Abstract
c-Jun N-terminal kinase (JNK) plays a key role in the regulation of neuronal apoptosis. Previous studies have revealed that forkhead transcription factor (FOXO3a) is a critical effector of JNK-mediated tumor suppression. However, it is not clear whether the JNK/FOXO3a pathway is involved in neuronal apoptosis in the developing rat brain after hypoxia-ischemia (HI). In this study, we generated an HI model using postnatal day 7 rats. Fluorescence immunolabeling and Western blot assays were used to detect the distribution and expression of total and phosphorylated JNK and FOXO3a and the pro-apoptotic proteins Bim and CC3. We found that JNK phosphorylation was accompanied by FOXO3a dephosphorylation, which induced FOXO3a translocation into the nucleus, resulting in the upregulation of levels of Bim and CC3 proteins. Furthermore, we found that JNK inhibition by AS601245, a specific JNK inhibitor, significantly increased FOXO3a phosphorylation, which attenuated FOXO3a translocation into the nucleus after HI. Moreover, JNK inhibition downregulated levels of Bim and CC3 proteins, attenuated neuronal apoptosis and reduced brain infarct volume in the developing rat brain. Our findings suggest that the JNK/FOXO3a/Bim pathway is involved in neuronal apoptosis in the developing rat brain after HI. Agents targeting JNK may offer promise for rescuing neurons from HI-induced damage.
Highlights
We found that both the p-Akt and total Akt levels were not changed at 48 h in the AS601245-treated cortex compared with the DMSO-treated cortex (Fig 3G and 3H), suggesting that the regulation of FOXO3a by Jun N-terminal kinase (JNK) is independent of Akt in this study
We found that the Bcl-2-interacting mediator of cell death (Bim) and Cleaved caspase-3 (CC3) protein levels were significantly reduced at 48 h in the AS601245-treated cortex compared with the DMSO-treated cortex (Fig 4A and 4B)
After normalization to GAPDH expression, we found that there was approximately a 43% decrease in Bim protein levels at 48 h (Fig 4B) and approximately a 61% decrease in CC3 protein levels at 48 h (Fig 4B) in the AS601245-treated cortex compared with the DMSO-treated cortex
Summary
All animal research was approved by the Sichuan University Committee on Animal Research. Pups were anesthetized with 2.5% halothane and intracerebroventricularly infused with vehicle (dimethyl sulfoxide [DMSO], Sigma-Aldrich, USA) or 150 nmol of AS601245 The sections were incubated overnight at 4°C in a mixture of two primary antibodies raised in separate species: rabbit polyclonal antibodies against p-JNK (Thr183/Tyr185; Cell Signaling, 1:100) or Bim (Abcam, 1:200); and mouse monoclonal antibodies against NeuN (Millipore, 1:100) or GFAP (Millipore, 1:100). Protein samples (100 μg per lane) were separated on 8% SDS-polyacrylamide gels, as described previously [23]. JNK activity was measured using a specific kit (Cell Signaling, USA), and glutathione Stransferase-Jun fusion peptides served as the substrate for JNK, as previously described [22]. Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred onto PVDF membranes, and incubated with an anti-phospho-c-Jun (Ser63) antibody (Cell Signaling, 1:1000).
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