Maternal Toxoplasma gondii infection affects proliferation, differentiation and cell cycle regulation of retinal neural progenitor cells in mouse embryo.

Background: Toxoplasmosis affects one third of the world population and has the protozoan Toxoplasma gondii as etiological agent. Congenital toxoplasmosis (CT) can cause severe damage to the fetus, including miscarriages, intracranial calcification, hydrocephalus and retinochoroiditis. Severity of CT depends on the gestational period in which infection occurs, and alterations at the cellular level during retinal development have been reported. In this study, we proposed a mouse CT model to investigate the impact of infection on retinal development. Methods: Pregnant females of pigmented C57BL/6 strain mice were infected intragastrically with two T. gondii cysts (ME49 strain) at embryonic day 10 (E10), and the offspring were analyzed at E18. Results: Infected embryos had significantly smaller body sizes and weights than the PBS-treated controls, indicating that embryonic development was affected. In the retina, a significant increase in the number of Ki-67-positive cells (marker of proliferating cells) was found in the apical region of the NBL of infected mice compared to the control. Supporting this, cell cycle proteins Cyclin D3, Cdk6 and pChK2 were significantly altered in infected retinas. Interestingly, the immunohistochemical analysis showed a significant increase in the population of ß-III-tubulin-positive cells, one of the earliest markers of neuronal differentiation. Conclusions: Our data suggests that CT affects cell cycle progression in retinal progenitor cells, possibly inducing the arrest of these cells at G2/M phase. Such alterations could influence the differentiation, anticipating/increasing neuronal maturation, and therefore leading to abnormal retinal formation. Our model mimics important events observed in ocular CT.

P2Y12 but not P2Y13 Purinergic Receptor Controls Postnatal Rat Retinogenesis In Vivo.

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.

Adenine Nucleotides Control Proliferation In Vivo of Rat Retinal Progenitors by P2Y1 Receptor.

Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y1 receptor, a G protein-coupled receptor. Here, we evaluated the function of adenine nucleotides in vivo during retinal development of newborn rats. Intravitreal injection of apyrase, an enzyme that hydrolyzes nucleotides, reduced cell proliferation in retinas at postnatal day 2 (P2). This decrease was reversed when retinas were treated together with ATPγ-S or ADPß-S, two hydrolysis-resistant analogs of ATP and ADP, respectively. During early postnatal days (P0 to P5), an increase in ectonucleotidase (E-NTPDase) activity was observed in the retina, suggesting a decrease in the availability of adenine nucleotides, coinciding with the end of proliferation. Interestingly, intravitreal injection of the E-NTPDase inhibitor ARL67156 increased proliferation by around 60 % at P5 rats. Furthermore, immunolabeling against P2Y1 receptor was observed overall in retina layers from P2 rats, including proliferating Ki-67-positive cells in the neuroblastic layer (NBL), suggesting that this receptor could be responsible for the action of adenine nucleotides upon proliferation of RPCs. Accordingly, intravitreal injection of MRS2179, a selective antagonist of P2Y1 receptors, reduced cell proliferation by approximately 20 % in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57KIP2 and cyclin D1 expression, a reduction in cyclin E and Rb phosphorylated expression and in BrdU-positive cell number. These data suggest that the adenine nucleotides modulate the proliferation of rat RPCs via activation of P2Y1 receptors regulating transition from G1 to S phase of the cell cycle.