Biología Celular

Activation of c-Jun N-Terminal Kinase (JNK) during Mitosis in Retinal Progenitor Cells
Vinicius Toledo Ribas, Bruno Souza Goncalves, Rafael Linden, Luciana Barreto Chiarini* ¸
´ Instituto de Biofısica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil

Abstract
Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In thisstudy, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy.Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosisof progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrantchromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.
Citation: Ribas VT, Goncalves BS, Linden R, Chiarini LB (2012) Activation of c-Jun N-Terminal Kinase (JNK) during Mitosis inRetinal Progenitor Cells. PLoS ONE 7(4): ¸ e34483. doi:10.1371/journal.pone.0034483 Editor: Thomas A. Reh, University of Washington, United States of America Received September 7, 2011; Accepted March 5, 2012; Published April 4, 2012 Copyright: ß 2012 Ribas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original author and source are credited. ´ ´ Funding: This study was supported by grants from Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq - www.cnpq.br), Fundacao de ¸˜ ` ˆncia-Rio (PRONEX-Rio), and Pensa Rio. The ´ Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ - www.faperj.br), Programa de Apoio a Nucleosde Excele funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chiarini@biof.ufrj.br

Introduction
The retina is part of the central nervous system and is widely used as a model to study mechanisms of neurogenesis [1], due to knowledgeof the spatio-temporal development of various retinal cell types. In newborn rats, the innermost (basal) cell layer is the ganglion cell layer (GCL) that contains only relatively differentiated ganglion cells [2], because displaced amacrine cells migrate postnatally into the GCL [3]. Further towards apical retina, there is an immature inner nuclear layer (INLi), followed by the proliferativeneuroblastic layer (NBL). The NBL of neonatal rats contains both proliferating progenitor and postmitotic cells, including early developing horizontal cells [4]. The cell cycle in the proliferative zone of the retina, similar to other parts of the CNS, is tightly controlled and proceeds in synchrony with interkinetic migration of the progenitor cell nuclei along the depth of the NBL [5]. The phases...