Neuroprotection of Ischemic Postconditioning by Downregulating the Postsynaptic Signaling Mediated by Kainate Receptors
Background and Purpose—Ischemic postconditioning, a brief episode of ischemia after a prolonged ischemic insult, has been found to reduce the delayed neuronal loss after stroke. However, the mechanisms underlying such endogenous neuroprotective strategy remain obscure. In this study, we try to explore the excitatory postsynaptic signal events associated with neuroprotective effect of ischemic postconditioning.
Methods—Global cerebral ischemia was induced for 15 minutes by the 4-vessel occlusion method in male Sprague–Dawley rats. Ischemic postconditioning was conducted 10 minutes later by a single reocclusion for 3 minutes.
Results—A severe global cerebral ischemia after 5 days of reperfusion destroyed almost all hippocampal CA1 pyramidal neurons. A brief ischemic postconditioning robustly reduced the neuronal loss after ischemia. Preadministration of phosphoinositide 3-kinase inhibitor LY294002 blocked the neuroprotection of postconditioning, whereas mitogen-activated protein kinase kinase 1 inhibitor PD98059 had no effect. Ischemic postconditioning significantly increased the Akt phosphorylation (Ser473). In addition, postconditioning not only perturbed the binding of postsynaptic density protein-95 with glutamatergic kainate receptor subunit 2 and mixed lineage kinase 3 but also suppressed the downstream activation of mixed lineage kinase 3, mitogen-activated protein kinase kinase 7, and c-Jun N-terminal kinase 3. LY294002, but not PD98059, abolished the postconditioning-induced decreases in the assembly of glutamatergic kainate receptor subunit 2–postsynaptic density protein-95–mixed lineage kinase 3 complex and in the mixed lineage kinase 3–c-Jun N-terminal kinase 3 signaling. Akt inhibitor IV, a specific Akt inhibitor, showed the same effects as LY294002.
Conclusions—Ischemic postconditioning protects neurons against stroke by attenuating the postsynaptic glutamatergic kainate receptor subunit 2–postsynaptic density protein-95–mixed lineage kinase 3–c-Jun N-terminal kinase 3 signal cascade via phosphoinositide 3-kinase–Akt pathway. (Stroke. 2013;44:2031-2035.)
- cerebral ischemia
- ischemic postconditioning
- kainate receptors
- mixed lineage kinase 3
- postsynaptic density protein-95
Ischemic postconditioning, which refers to a single or a series of brief interference in the cerebral blood supply performed after a prolonged severe form of ischemic insult, has been established as a novel neuroprotective strategy against ischemic stroke.1 However, the molecular mechanisms responsible for such intraneous neuroprotection largely remain elusive.
Excitatory postsynaptic signal events serve as the major mechanism responsible for ischemic neuronal loss.2 Recently, it has been revealed that postsynaptic density protein-95 (PSD-95) integrates the kainate receptors-evoked postsynaptic signaling by interacting with glutamatergic kainate receptor subunit 2 (GluK2) and downstream signal molecules, such as mixed lineage kinase 3 (MLK3). The formation of GluK2–PSD-95–MLK3 signal complex is consistent with the activation of MLK3, mitogen-activated protein kinase kinase 7 (MKK7), and c-Jun N-terminal kinase 3 (JNK3) signal cascade, which contributes to ischemic neuronal death.3,4 Here, we provide the first evidence that ischemic postconditioning prevents the kainate receptors-induced postsynaptic signaling.
Phosphoinositide 3-kinase (PI3K)–Akt and mitogen-activated protein kinase kinase 1 (MEK1)–extracellular signal-regulated kinases 1/2 (ERK1/2) are well documented as survival pathways in regulating neuronal fate. Ischemic postconditioning increases the level of phosphorylated Akt (p-Akt Ser473), and PI3K inhibitors suppress the beneficial effect of postconditioning, implicating a role of PI3K–Akt pathway in the neuroprotective effect of postconditioning.5,6 Nevertheless, signal events downstream of the PI3K–Akt pathway associated with neuroprotection of the postconditioning are rarely known. Recently, ischemic postconditioning has been found to alter the level of p-ERK1/2 in the brain, yet the contribution of ERK1/2 to neuronal survival is still highly debatable.5,6 In this study, we tested whether both PI3K–Akt and MEK1–ERK1/2 signal are involved in the neuroprotection of postconditioning and further investigated their roles in the inhibition of postsynaptic signal events evoked by postconditioning.
Materials and Methods
All experiments were performed in accordance with the guidelines of the local Animal Care Committee. Adult male Sprague–Dawley rats weighing 250 to 300 g were given free access to food and water before surgery. Global cerebral ischemia was induced for 15 minutes by the 4-vessel occlusion method, as previously described.7 The sham operation was performed using the same surgical procedures except for occlusion of carotid arteries. Ten minutes after the ischemia, both carotid arteries were reoccluded for 3 minutes as the ischemic postconditioning.
For methods of drug administration, histological assessment, immunoprecipitation, immunoblot and statistical analysis, and details of antibodies, please see http://stroke.ahajournals.org.
Nissl (cresyl violet) staining showed that global cerebral ischemia followed by 5 days of reperfusion destroyed ≈95% of pyramidal neurons in the vulnerable hippocampal CA1 subfield. Postconditioning, 3-minute reocclusion performed 10 minutes after ischemia, significantly decreased the delayed neuronal loss. Pretreatment of PI3K inhibitor LY294002 reduced the postconditioning-mediated neuronal survival, whereas MEK1 inhibitor PD98059 did not affect the number of surviving neurons (Figure 1A; Figure I in the online-only Data Supplement). Immunoblot analysis showed a similar change pattern of p-Akt at different times of reperfusion after ischemia with or without postconditioning, except a significant increase of p-Akt at 6 hours of reperfusion after postconditioning (Figure 1B). The data suggest that level of p-Akt up to a threshold value after reperfusion after ischemia may be required for the neuroprotection of postconditioning.
As shown in Figure 2A, postconditioning resulted in the decrease of p-MLK3 to the basal level at 6 and 24 hours of reperfusion. The data imply that the decline of MLK3 activity associates with the benefit of postconditioning. Next, postconditioning was found to disassociate the ischemia/reperfusion-induced binding of GluK2 and MLK3 with PSD-95, and to decrease the phosphorylation of MLK3, MKK7, and JNK3 (Figure 2B and 2C), suggesting a negative effect of postconditioning on the postsynaptic GluK2–PSD-95–MLK3 signal assembly and its downstream MLK3–JNK3 signal. LY294002 reversed the suppression of postconditioning in the association of GluK2 and MLK3 with PSD-95 and in the phosphorylation of MLK3, MKK7, and JNK3, whereas PD98059 did not play any effect (Figure 2B and 2C), which reveals a novel crosstalk between PI3K pathway and GluK2–PSD-95–MLK3–JNK3 signal induced by postconditioning.
Although Akt activity has been reported to be involved in the protection of postconditioning, the exact role of Akt signal has not been confirmed. Here, we found that Akt inhibitor IV abolished the postconditioning-mediated neuronal survival in the hippocampal CA1 subfield after global ischemia (Figure 3A). The binding of GluK2 and MLK3 with PSD-95 and the phosphorylation of MLK3, MKK7, and JNK3 stepped up in Akt inhibitor IV–treated postconditioning group (Figure 3B and 3C). These data indicate that the postconditioning-conferred inhibition of GluK2–PSD-95–MLK3–JNK3 signaling cascade is PI3K–Akt pathway dependent.
Ischemic stroke is one of the leading causes of death and disability worldwide.8 Rapid reperfusion, the only widely approved clinical treatment, mostly leads to further damage to the ischemic brain. It has been recently reported that ischemic postconditioning represents a novel neuroprotective strategy against ischemic brain damage. Exploring the signal events evoked by such endogenous neuroprotective approach may offer promising therapeutic targets for ischemic stroke. In this study, we confirm the efficacy of a single postconditioning ischemia in a global ischemic model and provide the first evidence that ischemic postconditioning prevents the excessive postsynaptic signaling.
PSD-95 acts as a postsynaptic organizer of excitatory signaling cascades.9,10 PSD-95 binds to NMDA and kainate receptor subunits, as well as to downstream signal molecules, such as MLK3, which facilitates the specificity and efficiency of signal transduction. Here, we found that ischemic postconditioning attenuates postsynaptic kainate receptors signaling by disassociating the GluK2–PSD-95–MLK3 signal complex. Whether postconditioning affects the NMDA receptors-triggered postsynaptic signaling still remains to be elucidated.
Our data further support a critical role of PI3K–Akt pathway in postconditioning neuroprotection. Also, we extend these data by establishing a novel association between PI3K–Akt pathway and GluK2–PSD-95–MLK3–JNK3 signal cascade. Previously, Gao et al6 showed a reduction of p-JNK level by postconditioning, although there is no reason to be mentioned. Our data suggest an important position of GluK2–PSD-95–MLK3 signal complex in the reverse correlation between prosurvival PI3K–Akt and prodeath MLK3–MKK7–JNK3 pathways. To date, we do not know how the activated PI3K–Akt signal acts on GluK2–PSD-95–MLK3 complex. It should be interesting to identify several Akt substrates in postsynaptic site.
In brief, postconditioning resets the balance between prosurvival PI3K–Akt and prodeath GluK2–PSD-95–MLK3–JNK3 pathways. Further work will be required to elucidate the molecular mechanisms involved in the correlation between PI3K–Akt pathway and excitatory postsynaptic signaling. Pharmacological approaches targeting postsynaptic signal events may provide a great protection when used after cerebral ischemia, which is more feasible clinically.
Sources of Funding
This work was supported by grants from the National Natural Science Foundation of China (81173030, 30873054), the Major Basic Research Project of Jiangsu Higher Education Institutions (11KJA310005), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the Qing Lan Project of Jiangsu Province.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.680181/-/DC1.
- Received October 13, 2012.
- Revision received February 25, 2013.
- Accepted March 18, 2013.
- © 2013 American Heart Association, Inc.
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