Angiotensin II Type 1 Receptor (AT1R)-Mediated Post-Transcriptional Reduction of Synaptophysin in Neuronal Cells: Mechanism of Neurodegeneration in the Diabetic Retina

Abstract

Purpose: : To investigate the detailed molecular mechanisms underlying the AT1R-mediated decrease in synaptophysin observed in the murine retina with streptozotocin (STZ)-induced diabetes. We have previously reported that increased levels of angiotensin II are involved in the retinal dysfunction caused by downregulation of synaptophysin protein in the diabetic retina at the ARVO 2007 annual meeting. In contrast, mRNA levels of synaptophysin were unaltered in diabetic mice compared with non-diabetic controls. In this study, we investigate the molecular mechanisms involved in the AT1R-mediated post-transcriptional decrease in synaptophysin utilizing the in vitro culture system with neuronal cells stimulated with angiotensin II.

Methods: : The neuronal cell line PC12D cells were incubated with nerve growth factor for differentiation to neurons for 2 days. The neuronal cells were pretreated with an AT1R blocker telmisartan, a proteasome inhibitor MG132, an ERK inhibitor U0126, or vehicle. The cells were subsequently stimulated with angiotensin II. The neurons were subjected to quantitative RT-PCR and immunoprecipitation for synaptophysin, and to immunoblot analyses for synaptophysin, ubiquitin and phosphorylated forms of ERK.

Results: : In agreement with the in vivo data (reported at ARVO 2007), in vitro application with angiotensin II significantly reduced synaptophysin protein, but not mRNA, levels in a post-transcriptional manner, which were significantly reversed by telmisartan. Multiubiquitinated synaptophysin, enhanced by stimulation with angiotensin II, was substantially attenuated by telmisartan. Application with MG132 led to significant suppression of angiotensin II-induced degradation of synaptophysin. Inhibition of ERK activation with U0126 led to significant suppression of angiotensin II-induced degradation of synaptophysin in a dose-dependent manner.

Conclusions: : AT1R-mediated post-transcriptional reduction of synaptophysin was regulated by activation of the ERK pathway and the ubiquitin-proteasome system in neuronal cells.

Keywords: diabetic retinopathy • neuroprotection • synapse