
==== Front
Stroke
Stroke
STR
Stroke
0039-2499
1524-4628
Lippincott Williams & Wilkins Hagerstown, MD

39224971
STROKE-2024-048163
00014
10.1161/STROKEAHA.124.048163
3
10014
10018
10033
10068
Original Contributions
Basic and Translational Sciences
Time-Dependent Potentiation of the PERK Branch of UPR by GPR68 Offers Protection in Brain Ischemia
https://orcid.org/0000-0002-3186-5002
Sun Wenyan PhD wzha@tulane.edu

https://orcid.org/0000-0002-5637-1076
Tiwari Virendra PhD virendratiwari20@gmail.com

Davis Grace BS g.e.davis@tcu.edu

https://orcid.org/0000-0002-5755-507X
Zhou Guokun PhD gkzhountu@163.com

https://orcid.org/0009-0007-5524-0406
Jonchhe Sarun MS sjzmd@umkc.edu

https://orcid.org/0000-0001-9490-2731
Zha Xiangming PhD
Division of Pharmacology and Pharmaceutical Sciences, University of Missouri-Kansas City (W.S., V.T., G.D., S.J., X.Z.).
Department of Physiology and Neuroscience, University of South Alabama, Mobile (G.Z.).
Now with: Tulane University, New Orleans, LA (W.S., V.T., X.Z.).
Nantong University, Nantong City, China (G.Z.).
Correspondence to: Xiangming Zha, PhD, Department of Neurosurgery, Clinical Neuroscience Research Center, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112. Email xzha@tulane.edu
03 9 2024
10 2024
55 10 25102521
12 6 2024
1 8 2024
7 8 2024
© 2024 The Authors.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Stroke is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

BACKGROUND:

In ischemia, acidosis occurs in/around injured tissue and parallels disease progression. Therefore, targeting an acid-sensitive receptor offers unique advantages in achieving the spatial and temporal specificity required for therapeutic interventions. We previously demonstrated that increased expression of GPR68 (G protein-coupled receptor 68), a proton-sensitive G protein-coupled receptor, mitigates ischemic brain injury. Here, we investigated the mechanism underlying GPR68-dependent protection.

METHODS:

We performed biochemical and molecular analyses to examine poststroke signaling. We used in vitro brain slice cultures and in vivo mouse transient middle cerebral artery occlusion (tMCAO) models to investigate ischemia-induced injuries.

RESULTS:

GPR68 deletion reduced PERK (protein kinase R-like ER kinase) expression in mouse brain. Compared with the wild-type mice, the GPR68-/- (knockout) mice exhibited a faster decline in eIF2α (eukaryotic initiation factor-2α) phosphorylation after tMCAO. Ogerin, a positive modulator of GPR68, stimulated eIF2α phosphorylation at 3 to 6 hours after tMCAO, primarily in the ipsilateral brain tissue. Consistent with the changes in eIF2α phosphorylation, Ogerin enhanced tMCAO-induced reduction in protein synthesis in ipsilateral brain tissue. In organotypic cortical slices, Ogerin reduced pH 6 and oxygen-glucose deprivation–induced neurotoxicity. Following tMCAO, intravenous delivery of Ogerin reduced brain infarction in wild-type but not knockout mice. Coapplication of a PERK inhibitor abolished Ogerin-induced protection. Delayed Ogerin delivery at 5 hours after tMCAO remained protective, and Ogerin has a similar protective effect in females. Correlated with these findings, tMCAO induced GPR68 expression at 6 hours, and Ogerin alters post-tMCAO proinflammatory/anti-inflammatory cytokine/chemokine expression profile.

CONCLUSIONS:

These data demonstrate that GPR68 potentiation leads to neuroprotection, at least in part, through enhancing PERK-eIF2α activation in ischemic tissue but has little impact on healthy tissue.

brain infarction
brain ischemia
endoplasmic reticulum
protons
stroke
HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS) 100000065R01NS102495 Not ApplicableHHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS) 100000065R01NS124722 Not ApplicableHHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS) 100000065R01NS135594 Not ApplicableAmerican Heart Association (AHA) 10000096823IPA1052359 Not ApplicableAmerican Heart Association (AHA) 10000096823CDA1051498 Not ApplicableOPEN-ACCESSTRUE
SDCT
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pmcAcidosis in the brain is prevalent in various neurological diseases.1 During ischemia, acidosis develops in and around ischemic loci and can persist for hours.2–4 The progression and severity of acidosis correlate with the development of brain injury in ischemia. Therefore, targeting acid-sensitive pathways would likely have a primary impact on the ischemic loci but have a diminished effect on healthy tissue. This injury site-specific effect, if achieved, would be advantageous for therapeutic interventions.

The mammalian brain expresses several acid-sensitive receptors, including the acid-sensing ion channels, proton-activated chloride channel, and several acid-sensitive G protein-coupled receptors, including GPR4, GPR65, and GPR68 (G protein-coupled receptor 68).4 The activation of acid-sensitive ion channels contributes to acidotoxicity.5,6 In contrast to the proinjury effect of these acid-sensitive ion channels, we previously showed that GPR68 activation is protective. Deleting the GPR68 gene in mice worsens acidotic and ischemic neuronal injury in vitro and in vivo.7,8 Conversely, GPR68 overexpression reduces brain infarct following transient middle cerebral artery occlusion (tMCAO). These data demonstrate that GPR68-dependent activities mediate a protective pathway in brain ischemia. Furthermore, these results suggest that enhancing GPR68-dependent acid signaling is a promising strategy for eliciting injury site-specific protection. While the previous findings are provocative, the mechanism for the GPR68 effect remains unclear.

In stroke, the disruption of endoplasmic reticulum function results in the accumulation of misfolded proteins.9–12 Following ischemia reperfusion, there is a rapid activation of the PERK (protein kinase R-like ER kinase) branch of unfolded protein responses (UPRs).13 PERK activation leads to eIF2α phosphorylation and consequently attenuates protein synthesis.10,14 Translational silencing during acute hours of ischemia reperfusion alleviates the load of misfolded proteins and provides one mechanism of protection in ischemia.14,15 On the other hand, prolonged PERK activation dysregulates protein homeostasis and promotes neurodegeneration.13,16,17 Thus, the effect of PERK depends on the timing and duration of the intervention. Identifying a mechanism to activate PERK during the acute phase of ischemia and preferentially in the injury site will be 2 key factors for a successful therapeutic intervention.

To start investigating how GPR68 contributes to neuroprotection, we performed a quantitative neurotoxicity profiling polymerase chain reaction array analysis. Among the genes that showed differential expression between wild-type (WT) and knockout brain tissues, we found the PERK/EIF2AK3 gene to be particularly intriguing because of the connection between PERK and ischemic protection. We analyzed the expression of key UPR proteins in WT and knockout mouse brains, asked whether GPR68 contributes to postischemia PERK-eIF2α signaling, and examined neuronal injury using in vitro and in vivo acidotoxicity and ischemic models.

METHODS

Data Sharing Plan

All data supporting the findings are within the article and its Supplemental Material. Additional details or data sets are available upon reasonable request.

Mice and In Vivo Studies

GPR68-/- mice were kindly provided by Dr Yan Xu at Indiana University. WT and knockout mice were maintained as breeding colonies at the University of Missouri-Kansas City as described previously.7,18 Animal care met the National Institutes of Health standards. The use of mouse and animal procedures was reviewed and approved by the University of Missouri-Kansas City Animal Care and Use Committee. All animal research adheres to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).19 The induction of tMCAO, drug delivery, and outcome analysis were performed as described earlier.7,20–22 For females, the estrus stage was determined by vaginal cytology23 (Supplemental Material).

Slice Culture, Histology, Biochemical, and Molecular Analysis

Organotypic brain slice culture and analysis were performed similarly as previously described.7,8 Immunofluorescence of brain cryosections, imaging, Western blot, and analysis were performed similarly as described.8 The cytokine profiler array and neurotoxicity array analysis were performed using commercial kits (Supplemental Material).

Statistical Analysis

Statistics were performed in GraphPad Prism and Microsoft Excel. Data normality was assessed to determine the downstream tests to use. For comparing 2 groups, depending on the normality test result, the 2-tailed t test, the Mann-Whitney U test, or the Wilcoxon matched pairs signed rank test was used. For multiple group comparison, we used ANOVA with the Tukey post hoc or Kruskal-Wallis test with the Dunn post hoc. The χ2 test was used for analyzing neurological scores. Data were reported as mean±SE. Differences were considered significant if P<0.05 (Supplemental Material).

RESULTS

GPR68 Deletion Reduces PERK Expression in Mouse Brain

In searching for potential GPR68-dependent mechanisms, we performed a profiling analysis using a Neurotoxicity RT2 Profiler PCR Array (Qiagen). As illustrated in Figure 1A, we isolated the tissue that covers a large portion of the middle cerebral artery territory for most in vitro analysis. We extracted total RNA from WT and knockout brain tissue. Following reverse transcription, we performed quantitative polymerase chain reaction analysis using the Neurotoxicity Array. Among the 84 neurotoxic response genes on the array, 19 exhibited significantly lower expression in the knockout compared with the WT (Figure 1B). Gene ontology analysis showed that these genes clustered in molecular function related to protein bindings (Figure 1C). Three genes clustered in heat shock protein binding. One of the genes in this function is EIF2AK3, which encodes PERK.

Figure 1. GPR68 (G protein-coupled receptor 68) deletion reduces PERK (protein kinase R-like ER kinase) and BIP (binding immunoglobulin protein) expression. A, Diagram shows the region of brain tissue used for reverse transcription-quantitative polymerase chain reaction analysis and most biochemical analysis in this study. Dashed lines illustrate the sections dissected: left (L) and right (R). B, Heatmap showing genes that exhibited reduced expression in GPR68-/- (knockout [KO]) brain tissue. A neurotoxicity gene array was used for screening of differentially expressed genes in wild-type (WT) vs GPR68-/- brain. The genes shown were statistically significant between the 2 genotypes with the 2-tailed t test. C, Plots showing the 2 molecular functions identified by gene ontology analysis. The dashed line indicates that P=0.05. D, Western blot and quantification of unfolded protein response proteins. See the Supplemental Methods for details. ATF indicates activating transcription factor; eIF2α, eukaryotic initiation factor-2α; and IRE1, serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α.

PERK is a key regulator of protein synthesis in stress conditions.13,24 The change in its RNA level prompted us to analyze its expression at the protein level. We analyzed WT and knockout brain lysates by Western blotting (Figure 1D). Knockout exhibited a 16% reduction of PERK (P=0.024). Activating transcription factor 4 (ATF4) and eIF2α, 2 downstream targets of PERK, were not different between the 2 genotypes. To gain a more complete picture of how GPR68 impacts UPR, we blotted for additional UPR regulators. BIP (binding immunoglobulin protein)/GRP78, a master regulator of the UPR, exhibited a 24% reduction (P=0.011) in knockout. IRE1 (serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α) and ATF6, which mediate 2 other branches of the UPR,24 showed no difference between the 2 genotypes.

GPR68 Deletion Time-Dependently Attenuates Postischemia eIF2α Phosphorylation

To test whether GPR68 activation contributes to poststroke PERK signaling, we examined the phosphorylation of eIF2α, a downstream effector of PERK, and a key regulator of translation. We performed tMCAO in mice, isolated the ipsilateral (left) and contralateral (right) brain tissues (Figure 1A), and conducted the Western blot. At 1 hour after reperfusion, the ipsilateral brain tissue of knockout showed a significantly reduced PERK level compared with either WT or the contralateral side of the knockout brain (Figure 2A). Both WT and knockout brains exhibited a similar increase in eIF2α phosphorylation on the ipsilateral side. At 3 hours after reperfusion, PERK protein level was not different between the groups although the knockout ipsilateral brain exhibited a trend of reduction (Figure 2B). eIF2α phosphorylation remained higher in ipsilateral brain tissues. However, the level of eIF2α phosphorylation was significantly lower in knockout compared with the WT. This result suggests that tMCAO-induced eIF2α phosphorylation does not require GPR68. Instead, GPR68-dependent signaling is necessary for a sustained elevation of eIF2α phosphorylation at ≈3 hours after reperfusion.

Figure 2. GPR68 (G protein-coupled receptor 68) deletion reduces postischemia eIF2α (eukaryotic initiation factor-2α) phosphorylation. At 1 and 3 hours after transient middle cerebral artery occlusion (tMCAO), ipsilateral and contralateral brain tissues were acutely isolated and subjected to the Western blot analysis. A and B, Representative blots and quantification for 1 (A) and 3 hours (B). See the Supplemental Methods for details. KO indicates knockout; L, left; PERK, protein kinase R-like ER kinase; R, right; and WT, wild-type.

Ogerin Potentiates GPR68-Dependent Signaling and Boosts PERK-eIF2α Activation

If GPR68-dependent activities prolong eIF2α phosphorylation, then we expect that stimulating GPR68 receptors will enhance this effect. Currently, there is no agonist for GPR68 other than protons. However, a small molecule positive modulator of GPR68, Ogerin, is available.25 We assessed here whether Ogerin potentiates GPR68 signaling in brain slices. We treated organotypic cortical slices with Ogerin (10 µmol/L) and performed Western blotting. We used a phospho-PKC (protein kinase C) substrate antibody because GPR68 activation in brain slices leads to PKC-dependent phosphorylation.7,8 In addition, we probed for CREB (cAMP response element-binding protein) phosphorylation because CREB acts as 1 convergence point for multiple signaling pathways. Ogerin increased the phosphorylation of PKC substrates and CREB in WT but not knockout slices (Figure 3A and 3B).

Figure 3. Ogerin (Og) potentiates transient middle cerebral artery occlusion (tMCAO)–induced eIF2α (eukaryotic initiation factor-2α) phosphorylation. A and B, Og on signaling in organotypic cortical slices cultured from wild-type (WT) and knockout (KO) mice. Organotypic cortical slices were treated with vehicle or Og (10 μM) for 30 minutes followed by the Western blot analysis. C, Western blot showing the time-dependent effect of Og on PERK (protein kinase R-like ER kinase)-eIF2α signaling following ischemia. Note that (see red box) Og had no apparent effect at 1 hour but increased eIF2α phosphorylation in ipsilateral tissue at 3 and 6 hours. D, Representative blots and quantification of eIF2α phosphorylation changes at 6 hours. E, Immunofluorescence images showing the pattern of puromycin incorporation after tMCAO. Note that a large area at and around the middle cerebral artery territory had little puromycin labeling, which indicates reduced protein synthesis. Central 2 figures show a high-magnification view of the striatum area. Note that vehicle-treated striatal neurons exhibit a low level of puromycin staining, while Og treatment diminished the puromycin signal in striatal neurons. The graph shows puromycin intensity in ipsilateral (left) striatal neurons. F, GPR68 (G protein-coupled receptor 68) expression at 6- and 24-hour after tMCAO. Relative expression was expressed as 2^(-ΔΔCT). See the Supplemental Material for details. CREB indicates cAMP response element-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; MERGE, merged image; NeuN, neuronal nuclei antigen; pCREB, phospho-CREB; p-PKCSS, phospho-PKC substrate; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; and V, vehicle.

After validating that Ogerin activates GPR68-dependent signaling in brain slices, we asked whether Ogerin induces eIF2α phosphorylation in ischemia. We performed tMCAO, intravenous injection of vehicle, or Ogerin at reperfusion, collected ipsilateral and contralateral brain tissue at 1, 3, 6, and 24 hours, and performed the Western blot analysis. Following tMCAO, ipsilateral brain tissue exhibited a reduction in PERK level compared with the contralateral brain tissue (Figure 3C). Ogerin attenuated this reduction. eIF2α exhibited elevated phosphorylation at 1 hour and then declined in a time-dependent manner (Figure 3C). Ogerin had no major effect on eIF2α phosphorylation at 1 and 24 hours but increased its phosphorylation at 3 and 6 hours. To gain a quantitative analysis, we focused on the 6-hour time point because 4 to 6 hours after reperfusion is when protein misfolding and neuronal injury start to become apparent.26,27 Vehicle-treated ipsilateral brain tissue showed a 58±13% increase in eIF2α phosphorylation (Figure 3D). In contrast, the Ogerin-treated group showed a significantly higher increase of 97±22%.

Higher eIF2α phosphorylation predicts increased translational silencing.17 To determine the effect of Ogerin on postischemia protein synthesis, we used a previously established approach of labeling newly synthesized proteins with puromycin.14,28 We performed tMCAO and puromycin labeling analysis, as illustrated in the diagram in Figure 3E. tMCAO reduced puromycin incorporation, which indicates a reduced protein synthesis, in the ipsilateral side of the brain (Figure 3E). The area of translational silencing was larger than the expected infarct core at this early time point.29,30 In the Ogerin-treated mouse brain, the ipsilateral brain area exhibited a similar pattern of protein silencing. We then quantified puromycin fluorescence intensity in striatal neurons. In vehicle-treated tMCAO animals, striatal neurons exhibited low levels of puromycin signal (Figure 3E, middle set of images). In contrast, the Ogerin-treated group showed diminished puromycin incorporation. Together, these data demonstrate that Ogerin potentiates GPR68-dependent signaling and stimulates PERK-eIF2α signaling to enhance translational silencing, and this effect occurs primarily in ipsilateral (ischemic) tissue.

Our findings above showed a positive relationship between GPR68 level and eIF2α activation. To better understand the regulation of GPR68 in ischemia, we asked whether ischemia alters GPR68 expression. At 6 and 24 hours after tMCAO, we isolated mouse brains and performed reverse transcription-quantitative polymerase chain reaction analysis. At 6 hours, both ipsilateral and contralateral sides exhibited ≈125% increase in GPR68 expression, significantly higher than that of the sham/control group (Figure 3F). At 24 hours, the ipsilateral side showed a 98% increase though it was not statistically significant.

GPR68 Potentiation Protects Against Acidotoxicity and Ischemic Brain Injury

At the acute phase of ischemia, stimulating eIF2α phosphorylation, which reduces the accumulation of misfolded proteins, leads to neuroprotection.14,31,32 Therefore, we hypothesized that Ogerin, through stimulating PERK-eIF2α, offers neuroprotection. We first assessed acidotoxicity in organotypic cortical slices. We treated slices with pH 6 for 1 hour and analyzed lactate dehydrogenase (LDH) release at 3 hours and live/dead staining at 6 hours (Figure 4A).7,20 We chose these time points based on our preliminary time course analysis for detecting acidosis-induced LDH release and propidium iodide (PI) staining. In WT slices, pH 6–treated slices exhibited elevated LDH release, while Ogerin attenuated this effect (Figure 4B). Similarly, pH 6 increased the uptake of PI; a result indicates increased injury (Figure 4C, top). Ogerin significantly reduced PI staining in pH 6. To determine whether the effect of Ogerin depends on GPR68, we performed a similar analysis in organotypic cortical slices isolated and cultured from knockout mice. Ogerin had no significant effect on pH 6–induced LDH release or PI staining in the knockout slices (Figure 4B and 4C, lower).

Figure 4. Ogerin (Og) protects against acidotoxicity and ischemia-induced brain injury. A, Diagram shows the experimental timeline. B, Summary of lactate dehydrogenase (LDH) release. C, Representative images and quantification for live/dead staining. D, Og on oxygen-glucose deprivation (OGD)–induced neuronal injury. E, Diagram on top shows the timeline. Figures at the bottom show neurological assessment and 2,3,5-triphenyltetrazolium chloride (TTC) analysis for 1-mg/kg Og (E1) and 2.5-mg/kg Og (E2). See the Supplemental Material for details. ACSF indicates artificial cerebral spinal fluid; Ctr, control; Veh, vehicle; and WT, wild-type.

Next, we asked whether Ogerin offers protection in ischemic conditions. We first examined in vitro following oxygen-glucose deprivation. In ischemia, delayed neuronal injury is one important readout.5,7 Therefore, we performed the live/dead analysis at 24 hours after oxygen-glucose deprivation. Oxygen-glucose deprivation increased the ratio of PI:Syto-13 (Figure 4D). Ogerin-treated WT slices exhibited a significant reduction in PI staining. In contrast, Ogerin had no significant effect on oxygen-glucose deprivation–induced injury in knockout slices.

To examine the effect of Ogerin in vivo, we performed tMCAO (see the diagram in Figure 4E for the timeline). We evaluated the neurological scores of the animals using a 5-point scoring scale, modified from previous reports.33,34 At 1-mg/kg dose, Ogerin reduced brain infarct size but had no significant effect on neurological scores (Figure 4E1). At 2.5-mg/kg dose, Ogerin improved neurological scores and reduced brain infarct size (Figure 4E2). We next examined the GPR68-/- mice. Vehicle- and Ogerin-treated knockout mice exhibited comparable neurological scores and brain infarct size (Figure 5A). Thus, in both in vitro slices and in vivo tMCAO analyses, Ogerin reduced neurotoxicity, and this effect depended on GPR68.

Figure 5. Ogerin-induced protection depends on GPR68 (G protein-coupled receptor 68) and PERK (protein kinase R-like ER kinase) and is effective at delayed time points. A, The effect of Ogerin in GPR68-/- mice. B, Coapplication of GSK2656157 (40 mg/kg) reversed the protection effect of Ogerin. C and D, The effect of delayed Ogerin intravenous (IV) at 3 (C) and 5 (D) hours after reperfusion. See the Supplemental Material for details. IP indicates intraperitoneal; KO, knockout; and tMCAO, transient middle cerebral artery occlusion.

PERK Dependence, the Effectiveness of Delayed Treatment, and the Effect on Females

If the effect of Ogerin in vivo depends on the PERK pathway, then we expect that PERK inhibition will abolish Ogerin-induced protection. To test whether this is the case, we examined the effect of GSK2656157, a PERK-specific inhibitor.35,36 We performed tMCAO, followed by Ogerin (intravenous) and GSK2656157 (intraperitoneal) treatment (Figure 5B). GSK2656157 did not change neurological performance but led to a significant increase in infarct size (P=0.019).

The effect of GPR68 activation on PERK signaling at 3 to 6 hours after tMCAO raises the question of whether delayed delivery of Ogerin is sufficient for protection. To assess this, we performed tMCAO surgery, intravenous delivery of Ogerin (2 mg/kg) at either 3 or 5 hours after reperfusion, and analyzed the outcome at 24 hours. Neither 3- nor 5-hour–delayed treatment improved neurological scores (Figure 5C and 5D). However, there was a trend of improved performance for both protocols. A pooled analysis of both delayed delivery experiments showed a significant improvement in neurological scores with Ogerin treatment (P=0.03; comparing neurological score >2 versus neurological score ≤2.0). For brain infarct size, Ogerin led to a significant reduction when given at either 3 or 5 hours after reperfusion (Figure 5C and 5D).

Sex is one important variable that influences stroke outcomes.37 We next asked whether GPR68 potentiation offers protection in female mice. For this experiment, we performed crystal violet staining of the vaginal smear to determine the estrus phase of each surgery mouse.23 Figure 6A shows the timeline and a representative set of images of vaginal cytology. For tMCAO analysis, we grouped the mice into 2 groups: one group in proestrus and estrus stages, which exhibit relatively higher estrogen levels, and a second group in metestrus and diestrus stages, which exhibit higher progesterone levels. Ogerin did not change the neurological scores but reduced brain infarct size in both proestrus/estrus and metestrus/diestrus stages (Figure 6B and 6C). This result suggests that the protective effect of Ogerin is not dependent on the estrogen/progesterone levels in females.

Figure 6. Ogerin (Og) protects females following transient middle cerebral artery occlusion (tMCAO) and changes postischemia cytokine expression. A, Top, The design of the experiment. Lower, A representative set of vaginal cytology images: leucocytes (yellow arrowhead), cornified epithelial cells (arrows), and nucleated cells (green filled arrowhead). B and C, Ischemia outcome in female mice in proestrus/estrus phase (B) or metestrus/diestrus phase (C). D, Representative blots showing the result of mouse cytokine proteome array. Blue boxes mark several targets that exhibited reduced expression in the vehicle-ipsilateral (left [L]) side but elevated expression in Og-L tissue. Red boxes mark the targets that exhibited lower expression in the Og-L. E, Heatmap showing the differential expression of the boxed targets in D. The result was an average of 3 separate experiments. See the Supplemental Material for details. IV indicates intravenous; and R, right.

Ogerin Treatment Alters Proinflammatory and Anti-Inflammatory Cytokine Expression After tMCAO

A change in proinflammatory and anti-inflammatory cytokines is important for ischemic outcomes. To gain more insights into the mechanism of Ogerin-induced protection, we performed a proteome profiler array analysis of cytokines. We prepared brain lysates from vehicle- and Ogerin-treated mice (see Figure 6D for timeline) and blotted using mouse Cytokine XL array membranes (Supplemental Methods). Figure 6D shows a representative set of blots. We quantified the signal intensity of each protein on the array (normalized to the positive controls on each membrane). Ogerin treatment led to an increase of several anti-inflammatory cytokines, including SDGF (Schwannoma-derived growth factor)/amphiregulin and TIG (tazarotene-induced gene)-2/chemerin in ipsilateral (left) tissue while reducing proinflammatory cytokines, including CXCL16 (C-X-C motif chemokine ligand 16), MMP (matrix metalloproteinase)-3, MMP-9, and PCSK9 (proprotein convertase subtilisin/kexin type 9) (Figure 6E).

DISCUSSION

GPR68 exhibits widespread expression in brain neurons and mediates a neuroprotective pathway in the brain.7,38,39 Our findings here connect GPR68 to the PERK-eIF2α branch of UPR. The initial phosphorylation of eIF2α after transient ischemia did not require GPR68. However, deleting GPR68 resulted in faster decline of eIF2α phosphorylation within 3 hours after reperfusion. Conversely, potentiating GPR68 with Ogerin boosted eIF2α phosphorylation during acute hours after reperfusion and offered neuroprotection both in vitro and in vivo. Of particular importance is that GPR68 exerted a stimulatory effect on eIF2α phosphorylation preferentially in ipsilateral ischemic brain tissue but had no significant effect in the rest of the brain areas. This preferential impact in injury tissue implicates a good therapeutic potential of GPR68 targeting for stroke therapy.

How does GPR68 activation or potentiation achieve the observed site- and time-specific effect on sustaining eIF2α phosphorylation? We speculate that the following reasons contribute to the phenotype. As a proton receptor, GPR68 exhibits low baseline activities. In both the WT versus knockout and the vehicle versus Ogerin comparisons, we did not observe a significant effect of Ogerin on eIF2α phosphorylation in the contralateral (ie, healthy) side of the brain. This result suggests that Ogerin, which is an allosteric positive modulator but not a ligand of GPR68,25 has little impact on healthy tissue. Following ischemia reperfusion, as a result of disrupted energy metabolism, the ischemic brain tissue exhibits a secondary phase of acidosis.2,3 At the fourth hour after a 60-minute tMCAO, ipsilateral brain tissue exhibits a pH value of ≈6.6.3 In addition, ischemia changes interstitial ionic concentration, which also modulates GPR68 function. We speculate that these changes in ipsilateral tissue after ischemia, together with the allosteric effect of Ogerin, stimulate GPR68 to achieve a significant impact on eIF2α phosphorylation. In addition, tMCAO elevated GPR68 expression at the sixth hour after reperfusion. This result supports an enhanced effect of GPR68 after transient ischemia. The exact mechanism for how GPR68 activation stimulates the PERK-eIF2α signaling warrants further investigation. Nevertheless, the 3- to 6-hour time window matches that needed for a protective silencing of translation after ischemia.12,27 This is consistent with the findings of delayed delivery of Ogerin at 5 hours remained effective.

The phosphorylation of eIF2α limits its availability for translational initiation and consequently attenuates new protein synthesis. Depending on the exact paradigm, this effect can be either protective or proapoptotic.40 In ischemia, acute translational silencing, typically within hours of reperfusion, reduces the load of misfolded proteins and leads to protection.12,27 PERK knockout worsens, while stimulating eIF2α phosphorylation with salubrinal alleviates ischemic brain injury.14 These findings are consistent with our data on GSK2656157, which demonstrated that PERK inhibition at reperfusion reversed the protective effect of Ogerin (Figure 6). On the other hand, persistent activation of the PERK pathway dysregulates protein homeostasis and leads to injury. In both neurodegenerative diseases and ischemia, inhibiting chronic PERK activation results in protection.16,41 Thus, for successful targeting of PERK to achieve neuroprotection, the timing and the location of the effect are pivotal. Here, we showed that Ogerin, an allosteric positive modulator of GPR68,42 stimulated eIF2α phosphorylation at 3 and 6 hours but had little effect at 24 hours (Figure 3). This time course matches a critical time window of 4 to 6 hours for the accumulation of misfolded proteins.12 The lack of effect at 24 hours suggests that Ogerin does not lead to a sustained potentiation of eIF2α phosphorylation and, thus, alleviates the concern associated with a worsened neuronal injury due to prolonged translational silencing.

We mainly examined the PERK-eIF2α axis here because GPR68 deletion reduced the PERK expression level at baseline (Figure 1). At 1 and 3 hours after tMCAO, PERK levels did not differ in the contralateral side of the WT and GPR68 null mouse brain (Figure 2). The reason for this result is unclear but could be a result of postischemia PERK regulation in the contralateral brain. Nevertheless, the knockout exhibited reduced PERK expression in ipsilateral brain tissue after tMCAO (Figure 2). This result is consistent with the changes in baseline and suggests that GPR68-dependent activities are important for PERK expression. Besides PERK, ATF6 and IRE1 mediate 2 other branches of UPR. Though we did not observe baseline expression change of ATF6 and IRE1 in the GPR68-/- mouse brain, it will be of future interest to determine whether GPR68 contributes to postischemia regulation of these 2 proteins.

It is interesting to observe that Ogerin treatment attenuated tMCAO-induced upregulation of MMP-9 and MMP-3, which are key regulators of postischemia inflammatory responses and blood-brain barrier dysfunction.43 This change in MMP-9 is consistent with the changes in several other cytokines, including the downregulation of proinflammatory IL (interleukin)-1α, CXCL16, Tim-1 (T-cell immunoglobulin and mucin domain 1), and upregulation of protective or anti-inflammatory cytokines, such as SDGF.44,45 The findings here together offer novel insights toward the connection between brain acid signaling, GPR68, UPR, and ischemic neuronal injury. Other than neurons, multiple cells at the neurovascular units play important roles in regulating cerebral vascular function and ischemic outcome. To gain a complete picture, it will be of future interest to examine proton signaling on neurovascular function. Additional limitations exist for the current study. First, age is one important biological variable in ischemic outcomes. The current study has focused on young adult mice. It will, thus, be important to examine the effectiveness of Ogerin in middle-aged and aged animals. Second, we evaluated here the acute injury at 24 hours after tMCAO. Further studies are warranted to assess the effectiveness of GPR68 potentiation at later time points, on both brain injury and long-term behavioral outcomes.

The findings here imply a potential translatable intervention through potentiating GPR68 function. The 2 desirable features of this approach include its preference impact on signaling in ischemic tissue with little impact in healthy tissue and the effectiveness of GPR68 potentiation at 5 hours after reperfusion. Given the prevalence of acidosis in conditions with brain injury and neuroinflammation, a better understanding of neuroprotective mechanisms through targeting an acid receptor, such as GPR68, would have broad implications for the treatment of multiple neurological diseases, which lead to acidosis.

ARTICLE INFORMATION

Acknowledgments

Dr Sun performed transient middle cerebral artery occlusion (tMCAO) surgery, array analysis, Western blot, live/dead analysis, and histology, analyzed the data, and wrote the article. Dr Tiwari performed a tMCAO study in female mice and part of the delayed Ogerin delivery surgery and analyzed the data. G. Davis performed Western blot. Dr Zhou performed oxygen-glucose deprivation in slices. S. Jonchhe performed pilot Western blot experiments. Dr Zha designed the study, performed in vitro experiments, analyzed the data, and wrote the article. All authors reviewed the article.

Sources of Funding

The study was supported in part by the National Institutes of Health grants R01NS102495, R01NS124722, and R01NS135594, an American Heart Association grant 23IPA1052359, and intramural support from the University of Missouri-Kansas City (to Dr Zha). Dr Sun was supported in part by an American Heart Association Career Development Award 23CDA1051498. The funders had no role in the study design, data collection, analysis, or the decision to publish the article.

Disclosures

None.

Supplemental Material

Supplemental Methods

Tables S1–S2

ARRIVE Checklist

Supplementary Material

Nonstandard Abbreviations and Acronyms

PERK protein kinase R-like ER kinase

tMCAO transient middle cerebral artery occlusion

UPR unfolded protein response

WT wild type

For Sources of Funding and Disclosures, see page 2520.

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.124.048163.
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