5 activity may also be due to glutamate released by astrocytes in response to ischemia. Indeed, ischemic conditions could result in glutamate release by astrocytes through a variety of mechanisms . The observed increases in InsP formation following OGD are consistent with previous work demonstrating that OGD results in increased InsP formation, likely via a metabotropic glutamate receptor .
We observe that OGD treatment activates a time-dependent increase in ERK1/2 phosphorylation that appears to be independent of mGluR5 activity, as increases in ERK1/2 phosphorylation were not antagonized by treatment of cultures with the mGluR5 selective antagonist MPEP. Furthermore, the genetic deletion of the mGluR5 gene did not result in diminished ERK1/2 phosphorylation in response to OGD. Indeed, in contrast to the InsP3 pathway, the mGluR5-induced activation of MAPK pathway occurs via mechanisms that are largely independent of PLC activation and calcium release in both striatal neurons (Homer protein dependent) and astrocytes (EGF receptor transactivation) [19, 37, 38]. The OGD-induced increase in ERK activation in astrocytes, as previously reported , did not significantly change in presence of the mGluR5 antagonist MPEP. Thus, it is possible that glutamate released in response to OGD may activate alternative mGluR subtypes that may be directly linked to the activation pro-survival signal transduction pathways in astrocytes. Consistent with this, mGluR3 is implicated in protecting against astrocytes apoptosis following OGD .
Interestingly, the mGluR5-dependent activation of PLC-mediated signaling leads to excessive calcium release and excitotoxic striatal neuronal death, which could be prevented by the down regulation of Group I mGluR expression following estradiol treatment . Thus, a selective increase in PLC signaling likely correlates with a pro-apoptotic role for astrocytic mGluR5, as OGD-induced apoptosis is reduced in astrocytes derived from mGluR5 null embryos. Moreover, selective mGluR5 blockade has been reported to be neuroprotective . Thus, it appears that increased mGluR5 signaling in astrocytes might lead to cell death following OGD-increased mGluR5a protein expression levels. Interestingly, mGluR5 has been shown to be upregulated in the spinal cord of ALS patients [22, 24] and astrocytic cell death in an amyotrophic lateral sclerosis transgenic model has been shown to be attenuated by blocking the mGluR5 receptor in vivo. Moreover, the disease onset was also delayed by this treatment. Similarly, the brain permeable mGluR5 antagonist MTEP provides neuroprotection in an in vivo ischemic model .
Because the effect of the InsP3 receptor inhibitor Xestospongin C on apoptosis is not observed in astrocytes lacking the mGluR5, we have confirmed that the mGluR5-PLC-InsP3-calcium signaling pathway is responsible for induction of apoptosis by releasing calcium from intracellular stores. As reviewed by Hanson et al. , calcium released through InsP3 receptors is sequestered by mitochondria and mitochondrial calcium overload can induce the apoptotic cascade. In addition, calcium release can also stimulate calcium-sensitive enzymes such as the phosphatase calcineurin, which is involved in other apoptotic mechanisms . Furthermore, we show that the formation of a molecular complex between Homer proteins and mGluR5 contributes to the activation of a pro-apoptotic pathway, as the treatment of astrocytes with a Tat-Homer interference peptide reduced OGD-induced astrocytes apoptosis. This Tat-peptide was previously shown to block mGluR5/Homer interactions but also blocks Homer-dependent activation of ERK1/2 . Thus, although we did not observe reduced ERK1/2 phosphorylation in response to OGD following either MPEP treatment or the loss of mGluR5 expression, it is possible that mGluR5-dependent activation of the ERK1/2 pathway via a Homer-dependent mechanism may also function to antagonize mGluR5-mediated apoptosis of astrocytes that is induced as the consequence of PLC-mediated mGluR5 signaling.
In conclusion, because of the neuroprotective role of astrocytes, astrocytic cell death could have a huge impact on acute or chronic neurodegenerative conditions. Thus, targeting mGluR5-signaling pathway involved in the induction of astrocyte apoptosis may have therapeutic benefits following ischemic insults such as stroke. For example, selective blockade of mGluR5-dependent PLC signaling in astrocytes might help limit cellular loss in a variety of neurodegenerative diseases. In contrast, the selective activation of mGluR5 G protein-mediated PLC signaling might promote apoptotic cell death in brain tumors, such as astrocytomas. This is of particular importance considering that mutations in mGluR1a have been associated with a variety of tumors, and that many of the mutations bias receptor signaling via the activation of ERK1/2 at the expense of G protein signaling [44–46] suggesting it may be possible to design biased allosteric agonists to prevent both astrocytic and neuronal cell death.
2-Methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was purchased from Tocris Cookson (Ellisville, MO). All cell culture reagents were from Invitrogen (Burlington, ON). Cytotoxicity detection kit plus (lactose dehydrogenase, LDH) was from Roche (Mississauga, ON). Rabbit anti-mGluR5 antibodies were from Millipore (Billerica, MA). Rabbit anti-phospho-p44/42ERK and anti-p44/42 ERK antibodies were from Cell Signaling Technology (Danvers, MA). Rabbit anti-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-rabbit antibody, Dowex 1-X8 (formate form) resin with 200–400 mesh and Bradford assay were purchased from Bio-Rad (Mississauga, ON). Anti-mouse IgG secondary antibody and ECL Western blotting detection reagents were from GE Healthcare (Oakville, ON). myo-3H]Inositol was acquired from PerkinElmer Life Sciences (Waltham, MA). The QuantiTect SYBR green single-step RT-PCR was from Qiagen (Toronto, ON). Alexa-488 anti-mouse was purchased from Molecular Probes (Burlington, ON). GenElute Mammalian Total RNA miniprep kit, Hoechst 33342, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Xestospongin C, and all other biochemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). The Tat-peptides corresponding to the mGluR5 binding motif for Homer proteins (Homer peptide: YGRKKRRQRRRALTPPSPFRDS and Scramble peptide: YGRKKRRQRRRTRSLPSDPPAF  were purchased from CanPeptide (Pointe Claire, PQ).
CD1 and BL6/129-Grm5tm1Rod/J  mice were purchased from Charles River Labs and Jackson Laboratory, respectively. Mice were housed in an animal care facility at 23°C on a 12 h light/12 h dark cycle with food and water provided ad libitum. Animal care was in accordance with the University of Western Ontario Animal Care Committee.
Primary astrocyte cultures
Cortices were dissected from CD1 or BL6/129Grm5 individual littermate embryos (E15-16) and separated from the striatum, as previously described . After dissociation in D-MEM/glucose/10%FBS/Gentamycin/L-glutamine media, the cells were plated onto poly-D-lysine coated culture flasks and maintained at 37°C and 5%CO2. Plating media was replaced by fresh media the following day and every 4 days thereafter. At DIV 7, the primary cultures were shaken overnight at 260 rpm at 37°C and washed three times with PBS to remove microglia. Cells were allowed to recover in fresh media and after 24 hours cells were trypsinized and re-plated onto poly-D-lysine coated plates at a density of 12,000 cells/cm2. Experiments were performed at DIV 14-19. The purity of the astrocyte cultures was assessed by immunocytochemistry with mouse anti-GFAP (1:400) and Alexa-488 anti-mouse (1:500) by microscopy (with nuclear staining with Hoechst 33258) as adapted from .
Oxygen/glucose deprivation experiments
After one wash with glucose/ L-glutamine-free D-MEM, astrocyte cultures were incubated for different periods of time with the same media in a hypoxic chamber (COY Laboratory Products Inc.) at 37°C, 5% CO2 and 0% O2, as described previously . Control cell plates (0 h) were maintained in normoxic conditions. After OGD, equal volume of DMEM/2X glucose/10%FBS/Gentamycin/L-glutamine media was added to the cells. The media of control astrocyte cultures was replaced by equal volumes of D-MEM with 2X glucose and without glucose. The cells were incubated at 37°C, 5% CO2 for 24 h (reperfusion).
Cells were harvested either immediately after the incubation in the hypoxic chamber (OGD), or after the 24 h reperfusion period with RIPA buffer (10 mM Tris-HCl pH=7.5, 140 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS). After solublization, insoluble material was removed by centrifugation and supernatant was used to determine protein concentration by Bradford assay. After dilution in SDS sample buffer, 75-100 μg of protein was loaded into a 10% acrylamide SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane. After blocking non-specific binding with 10% non-fat milk in TBS with 0.05% Tween-20 (TBS-T), membranes were incubated with rabbit anti-mGluR5 (1:4000), rabbit anti-actin (1:10000), rabbit anti-phospho-pERK44/42 (1:1000), or rabbit anti-pERK44/42 (1:1000, after anti-phosphoERK stripping) antibodies overnight at 4°C. After three washes with TBS-T, blots were incubated 1 hour at room temperature with HRP-conjugated anti-rabbit (1:10000) or anti-mouse (1:5000) antibodies . After three washes with TBS-T, blots were developed with ECL and exposed to films.
Quantitative real time reverse-transcript polymerase chain reaction (qRT-PCR)
RNA samples were isolated with the GenElute Mammalian Total RNA miniprep kit and treated with DNAse I following the manufacturer instructions. 30 ng of total RNA was used per reaction and mouse mGluR5 mRNA and the mitochondrial ribosomal protein S12 (endogenous housekeeping gene) mRNAs were amplified separately using the following primers: 5′ mGluR5: 5′GGTGGTAGCCTGCTTCTGTGA3′ (exon2), 3′mGluR5: 5′CGCTGATACCCATCTGTCACG3′ (exon3), 5′S12:, 5′GGAAGGCATAGCTGCTGG3′, 3′S12: 5′CCTCGATGACATCCTTGG3′ using the QuantiTect SYBR green single-step RT-PCR kit as per manufacturers’ instructions. The comparative Ct method (2–[delta][delta]Ct) was used for quantification .
Inositol phosphate formation assay
Inositol lipids were radiolabeled by incubating the cortical astrocyte cultures overnight with 1 μCi/ml myo-3H]inositol in D-MEM, as previously described by Ribeiro et al. . In brief, incorporated myo-3H]inositol was removed by washing cells with Hank’s balanced salt solution (HBSS). Cells were transferred in D-MEM without glucose and placed in the hypoxic chamber or not (0 h OGD control) for 4 h. After ischemia, LiCl was added to the wells to achieve a final concentration of 10 mM and the cells were incubated for 30 min in presence or in absence of 10 μM MPEP. The reactions were stopped on ice by the addition of 500 μl of perchloric acid and then neutralized with 400 μl of 0.72 M KOH, 0.6 M KHCO3. Total 3H]inositol incorporated into cells was determined by counting the radioactivity present in 50 μl of cell lysate. Total inositol phosphate was purified from cell extracts by anion exchange chromatography using Dowex 1-X8 (formate form) 200–400 mesh anion exchange resin. 3H]Inositol phosphate formation was determined by liquid scintillation.
MTT cell viability assay
After reperfusion, 1/10 of media volume of 5 mg/ml MTT was added to the culture dishes and incubated at 37°C, 5% CO2 for 3 h. The reaction was then stopped by adding an equal volume of isopropanol. After trituration, the crystal deposits (converted dye) were dissolved by shaking for 20 min. Absorbances at 570 nm and 690 nm (background) were read in triplicate and absorbance values were corrected for the absorbance due to the culture media in absence of cells .
As described by Steckley et al.,  Hoechst 33342 dye was added in triplicate directly to live cells and incubated for 30 min at 37°C. The media was then removed and the cells fixed in 3.7% formaldehyde for 60 min at RT. After three PBS washes, the cells were kept at 4°C until imaged and apoptotic cells (condensed nuclei) were quantified using ImageJ software. For each experimental data point, 1000-2000 cells were counted from triplicate wells of sister cultures from at least 3 independent experiments.
LDH release assay
After OGD/reperfusion experiments, lysis buffer (10 mM Tris-HCl pH=7.5, 140 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) was added to culture wells to determine the maximum LDH release. After 15 min incubation at 37°C and 5% CO2, 50 μl aliquots of triplicate samples or cell-free culture media (background) were transferred to wells of a 96 well plate containing 50 μl of PBS/well and experiments were performed according to the manufacturer instructions (Cytotoxicity detection kit plus, Roche). LDH release was determined by absorbance (A492 nm-A690 nm) measured using a SPECTRAmax M5 plate reader and expressed as percentage of the maximum release.
GraphPad Prism software was used to analyze data for statistical significance and for curve fitting. Statistical significance was determined by analysis of variance (ANOVA) testing followed by post-hoc Multiple Comparison testing.
MP was the recipient of a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship award. FMR is the recipient of a Heart and Stroke Foundation of Canada Postdoctoral Fellowship. SPC and SSGF hold Canada Research Chairs and SSGF is a Career Investigator of the Heart and Stroke Foundation of Ontario. This work was supported by CIHR Grants MOP 119437 and MOP 111093 (SSGF) and CIHR Grant MOP 68995 and HSFO grant in aid NA 7380 (SPC).
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