Platelet-activating factor receptor (PAFR) regulates neuronal maturation and synaptic transmission during postnatal retinal development.

Introduction: Platelet-activating factor (PAF), PAF receptor (PAFR), and PAF- synthesis/degradation systems are involved in essential CNS processes such as neuroblast proliferation, differentiation, migration, and synaptic modulation. The retina is an important central nervous system (CNS) tissue for visual information processing. During retinal development, the balance between Retinal Progenitor Cell (RPC) proliferation and differentiation is crucial for proper cell determination and retinogenesis. Despite its importance in retinal development, the effects of PAFR deletion on RPC dynamics are still unknown. Methods: We compared PAFR knockout mice (PAFR-/-) retinal postnatal development proliferation and differentiation aspects with control animals. Electrophysiological responses were analyzed by electroretinography (ERG). Results and discussion: In this study, we demonstrate that PAFR-/- mice increased proliferation during postnatal retinogenesis and altered the expression of specific differentiation markers. The retinas of postnatal PAFR-/- animals decreased neuronal differentiation and synaptic transmission markers, leading to differential responses to light stimuli measured by ERG. Our findings suggest that PAFR signaling plays a critical role in regulating postnatal RPC cell differentiation dynamics during retinal development, cell organization, and neuronal circuitry formation.

Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells.

The retina is a central nervous tissue essential to visual perception and highly susceptible to environmental damage. Lower vertebrate retinas activate intrinsic regeneration mechanisms in response to retinal injury regulated by a specialized population of progenitor cells. The mammalian retina does not have populations of progenitor/stem cells available to activate regeneration, but contains a subpopulation of differentiated cells that can be reprogrammed into retinal stem cells, the ciliary epithelium (CE) cells. Despite the regenerative potential, stem cells derived from CE exhibit limited reprogramming capacity probably associated with the expression of intrinsic regulatory mechanisms. Platelet-activating factor (PAF) is a lipid mediator widely expressed in many cells and plays an important role in stem cell proliferation and differentiation. During mammalian development, PAF receptor signaling showed important effects on retinal progenitors' cell cycle regulation and neuronal differentiation that need to be further investigated. In this study, our findings suggested a dynamic role for PAF receptor signaling in CE cells, impacting stem cell characteristics and neurosphere formation. We showed that PAF receptors and PAF-related enzymes are downregulated in retinal progenitor/stem cells derived from PE cells. Blocking PAFR activity using antagonists increased the expression of specific progenitor markers, revealing potential implications for retinal tissue development and maintenance.

New Perspectives in Stem Cell Transplantation and Associated Therapies to Treat Retinal Diseases: From Gene Editing to 3D Bioprinting.

Inherited and non-inherited retinopathies can affect distinct cell types, leading to progressive cell death and visual loss. In the last years, new approaches have indicated exciting opportunities to treat retinopathies. Cell therapy in retinitis pigmentosa, age-related macular disease, and glaucoma have yielded encouraging results in rodents and humans. The first two diseases mainly impact the photoreceptors and the retinal pigmented epithelium, while glaucoma primarily affects the ganglion cell layer. Induced pluripotent stem cells and multipotent stem cells can be differentiated in vitro to obtain specific cell types for use in transplant as well as to assess the impact of candidate molecules aimed at treating retinal degeneration. Moreover, stem cell therapy is presented in combination with newly developed methods, such as gene editing, Müller cells dedifferentiation, sheet & drug delivery, virus-like particles, optogenetics, and 3D bioprinting. This review describes the recent advances in this field, by presenting an updated panel based on cell transplants and related therapies to treat retinopathies.

Let-7, Lin28 and Hmga2 Expression in Ciliary Epithelium and Retinal Progenitor Cells.

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues. Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2. Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2. Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Age-related increase of let-7 family microRNA in rat retina and vitreous.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.

Platelet activating factor in the eye: Physiological roles, diseases and future perspectives.

Platelet Activating Factor (PAF) is a known phospholipid mediator of inflammation. Since its first description in 1972, it has emerged as a key regulator of vital cellular signaling functions, as proliferation, cell adhesion, and apoptosis. Evidence suggests that interactions between PAF and its receptor (PAFR) play a critical role in nervous system tissues, including the retina. The retina is a very important constituent of the visual system, along with the cornea, sclera, choroid, iris, and ciliary body, that acts synergistically to provide vision and to maintain optical homeostasis. There is evidence that PAF may regulate a wide range of physiological functions in the visual system tissues, such as eye development, inflammation, epithelial wound healing, and synapsis. Due to their multiple functions, PAF and PAFR also have important pathological and clinical implications in ocular disorders such as Choroidal Neovascularization (CNV), Age Macular Degeneration, (AMD), Diabetic Retinopathy (DR), transplant responses, and pharmacological interactions. Studies with PAFR antagonists have shown promising results such as inhibition of neovascularization and chloroquine-induced retinopathies, as well as reducing inflammation and retinal cell death. Due to the importance of PAFR signaling in the visual system and ophthalmology research, this review aims to provide a general overview of current and future perspectives about PAF in eye biology.

Small Molecule GSK-J1 Affects Differentiation of Specific Neuronal Subtypes in Developing Rat Retina.

Histone post-translational modification has been shown to play a pivotal role in regulating gene expression and fate determination during the development of the central nervous system. Application of pharmacological blockers that control histone methylation status has been considered a promising avenue to control abnormal developmental processes and diseases as well. In this study, we focused on the role of potent histone demethylase inhibitor GSK-J1 as a blocker of Jumonji domain-containing protein 3 (Jmjd3) in early postnatal retinal development. Jmjd3 participates in different processes such as cell proliferation, apoptosis, differentiation, senescence, and cell reprogramming via demethylation of histone 3 lysine 27 trimethylation status (H3K27 me3). As a first approach, we determined the localization of Jmjd3 in neonate and adult rat retina. We observed that Jmjd3 accumulation is higher in the adult retina, which is consistent with the localization in the differentiated neurons, including ganglion cells in the retina of neonate rats. At this developmental age, we also observed the presence of Jmjd3 in undifferentiated cells. Also, we confirmed that GSK-J1 caused the increase in the H3k27 me3 levels in the retinas of neonate rats. We next examined the functional consequences of GSK-J1 treatment on retinal development. Interestingly, injection of GSK-J1 simultaneously increased the number of proliferative and apoptotic cells. Furthermore, an increased number of immature cells were detected in the outer plexiform layer, with longer neuronal processes. Finally, the influence of GSK-J1 on postnatal retinal cytogenesis was examined. Interestingly, GSK-J1 specifically caused a significant decrease in the number of PKCα-positive cells, which is a reliable marker of rod-on bipolar cells, showing no significant effects on the differentiation of other retinal subtypes. To our knowledge, these data provide the first evidence that in vivo pharmacological blocking of histone demethylase by GSK-J1 affects differentiation of specific neuronal subtypes. In summary, our results indisputably revealed that the application of GSK-J1 could influence cell proliferation, maturation, apoptosis induction, and specific cell determination. With this, we were able to provide evidence that this small molecule can be explored in therapeutic strategies for the abnormal development and diseases of the central nervous system.

Notch Signaling Activates Stem Cell Properties of Müller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27Kip1.

Müller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27Kip1, which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27Kip1 expression. This is likely achieved through the Notch- p27Kip1 and Notch-Skp2-p27Kip1 axes, the former inhibiting the expression of p27Kip1 transcripts and the latter levels of p27Kip1 proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27Kip1 expression both transcriptionally and post-translationally.

Rho GTPases control ciliary epithelium cells proliferation and progenitor profile induction in vivo.

PURPOSE: Rho GTPases play a central role in actin-based cytoskeleton reorganization and regulate multiple signaling pathways that control gene transcription, cell survival, and proliferation. We investigated the effect of Rho GTPases on cell cycle regulation and progenitor genes expression on mouse ciliary epithelium (CE), a potential source of progenitor/stem cells in the adult retina. METHODS: Rho GTPases were activated by intraocular injection of lysophosphatidic acid and inactivated by Clostridium difficile Toxin A (general Rho GTPase inhibitor), NSC23766 (Rac1 activation inhibitor), or Y27632 (Rho-associated protein kinase [ROCK] inhibitor). Thereafter, we assayed for RhoA, RhoB, and Rac1 protein localization in CE cells. Proliferation was examined by the expression levels of cell cycle regulators p27(kip), p16(INK4a), and Ki67 and the effects on progenitors by determining the changes in Pax6 and Chx10 progenitor markers expression. RESULTS: All GTPases investigated were expressed in mouse CE cells. Activation increased the coexpression of Pax6 and Chx10, but had no significant effect on the proliferation of CE cells. In contrast, Rho GTPases inactivation increased cell proliferation and potentiated the proliferative effect of growth factors. Specific inactivation of Rac1 or ROCK increased the levels of Ki67 and decreased the expression of the cell cycle inhibitors p27(kip) and p16(INK4a). CONCLUSIONS: This study reports that Rho GTPase modulation (activation and inactivation) controls the expression of retinal progenitor genes and proliferation, respectively, in the adult ciliary epithelial progenitor/stem cells of rodent eyes. The modulation of these two different mechanisms (proliferation and reprogramming) may provide a potential new approach in retinal repair.

Adult ciliary epithelial stem cells generate functional neurons and differentiate into both early and late born retinal neurons under non-cell autonomous influences.

BACKGROUND: The neural stem cells discovered in the adult ciliary epithelium (CE) in higher vertebrates have emerged as an accessible source of retinal progenitors; these cells can self-renew and possess retinal potential. However, recent studies have cast doubt as to whether these cells could generate functional neurons and differentiate along the retinal lineage. Here, we have systematically examined the pan neural and retinal potential of CE stem cells. RESULTS: Molecular and cellular analysis was carried out to examine the plasticity of CE stem cells, obtained from mice expressing green fluorescent protein (GFP) under the influence of the promoter of the rod photoreceptor-specific gene, Nrl, using the neurospheres assay. Differentiation was induced by specific culture conditions and evaluated by both transcripts and protein levels of lineage-specific regulators and markers. Temporal pattern of their levels were examined to determine the expression of genes and proteins underlying the regulatory hierarchy of cells specific differentiation in vitro. Functional attributes of differentiation were examined by the presence of current profiles and pharmacological mobilization of intracellular calcium using whole cell recordings and Fura-based calcium imaging, respectively. We demonstrate that stem cells in adult CE not only have the capacity to generate functional neurons, acquiring the expression of sodium and potassium channels, but also respond to specific cues in culture and preferentially differentiate along the lineages of retinal ganglion cells (RGCs) and rod photoreceptors, the early and late born retinal neurons, respectively. The retinal differentiation of CE stem cells was characterized by the temporal acquisition of the expression of the regulators of RGCs and rod photoreceptors, followed by the display of cell type-specific mature markers and mobilization of intracellular calcium. CONCLUSIONS: Our study demonstrates the bonafide retinal potential of adult CE stem cells and suggests that their plasticity could be harnessed for clinical purposes once barriers associated with any lineage conversion, i.e., low efficiency and fidelity is overcome through the identification of conducive culture conditions.

Notch and Wnt signaling mediated rod photoreceptor regeneration by Müller cells in adult mammalian retina.

BACKGROUND: Evidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception. CONCLUSION/SIGNIFICANCE: In summary, our results provide a proof of principle for non-neurotoxin-mediated activation of Müller cells through Notch and Wnt signaling toward the regeneration of rod photoreceptors.

Jararhagin, a snake venom metalloprotease-disintegrin, activates the Rac1 GTPase and stimulates neurite outgrowth in neuroblastoma cells.

It has been shown previously that the snake venom metalloprotease-disintegrin jararhagin stimulates cell migration and cytoskeletal rearrangement, independently of its effects on cellular adhesion but possibly associated with the activation of small GTP-binding proteins from the Rho family [Costa, E.P., Santos, M.F., 2004. Toxicon 44(8), 861-870.] Here we show that jararhagin stimulates spreading, actin dynamics and neurite outgrowth in neuroblastoma cells, and that this effect is accompanied by the translocation of the Rac1 small GTPase to the membrane fraction, suggesting its activation. Stimulation of neurite outgrowth was observed within minutes and was dependent on the proteolytic activity of the toxin. These results suggest that jararhagin may stimulate neuronal differentiation, being a potential tool for neuronal regeneration studies.

[Monoclonal antibody therapy: a literature review]. / Terapia com anticorpos monoclonais: uma revisão de literatura.

Monoclonal antibodies (MA) are an important group of drugs used in oncology. The objective of this study was to identify MAs used in oncology, and to describe their pharmacological characteristics. This literature review was based on database, index, and library collections. Eight MA were identified, out of which, 37.4% are immunoconjugated, 62.5% are recommended for hematological neoplasms, 75.0% are diluted in saline solution and should not be mixed with other drugs, 100.0% cause digestive side effects, and 87.5% affect the hematopoietic system. In order to guarantee MA's therapeutic efficacy and safety, professionals must fully understand their pharmacological characteristics.