Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin‑induced diabetic rats

Silva,Viviane Aline Oliveira; André,Nayara Delgado; Sousa,Thaís Amaral E; Alves,Vâni Maria; Kettelhut,Isis Do Carmo; De Lucca,Fernando Luiz

doi:10.3892/mmr.2021.12253

Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin‑induced diabetic rats

Authors:
- Viviane Aline Oliveira Silva
- Nayara Delgado André
- Thaís Amaral E Sousa
- Vâni Maria Alves
- Isis Do Carmo Kettelhut
- Fernando Luiz De Lucca
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Affiliations: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo 14784‑400, Brazil, University of São João Del Rei, Divinópolis, Minas Gerais 35501‑296, Brazil, Federal Institute of Education, Science and Technology of Goiás, Formosa, Goiás 73813‑816, Brazil, Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo 14049‑900, Brazil
Published online on: June 28, 2021 https://doi.org/10.3892/mmr.2021.12253
Article Number: 614
Copyright: © Silva et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)‑induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA‑dependent protein kinase (PKR) is a stress‑sensitive pro‑apoptotic kinase. Our previous study indicated that PKR‑associated protein X, a stress‑sensitive activator of PKR, is upregulated in the early stage of STZ‑induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ‑induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p‑eIF2‑α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p‑eIF2‑α expression and cellular localization followed the trend of PKR, suggesting that this pro‑apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

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