The Healthy and Diseased Retina Seen through Neuron-Glia Interactions.

The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.

Apoptosis in the neuroprotective effect of α7 nicotinic receptor in neurodegenerative models.

The α7 subtype of nicotinic receptors (α7 nAChRs) is one of the most abundant nicotinic receptor subtypes in the central nervous system (CNS) and both neurons and nonneuronal cells express α7 nAChRs. When activated, α7 nAChRs become permeable to cations and promote cellular responses such as anti-apoptotic signaling by modulating the caspases and proteins of the Bcl-2 family. Neuroprotection is an important function of these receptors, promoting neuronal survival under pathological conditions, including situations of stress and neuronal degeneration. Studies have demonstrated the relationship between the activation of these receptors and the reduction of neuronal or glial cell injury, by controlling apoptotic processes in different models, including neurodegenerative diseases such as Alzheimer's disease. Therefore, one of the most important signaling pathways activated by α7 nAChRs is the PI3K/Akt signaling cascade, which promotes the stimulation of anti-apoptotic molecules of the Bcl-2 family, Bcl-2 and Bcl-xl, and reduces the expression of caspases and proapoptotic molecules, resulting in cell survival. In Alzheimer's models, the literature shows that α7 nAChR activation attenuates Aß-induced neurotoxicity through modulation of different intrinsic apoptotic pathways via PI3K/Akt and mitogen-activated protein kinase (MAPK). In this review, we provide an up-to-date summary of the current evidence on the relationship between the activation of α7 nAChRs, a subtype of nicotinic acetylcholine receptor, and its role in neuroprotection by modulating apoptotic pathways.

Insulin-like growth factor-1 stimulates retinal cell proliferation via activation of multiple signaling pathways.

Insulin-like growth factor-1 (IGF-1) plays critical roles in the development of the central nervous system (CNS), including the retina, regulating cell proliferation, differentiation, and survival. Here, we investigated the role of IGF-1 on retinal cell proliferation using primary cultures from rat neural retina. Our data show that IGF-1 stimulates retinal cell proliferation and regulates the expression of neurotrophic factors, such as interleukin-4 and brain-derived neurotrophic factor. In addition, our results indicates that IGF-1-induced retinal cell proliferation requires activation of multiple signaling pathways, including phosphatidylinositol 3-kinase, protein kinase Src, phospholipase-C, protein kinase C delta, and mitogen-activated protein kinase pathways. We further show that activation of matrix metalloproteinases and epidermal growth factor receptor is also necessary for IGF-1 enhancing retinal cell proliferation. Overall, these results unveil potential mechanisms by which IGF-1 ensures retinal cell proliferation and support the notion that manipulation of IGF-1 signaling may be beneficial in CNS disorders associated with abnormal cell proliferation.

Interleukin-4 activates divergent cell-intrinsic signals to regulate retinal cell proliferation induced by classical growth factors.

In the developing retina, precise coordination of cell proliferation, differentiation, and survival is essential for proper retinal maturation and function. We have previously reported evidence that interleukin-4 (IL-4) plays critical roles in neuronal differentiation and survival during retinal development. However, little is known about the role of IL-4 on retinal cell proliferation. In the current study, we investigated if IL-4 regulates cell proliferation induced by epidermal growth factor (EGF) and by fibroblast growth factor 2 (FGF2) in primary retinal cell cultures obtained from newborn rats. First, we show that EGF and FGF2 act as mitogens for glial cells, increasing proliferation of these cells in the retina. EGF- and FGF2-induced mitogenesis requires activation of distinct cell-intrinsic signals. In retinal cells exposed to FGF2, IL-4 downregulates p53 levels (a protein whose activation induces cell-cycle arrest) and increases mitogenic responsiveness to FGF2 through activation of protein kinase A (PKA) pathway. Conversely, in retinal cells exposed to EGF, IL-4 downregulates cyclin D1 levels (a protein required for cell-cycle progression), upregulates p53 levels, and decreases mitogenic responsiveness to EGF. The inhibitory effect induced by IL-4 on retinal cells exposed to EGF requires activation of Janus kinase 3 (JAK3), but not activation of PKA. Based on previous and current findings, we propose that IL-4 serves as a node of signal divergence, modulating multiple cell-intrinsic signals (e.g., cyclin D1, p53, JAK3, and PKA) and mitogenic responsiveness to cell-extrinsic signals (e.g., FGF2 and EGF) to control cell proliferation, differentiation, and survival during retinal development.

Ouabain-Na+/K+-ATPase Signaling Regulates Retinal Neuroinflammation and ROS Production Preventing Neuronal Death by an Autophagy-Dependent Mechanism Following Optic Nerve Axotomy In Vitro.

Ouabain is a classic Na+K+ATPase ligand and it has been described to have neuroprotective effects on neurons and glial cells at nanomolar concentrations. In the present work, the neuroprotective and immunomodulatory potential of ouabain was evaluated in neonatal rat retinal cells using an optic nerve axotomy model in vitro. After axotomy, cultured retinal cells were treated with ouabain (3 nM) at different periods. The levels of important inflammatory receptors in the retina such as TNFR1/2, TLR4, and CD14 were analyzed. We observed that TNFR1, TLR4, and CD14 were decreased in all tested periods (15 min, 45 min, 24 h, and 48 h). On the other hand, TNFR2 was increased after 24 h, suggesting an anti-inflammatory potential for ouabain. Moreover, we showed that ouabain also decreased Iba-1 (microglial marker) density. Subsequently, analyses of retrograde labeling of retinal ganglion cells (RGC) were performed after 48 h and showed that ouabain-induced RGC survival depends on autophagy. Using an autophagy inhibitor (3-methyladenine), we observed a complete blockage of the ouabain effect. Western blot analyses showed that ouabain increases the levels of autophagy proteins (LC3 and Beclin-1) coupled to p-CREB transcription factor and leads to autophagosome formation. Additionally, we found that the ratio of cleaved/pro-caspase-3 did not change after ouabain treatment; however, p-JNK density was enhanced. Also, ouabain decreased reactive oxygen species production immediately after axotomy. Taken together, our results suggest that ouabain controls neuroinflammation in the retina following optic nerve axotomy and promotes RGC neuroprotection through activation of the autophagy pathway.

PMA treatment fosters rat retinal ganglion cell survival via TNF signaling.

An insult can trigger a protective response or even cell death depending on different factors that include the duration and magnitude of the event and the ability of the cell to activate protective intracellular signals, including inflammatory cytokines. Our previous work showed that the treatment of Lister Hooded rat retinal cell cultures with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, increases the survival of retinal ganglion cells (RGCs) kept in culture for 48 h after axotomy. Here we aim to analyze how PMA modulates the levels of TNF-α and IL-1ß (both key inflammatory mediators) and the impact of this modulation on RGCs survival. We hypothesize that the increase in RGCs survival mediated by PMA treatment depends upon modulation of the levels of IL-1ß and TNF-α. The effect of PMA treatment was assayed on cell viability, caspase 3 activation, TNF-α and IL-1ß release and TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) levels. PMA treatment increases IL-1ß and TNF-α levels in 15 min in culture and increases the release of both cytokines after 30 min and 24 h, respectively. Both IL-1ß and TNF-α levels decrease after 48 h of PMA treatment. PMA treatment also induces an increase in TNFRII levels while decreasing TNFRI after 24 h. PMA also inhibited caspase-3 activation, and decreased ROS production and EthD-1/calcein ratio in retinal cell cultures leading to an increase in cell viability. The neutralization of IL-1ß (anti-IL1ß 0,1ng/mL), the neutralization of TNF-α (anti-TNF-α 0,1ng/mL) and the TNF-α inhibition using a recombinant soluble TNFRII abolished PMA effect on RGCs survival. These data suggest that PMA treatment induces IL1ß and TNF-α release and modulation of TNFRI/TNFRII expression promoting RGCs survival after axotomy.

Increased Retinal Ganglion Cell Survival by Exogenous IL-2 Depends on IL-10, Dopamine D1 Receptors, and Classical IL-2/IL-2R Signaling Pathways.

Interleukin-2 (IL-2) is a classical pro-inflammatory cytokine known to display neuroprotective roles in the central nervous system including the retina. In the present study, we investigate the molecular targets involved in the neurotrophic effect of IL-2 on retinal ganglion cells (RGC) after optic nerve axotomy. Analysis of retrograde labeling of RGC showed that common cell survival mediators, as Trk receptors, Src, PI3K, PKC, and intracellular calcium do not mediate the neurotrophic effect of IL-2 on RGC. No involvement of MAPK p38 was also observed. However, other MAPKs as MEK and JNK appear to be mediating this IL-2 effect. Our data also indicate that JAK2/3 are important intracellular proteins for the IL-2 effect. Interestingly, we demonstrate that the IL-2 effect depends on dopamine D1 receptors (D1R), the cAMP/PKA pathway, interleukin-10 (IL-10), and NF-κB, suggesting that RGC survival induced by IL-2 encompasses a molecular network of major complexity. In addition, treatment of retinal cells with recombinant IL-10 or 6-Cl-pb (D1R full agonist) was able to increase RGC survival similar to IL-2. Taken together, our results suggest that after optic nerve axotomy, the increase in RGC survival triggered by IL-2 is mediated by IL-10 and D1R along with the intracellular pathways of MAPKs, JAK/STAT, and cAMP/PKA.

COVID-19 pandemic impact on children and adolescents' mental health: Biological, environmental, and social factors.

Since the Coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was announced, we had an unprecedented change in the way we organize ourselves socially and in our daily routine. Children and adolescents were also greatly impacted by the abrupt withdrawal from school, social life and outdoor activities. Some of them also experienced domestic violence growing. The stress they are subjected to directly impacts their mental health on account of increased anxiety, changes in their diets and in school dynamics, fear or even failing to scale the problem. Our aim is to bring up a discussion under different aspects and to alert public health and government agents about the need for surveillance and care of these individuals. We hope that the damage to their mental health as a result of the side effect of this pandemic can be mitigated by adequate and timely intervention.

PKC delta activation increases neonatal rat retinal cells survival in vitro: Involvement of neurotrophins and M1 muscarinic receptors.

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.

Ouabain and BDNF Crosstalk on Ganglion Cell Survival in Mixed Retinal Cell Cultures.

Brain-derived neurotrophic factor (BDNF) is a well-known and well-studied neurotrophin. Most biological effects of BDNF are mediated by the activation of TrkB receptors. This neurotrophin regulates several neuronal functions as cell proliferation, viability, and differentiation. Ouabain is a steroid that binds to the Na(+)/K(+) ATPase, inducing the activation of several intracellular signaling pathways. Previous data from our group described that ouabain treatment increases retinal ganglion cells survival (RGC). The aim of the present study was to evaluate, if this cardiac glycoside can have a synergistic effect with BDNF, the classical trophic factor for retinal ganglion cells, as well as investigate the intracellular signaling pathways involved. Our work demonstrated that the activation of Src, PLC, and PKCδ participates in the signaling cascade mediated by 50 ng/mL BDNF, since their selective inhibitors completely blocked the trophic effect of BDNF. We also demonstrated a synergistic effect on RGC survival when we concomitantly used ouabain (0.75 nM) and BDNF (10 ng/mL). Moreover, the signaling pathways involved in this synergistic effect include Src, PLC, PKCδ, and JNK. Our results suggest that the synergism between ouabain and BDNF occurs through the activation of the Src pathway, JNK, PLC, and PKCδ.

IL-4 Induces Cholinergic Differentiation of Retinal Cells In Vitro.

Interleukin-4 (IL-4) is a pleiotropic cytokine that regulates several phenomena, among them survival and differentiation of neuronal and glial cells. The aim of this work was to investigate the effect of IL-4 on the cholinergic differentiation of neonatal rat retinal cells in vitro, evaluating its effect on the levels of cholinergic markers (CHT1-high-affinity choline transporter; VAChT-vesicular acetylcholine transporter, ChAT-choline acetyltransferase, AChE-acetylcholinesterase), muscarinic receptors, and on the signaling pathways involved. Lister Hooded rat pups were used in postnatal days 0-2 (P0-P2). Our results show that IL-4 treatment (50 U/mL) for 48 h increases the levels of the cholinergic transporters VAChT and CHT1, the acetylcholinesterase activity, and the number of ChAT-positive cells. It also induces changes in muscarinic receptor levels, leading to a small decrease in M1 levels and a significant increase in M3 and M5 levels after 48 h of treatment. We also showed that IL-4 effect on M3 receptors is dependent on type I IL-4 receptor and on an increase in NFκB phosphorylation. These results indicate that IL-4 stimulates cholinergic differentiation of retinal cells.

PMA increases M3 muscarinic receptor levels and decreases retinal cells proliferation through a change in the levels of cell-cycle regulatory proteins.

Protein kinase C (PKC) pathway plays important roles in different phenomena in nervous system development. Our previous data demonstrated that phorbol 12-myristate 13-acetate (PMA) treatment, a PKC activator, for 48 h decreases retinal cells proliferation by a mechanism mediated by muscarinic receptor activation, involving a decrease in M1 receptors levels. The aim of this work was to analyze how PMA interferes in the levels of cell cycle control proteins p53, p21 and cyclin D1 and also to investigate its influence on M3 receptor levels. Our results show that PMA (50 ng/mL) produces a significant increase in p21 and p53 levels, decreases cyclin D1 levels, and also enhances M3 receptors levels in cell cultures. Evaluating the postnatal retinal tissue development until 30 days, we observed that tissue differentiation is accompanied by an increase in M3 and p21 levels. Based on our results we suggest that PMA treatment is promoting a change in muscarinic receptors expression mimicking the pattern observed during tissue differentiation, indicating that PMA is probably accelerating the cholinergic differentiation in rat retinal cell cultures.

Treatment in vitro of retinal cells with IL-4 increases the survival of retinal ganglion cells: the involvement of BDNF.

Interleukin 4 (IL-4) is a pleiotropic cytokine involved in many functions during the development as well as in adult life. Previous work from our group demonstrated, in vitro, that this interleukin is able to prevent rat retinal ganglion cells death after axotomy. The aim of the present study was to investigate the signaling pathways involved in this trophic effect, particularly the cAMP pathway and also to demonstrate the expression of IL-4 in retinas at different stages of post natal development. Our results show that the trophic effect of IL-4 on rat retinal ganglion cells is dependent on the activation of Janus Kinase 3, Protein Kinase A, c-Jun N-terminal Kinase and Tropomyosin related Kinase receptors, on the increase in intracellular calcium levels, on polypeptide release and on the endogenous Brain Derived Neurotrophic Factor (BDNF). We also observed that treatment with IL-4 enhances c-AMP response element binding and Mitogen Activated Protein Kinase phosphorylation and increases the expression of BDNF. Concerning the IL-4 expression our data show an increase in IL-4 levels during post natal development. Taken together our results demonstrate that the trophic effect of IL-4 on retinal ganglion cells of newborn rats is mediated by cAMP pathway and BDNF release.

The trophic effect of ouabain on retinal ganglion cell is mediated by EGF receptor and PKC delta activation.

It was already shown that ouabain treatment can stimulate PKC isoenzymes leading to the activation of intracellular pathways involved in cell survival, growth and proliferation. We have previously demonstrated that ouabain or PMA treatment increases retinal ganglion cell survival, an effect mediated by PKC activation. The aim of this work was to investigate the role of EGF receptors in the ouabain effect and also to study which PKC isoform is activated by treatment with ouabain and PMA. Our results show that 2.5 microM tyrphostin, 1.0 microM PP1, 4.0 microM U73122, 1.0 microM JNK inhibitor V and 2.0 microM rottlerin blocked the ouabain effect indicating an involvement of receptors for EGF, Src, PLC, JNK and PKC delta respectively. The effect of PMA was only abolished when cultures were treated with rottlerin or with the JNK inhibitor suggesting the involvement of PKC delta and JNK. These results indicate that PKC delta could be a key regulator of retinal ganglion cell survival.

Neuronal cell survival: the role of interleukins.

One of the central issues in neuroscience today is the study of the mechanisms of neuronal survival. Since the discovery of nerve growth factor (almost 60 years ago), many groups have clearly demonstrated the central role of neurotrophins on the regulation of neuronal cell survival during developmental stages as well as during adult life. However, neurotrophins are not alone in regulating neuronal survival, and many groups have demonstrated the effect of different cytokines on this phenomenon. In this brief review we will address the effect of interleukins (IL), particularly IL-2, IL-6, and IL-4, on the survival of neuronal cells.

Protein kinase C regulates the expression of M1 receptors and BDNF in rat retinal cells.

Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation. This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells.

The increase in retinal cells proliferation induced by FGF2 is mediated by tyrosine and PI3 kinases.

Since 1973, multiple effects of basic fibroblast growth factor have been described in a large number of cells. These effects include proliferation, survival and differentiation. The aim of this work was to study the intracellular pathways involved in the basic fibroblast growth factor (FGF2) effect on rat retinal cells proliferation in vitro. Our data show that treatment with FGF2 increases proliferation in a concentration- and time-dependent manner. The effect of 25 ng/ml FGF2 was blocked by 10 microM genistein, a tyrosine kinase inhibitor and by 25 microM LY294002, a PI3 kinase inhibitor. The concomitant treatment with 0.3 microM chelerythrine chloride, a protein kinase C inhibitor, and 6.25 microM LY294002 also inhibited the effect of FGF2. Our results suggest that the proliferative effect of FGF2 on retinal cell cultures involves the activation of distinct kinases.

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.