Aline Araujo dos Santos

Retinal ganglion cell (RGC) death profoundly impacts vision because RGC axons form the optic nerve, which transmits information to central visual areas. The α7 nicotinic acetylcholine receptor (α7nAChR) participates in the cholinergic anti‐inflammatory pathway and plays a neuroprotective role in the central nervous system. Previously, we showed that protein kinase C activation by phorbol 12‐myristate 13‐acetate (PMA) treatment for 48 h increases the survival of neonatal rat RGCs by modulating muscarinic receptor levels. Herein, we aimed to investigate the effects of the selective α7nAChR agonist PNU‐282987 in rat retinal cell cultures and analyse whether the activation of this receptor is involved in PMA‐mediated RGC survival. Our results showed that α7nAChR inhibition using methyllycaconitine (MLA) abolished the effects of selected cholinergic agonists on RGC survival. We also observed that PNU‐282987 regulates TNF‐α and IL‐1β levels and release. Moreover, PNU‐282987 promoted RGC survival, and its neuroprotection was partially mediated by the induction of TNF‐α and IL‐1β during the initial stages of culture. MLA blocked the effect of PMA (50 ng/mL) on RGC, whereas PMA slightly increased the α7 subunit levels at 48 h. Further, PMA treatment decreased intracellular TNF‐α and p‐NF‐κB p50 levels through α7nAChR activation. In conclusion, we provide evidence that α7nAChR activation leads to the modulation of pro‐inflammatory cytokines in rat retinal cell cultures, thereby increasing RGC survival. Furthermore, activated α7nAChR enhances PKC activation and increases RGC survival after axotomy, corroborating the role of this receptor in neuroprotection.

Background: Ouabain is a steroid hormone that binds to the sodium pump (Na⁺, K⁺-ATPase) at physiological (nanomolar) concentrations, activating different signaling pathways. This interaction has been shown to prevent the axotomy-induced death of retinal ganglion cells (RGCs), although the underlying mechanisms remain unclear. Objective: In this study, we investigated potential mechanisms by which ouabain promotes RGC survival using primary cultures of rat neural retina. Results: Our findings indicate that ouabain regulates brain-derived neurotrophic factor (BDNF) signaling in retinal cells via matrix metalloproteinase-9-mediated processing of proBDNF to mature BDNF (mBDNF) and by increasing the phosphorylation of the mBDNF receptor, tropomyosin-related receptor kinase B. Ouabain also enhances the maturation of interleukin (IL)-1β through the increased activation of caspase-1, which mediates the processing of proIL-1β into IL-1β, and transiently upregulates both IL-1 receptor and IL-1 receptor antagonist (IL-1Ra). Treatment using either IL-1β or IL-1Ra alone is sufficient to enhance RGC survival similarly to that achieved with ouabain. Finally, we further show that ouabain prevents RGC death through a complex signaling mechanism shared by BDNF and IL-1β, which includes the activation of the Src and protein kinase C pathways. Conclusions: Collectively, these results suggest that ouabain stimulates the maturation and signaling of both BDNF and IL-1β, which act as key mediators of RGC survival.

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.

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. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres or as organoids are valuable to understand both neuronal and glial compartments which have contributed to reveal roles and mechanisms of 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 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.

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 Aβ‐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.

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.