Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment

Jia Zhang,Hui Yan,Yong-Ping Wu,Chong Li,Guang-Yi Zhang

doi:10.1074/jbc.m110.156299

Abstract

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.

Highlights

Receptors: NMDA, AMPA, and kainate (KA) receptors
We demonstrate that KA induces Bcl-2 denitrosylation through the GluR6-KA receptor pathway, which is proposed to facilitate Bcl-2 ubiquitination and downregulate its protein level
The Degradation of Bcl-2 in the Hippocampal CA1 and CA3/DG Regions Is Induced by KA through the Activation of GluR6-containing Kainate Receptors—We previously reported that KA treatment through intracerebroventricular infusion or intraperitoneal injection down-regulates the Bcl-2 protein level gradually in hippocampal CA1 and CA3/DG regions in a concentration-dependent manner [3, 24]

Summary

Introduction

It has been well established that the Bcl-2 protein levels are essential for its anti-apoptotic function. The regulation of these levels mainly occurs via post-translational modifications and degradation (9 –14). Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation protein (Cys-158 and Cys-229) are important for the S-nitrosylation process [18]. A more recent report has indicated that the S-nitrosylation of Bcl-2 prevents its ubiquitin-proteasomal degradation during the apoptotic cell death induced by chromium (VI) in lung cancers [18]. It has been shown that the mechanism of cisplatin resistance involves the up-regulation of Bcl-2 expression by NO, which occurs by preventing its ubiquitin-dependent degradation in human lung carcinoma H-460 cells [20]. The GluR6 antagonist NS102, NO donor S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and the proteasome inhibitor MG132 prevent the denitrosylation and the degradation of Bcl-2, playing an important role in neuron protection in hippocampal CA1 and CA3/DG regions

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Results

Conclusion