Abstract
Adenine nucleotides through P2Y1 receptor stimulation are known to control retinal progenitor cell (RPC) proliferation by modulating expression of the p57KIP2, a cell cycle regulator. However, the role of Gi protein-coupled P2Y12 and P2Y13 receptors also activated by adenine nucleotides in RPC proliferation is still unknown. Gene expression of the purinergic P2Y12 subtype was detected in rat retina during early postnatal days (P0 to P5), while expression levels of P2Y13 were low. Immunohistochemistry assays performed with rat retina on P3 revealed P2Y12 receptor expression in both Ki-67-positive cells in the neuroblastic layer and Ki-67-negative cells in the ganglion cell layer and inner nuclear layer. Nonetheless, P2Y13 receptor expression could not be detected in any stratum of rat retina. Intravitreal injection of PSB 0739 or clopidogrel, both selective P2Y12 receptor antagonists, increased by 20 and 15%, respectively, the number of Ki-67-positive cells following 24 h of exposure. Moreover, the P2Y12 receptor inhibition increased cyclin D1 and decreased p57KIP2 expression. However, there were no changes in the S phase of the cell cycle (BrdU-positive cells) or in mitosis (phospho-histone-H3-positive cells). Interestingly, an increase in the number of cyclin D1/TUNEL-positive cells after treatment with PSB 0739 was observed. These data suggest that activation of P2Y12 receptors is required for the successful exit of RPCs from cell cycle in the postnatal rat retina.
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This study received grant support from the Brazilian funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (São Paulo Research Foundation, FAPESP Proj. Nr. 2012/50880-4), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ Proj. Nr.110.145/2014), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowship support.
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Supplementary Figure 1
P2Y12 receptor was expressed in P2 rat retina. Projection of confocal optical sections showing that P2Y12 labeling was found in Ki-67 positive and negative cells in the NBL. The RPCs nuclei were stained with DAPI (blue), P2Y12 in red and Ki-67 in green. Scale bar = 50 μm. (GIF 395 kb)
Supplementary Figure 2
P2Y12 receptor blockage receptor in rat retina in vitro increased proliferation of RPC. Explants at P2 rat retina were treated with PSB 0739 at concentrations of 0.05, 0.1 and 1 μM for 24 h. Treatment at 1 μM concentration increased the number of Ki-67 positive cells relative to the control (Control = 6552 ± 185; 0.05 μM = 7506 ± 328.3; 0.1 μM = 7281 ± 370.2; 1 μM = 8384 ± 644). Data are mean values ± S.E.M. from three independent experiments. *p < 0.05 as compared to control. (GIF 11 kb)
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de Almeida-Pereira, L., Repossi, M.G., Magalhães, C.F. et al. P2Y12 but not P2Y13 Purinergic Receptor Controls Postnatal Rat Retinogenesis In Vivo. Mol Neurobiol 55, 8612–8624 (2018). https://doi.org/10.1007/s12035-018-1012-1
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DOI: https://doi.org/10.1007/s12035-018-1012-1