Alfred Sholl-Franco

ATP controls cell cycle and induces proliferation in the mouse developing retina

Previous data suggest that nucleotides are important mitogens in the developing chick retina. Here, we extended the study on the mitogenic effect of ATP to newborn mouse retinal explants. Our results showed that P2Y1 receptors were widely distributed in C57bl/6 mice retina and that the majority of PCNA positive cells co‐localized with P2Y1 receptor. To evaluate proliferation, retinal explants obtained from newborn mice were incubated with 0.5 μCi [3H]‐thymidine or 3 μM BrDU 1 h before the end of culture. Our data showed that ATP induced a dose‐dependent increase in [3H]‐thymidine incorporation, an effect that was mimicked by ADP but not by UTP and was blocked by the P2 antagonist PPADS in a dose‐dependent manner. The increase in [3H]‐thymidine incorporation induced by ATP was only observed in explants cultured for 3 days or less and was mimicked by the ectoapyrase inhibitor ARL 67156. It corresponded to an increase in the number of BrdU+ cells in the neuroblastic layer (NL) of the tissue, suggesting that ATP, through activation of P2Y1 receptors, induced proliferation of late developing progenitors in retinal explants of newborn mice. The increase in the number of BrdU+ cells was observed across the whole NL when explants were incubated with ATP for 24 h and no increase in the number of p‐histone H3 labeled cells could be noticed at this time point. In longer incubations of 48 h with ATP or 24 h with ATP followed by a period of 24 h in fresh medium, an increase in the number of BrdU+ cells promoted by ATP was observed only in the middle and outer, but not in the inner NL. In these conditions, an increase in the number of p‐histone H3 labeled cells was detected in the outer NL, suggesting that ATP induced cells to enter S and progress to G2 phase of the cell cycle in the first 24 h period of incubation. ATP also induced an increase and a decrease in the expression of cyclin D1and p27kip1, respectively, in retinal progenitors of the NL. While the increase in the expression of cyclin D1 was observed when retinal explants were incubated for 3 h or longer periods of time, the decrease in the expression of p27kip1 was noticed only after 6 h incubation with ATP. Both effects were blocked by the P2 receptor antagonist PPADS. These data suggest that ATP induces cell proliferation in retinal explants by inducing late developing progenitors to progress from G1 to S phase of cell cycle.

Signal transduction pathways associated with ATP-induced proliferation of cell progenitors in the intact embryonic retina

ATP and ADP induce retinal cell proliferation through activation of PKC and extracellular signal‐regulated kinases (ERKs). Here, we characterized the effect of purinergic agonists on the turnover of phosphoinositides and activation of ERKs during development of the chick embryo retina. When intact retinas were incubated with ATP, ADP or UTP, a dose‐dependent accumulation of [3H]‐phosphoinositides was observed (% of control, EC50: 548 ± 20.5%, 0.18 mM; 314 ± 53.8%, 0.51 mM; 704 ± 139.9%, 0.018 mM, respectively). Only the response promoted by ADP was completely inhibited by the P2 receptor antagonists, PPADS and suramin. All the responses decreased with the progression of retinal development. Western blot assays revealed that ATP, ADP and UTP stimulated the phosphorylation of ERKs in the chick embryo retina very early during development (% of control: 174 ± 16; 199 ± 16.4 and 206 ± 37, respectively). The responses to ADP and UTP were transient and dose‐dependent, showing EC50 values of 0.12 mM and 0.009 mM. The response to ADP was inhibited by the antagonists PPADS and suramin and by U73122 and chelerythrine chloride, which block PLC and PKC, respectively. Conversely, chelerythrine chloride did not block the response induced by UTP. Immunohistochemical analysis revealed that ATP and ADP induced the phosphorylation of ERKs in cells of the neuroblastic layer of retinas from embryos at E8. Our data showed that ATP, ADP and UTP stimulate the turnover of InsPs and promoted the activation of ERKs in the chick embryo retina. ADP, through activation of P2Y1 receptors, activated ERK pathway through PLC and PKC and UTP, via P2Y4‐like receptors, induced the phosphorylation of ERKs through a pathway that did not involve PKC.

Interleukin-2 and interleukin-4 increase the survival of retinal ganglion cells in culture

Natural cell death is a degenerative phenomenon observed during the normal development of the nervous system. The neuroprotective effects of cytokines produced by neuronal, glial or infiltrating cells on neurons have been extensively studied. In this work we studied the role of interleukin (IL)-2 and IL-4 on the survival of retinal ganglion cells (RGC) after 48 h in culture. Our results demonstrate that the effect of both ILs was dose-dependent and the treatment with either IL-2 (50 U/ml) or IL-4 (5 U/ml) induced a 2-fold increase in RGC survival. The effect of IL-4, but not of IL-2, was totally abolished by either 20 μM 5-fluoro-2′-deoxyuridine, an inhibitor of cell proliferation, or by 1 μM telenzepine, an inhibitor of M1 muscarinic receptor. Our results suggest that both cytokines could play an important role during the development of retinal tissue as well as during retina trauma.

IL-4 increases GABAergic phenotype in rat retinal cell cultures: involvement of muscarinic receptors and protein kinase C

Interleukin-4 (IL-4) is an anti-inflammatory cytokine. During injuries, infections and neurodegenerative diseases, high levels of this molecule are expressed in the brain. In the present work, we investigated the effect of IL-4 on GABAergic differentiation of retinal cells kept in vitro. We analyzed either the uptake of [3H]-gamma-aminobutyric acid (GABA) or the expression of glutamic acid decarboxylase (GAD-67) following IL-4 treatment. We have also investigated the pharmacological modulation of the [3H]-GABA uptake by cholinergic activation. Our results demonstrate that IL-4 increases the uptake of [3H]-GABA after 48 h in culture in a dose-dependent manner (0.5-100 U/ml). The maximal effect was obtained with 5 U/ml (75% increase). This effect was blocked by 1 mM of nipecotic acid, demonstrating the involvement of the GAT-1 subtype of GABA transporter. The IL-4 effect depends on M1 muscarinic activity, an increase in intracellular calcium levels, tyrosine kinase activity and protein kinase C (PKC) activity. Treatment with IL-4 for 48 h induced an increase of 90% in the number of GAD- and GABA-immunoreactive cells when compared with control cultures. Our results indicate that IL-4 modulates the GABAergic phenotype of retinal cells in culture. This result can suggest an important role for this cytokine either during the normal development of retinal circuitry or during neuroprotection after injuries.

Interleukin-4 blocks proliferation of retinal progenitor cells and increases rod photoreceptor differentiation through distinct signaling pathways.

Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been related to the differentiation of the rodent retina in vitro, but constitutive presence of either IL-4 or of IL-4 receptor in the retina has not been reported. In this work we examined the expression of IL-4 and its specific receptor alpha subunit (IL-4Ralpha). IL-4Ralpha is expressed both in neural retina and non-neural ocular tissue, while IL-4 was found mainly in non-neural tissue. We characterized a novel trophic effect of IL-4 upon the retina. We showed that IL-4 can inhibit the proliferation of retinal cells (approximately 40%) through the cAMP-PKA pathway and associated with a reduction of cyclin D1 and increase of p27(kip1). IL-4 also promotes the differentiation of rod photoreceptors. Activation of tyrosine kinases, protein kinase C, and mitogen-activated kinases of the Erk family were required for IL-4-induced rod photoreceptor differentiation, independent of the release of other trophic factors in culture. Taken together, our results show, for the first time, that IL-4 directly modulates proliferation of retinal cells and rod photoreceptor differentiation, through distinct signaling pathways.

Expression of GAP-43 during development and after monocular enucleation in the rat superior colliculus

The retinotectal projection of rodents presents a precise retinotopic organization that develops, from diffuse connections, from the day of birth to post-natal day 10. Previous data had demonstrated that these projections undergo reorganization after retinal lesions, nerve crush and monocular enucleation. The axonal growth seems to be directly related to growth-associated protein-43 (GAP-43) expression, a protein predominantly located in growth cones, which is regulated throughout development. GAP-43 is presented both under non-phosphorylated and phosphorylated (pGAP-43) forms. The phosphorylated form, has been associated to axon growth via polymerization of F-actin, and synaptic enhancement through neurotransmitter release facilitation. Herein we investigated the spatio-temporal expression of GAP-43 in the rat superior colliculus during normal development and after monocular enucleation in different stages of development. Lister Hooded rats ranging from post-natal day 0 to 70 were used for ontogeny studies. Another group of animals were submitted to monocular enucleation at post-natal day 10 (PND10) or PND21. After different survival-times, the animals were sacrificed and the brains processed for either immunohistochemistry or western blotting analysis. Our data show that GAP-43 is expressed in retinotectal axons in early stages of development but remains present in adulthood. Moreover, monocular enucleation leads to an increase in pGAP-43 expression in the deafferented colliculus. Taken together these results suggest a role for pGAP-43 in retinotectal morphological plasticity observed both during normal development and after monocular enucleation.

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF.

Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca(2+) increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.

In vitro effects of bevacizumab treatment on newborn rat retinal cell proliferation, death, and differentiation.

PURPOSE Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. METHODS Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared. RESULTS No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls. CONCLUSIONS Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Protein kinases JAK and ERK mediate protective effect of interleukin-2 upon ganglion cells of the developing rat retina

Interleukin-2 (IL-2), a prototypical pro-inflammatory cytokine firstly related to T cells differentiation, exerts pleiotrophic functions in several areas of the central nervous system. Previously we had described the neurotrophic roles of this interleukin upon retinal neurons. Therefore, the aim of this work was to investigate the signaling pathways involved in the neuroprotective effect of IL-2 on axotomized RGC. Herein we demonstrated that at postnatal day 2 IL-2 receptor α subunit (IL-2Rα) is expressed in inner plexiform layer, retinal ganglion cells layer and retinal nerve fibers layer. Moreover, using a model of organotypic retinal explants and rhodamine dextran retrograde labeling for specifically quantify RGC, we showed that IL-2 increased the survival of axotomized RGC after 2 (85.43±5.43%) and 5 (50.23%±5.32) days in vitro. Western blot analysis demonstrated that IL-2 treatment increased the phosphorilation of both extracellular signal-regulated kinases (ERK)1/2 and AKT (~two fold). However, its neuroprotective effect upon RGC was dependent of Janus kinase (JAK) and ERK1/2 activity but not of AKT activity. Taken together our results showed that the IL-2 neuroprotective action upon RGC in vitro is mediated by JAK and ERK1/2 activation.

Intravitreous interleukin-2 treatment and inflammation modulates glial cells activation and uncrossed retinotectal development

Interleukin-2 (IL-2) plays regulatory functions both in immune and nervous system. However, in the visual system, little is known about the cellular types which respond to IL-2 and its effects. Herein, we investigated the influence of IL-2 in the development of central visual pathways. Lister Hooded rats were submitted to multiple (at postnatal days [PND]7/10/13) or single (at PND10) intravitreous injections of phosphate-buffered saline (PBS) (vehicle), zymosan, or IL-2. IL-2 receptor α subunit was detected in the whole postnatal retina. Chronic treatment with either PBS or IL-2 increases retinal glial fibrillary acidic protein (GFAP) expression, induces intravitreous inflammation revealed by the presence of macrophages, and results in a slight rearrangement of retinotectal axons. Acute zymosan treatment disrupts retinotectal axons distribution, confirming the influence of inflammation on retinotectal pathway reordering. Furthermore, acute IL-2 treatment increases GFAP expression in the retina without inflammation and produces a robust sprouting of the intact uncrossed retinotectal pathway. No difference was observed in glial cells activity in superior colliculus. Taken together, these data suggest that inflammation and interleukin-2 modulate retinal ganglion cells development and the distribution of their axons within central targets.