Immunohistochemical Characterisation of the Whale Retina

[EN] The eye of the largest adult mammal in the world, the whale, offers a unique opportunity to study the evolution of the visual system and its adaptation to aquatic environments. However, the difficulties in obtaining cetacean samples mean these animals have been poorly studied. Thus, the aim of this study was to characterise the different neurons and glial cells in the whale retina by immunohistochemistry using a range of molecular markers. The whale retinal neurons were analysed using different antibodies, labelling retinal ganglion cells (RGCs), photoreceptors, bipolar and amacrine cells. Finally, glial cells were also labelled, including astrocytes, Muller cells and microglia. Thioflavin S was also used to label oligomers and plaques of misfolded proteins. Molecular markers were used to label the specific structures in the whale retinas, as in terrestrial mammalian retinas. However, unlike the retina of most land mammals, whale cones do not express the cone markers used. It is important to highlight the large size of whale RGCs. All the neurofilament (NF) antibodies used labelled whale RGCs, but not all RGCs were labelled by all the NF antibodies used, as it occurs in the porcine and human retina. It is also noteworthy that intrinsically photosensitive RGCs, labelled with melanopsin, form an extraordinary network in the whale retina. The M1, M2, and M3 subtypes of melanopsin positive-cells were detected. Degenerative neurite beading was observed on RGC axons and dendrites when the retina was analysed 48 h post-mortem. In addition, there was a weak Thioflavin S labelling at the edges of some RGCs in a punctuate pattern that possibly reflects an early sign of neurodegeneration. In conclusion, the whale retina differs from that of terrestrial mammals. Their monochromatic rod vision due to the evolutionary loss of cone photoreceptors and the well-developed melanopsin-positive RGC network could, in part, explain the visual perception of these mammals in the deep sea

Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells

Retinal neurons, particularly retinal ganglion cells (RGCs), are susceptible to the degenerative damage caused by different inherited conditions and environmental insults, leading to irreversible vision loss and, ultimately, blindness. Numerous strategies are being tested in different models of degeneration to restore vision and, in recent years, stem cell technologies have offered novel avenues to obtain donor cells for replacement therapies. To date, stem cell–based transplantation in the retina has been attempted as treatment for photoreceptor degeneration, but the same tools could potentially be applied to other retinal cell types, including RGCs. However, RGC-like cells are not an abundant cell type in stem cell–derived cultures and, often, these cells degenerate over time in vitro. To overcome this limitation, we have taken advantage of the neuroprotective properties of Müller glia (one of the main glial cell types in the retina) and we have examined whether Müller glia and the factors they secrete could promote RGC-like cell survival in organoid cultures. Accordingly, stem cell-derived RGC-like cells were co-cultured with adult Müller cells or Müller cell-conditioned media was added to the cultures. Remarkably, RGC-like cell survival was substantially enhanced in both culture conditions, and we also observed a significant increase in their neurite length. Interestingly, Atoh7, a transcription factor required for RGC development, was up-regulated in stem cell-derived organoids exposed to conditioned media, suggesting that Müller cells may also enhance the survival of retinal progenitors and/or postmitotic precursor cells. In conclusion, Müller cells and the factors they release promote organoid-derived RGC-like cell survival, neuritogenesis, and possibly neuronal maturation.This work was supported by National Institutes of Health Grant R01EY026942 to A.L.T., and by the National Institutes of Health T32 Vision Science Training grant 4T32EY015387 to A.M.M. We also benefit from the National Eye Institute Core Facilities grant P30 EY012576. ELKARTEK KK-2019/00086 to E.V., Research groups of the UPV/EHU (GIU 2018/50) to E.V., Movilidad de personal de investigación UPV/EHU to X.P. and Programa de perfeccionamiento de personal Investigador Doctor, Gobierno Vasco (POS_2019_1_0027) to X.P

Differential Distribution of RBPMS in Pig, Rat, and Human Retina after Damage

RNA binding protein with multiple splicing (RBPMS) is expressed exclusively in retinal ganglion cells (RGCs) in the retina and can label all RGCs in normal retinas of mice, rats, guinea pigs, rabbits, cats, and monkeys, but its function in these cells is not known. As a result of the limited knowledge regarding RBPMS, we analyzed the expression of RBPMS in the retina of different mammalian species (humans, pigs, and rats), in various stages of development (neonatal and adult) and with different levels of injury (control, hypoxia, and organotypic culture or explants). In control conditions, RBPMS was localized in the RGCs somas in the ganglion cell layer, whereas in hypoxic conditions, it was localized in the RGCs dendrites in the inner plexiform layer. Such differential distributions of RBPMS occurred in all analyzed species, and in adult and neonatal retinas. Furthermore, we demonstrate RBPMS localization in the degenerating RGCs axons in the nerve fiber layer of retinal explants. This is the first evidence regarding the possible transport of RBPMS in response to physiological damage in a mammalian retina. Therefore, RBPMS should be further investigated in relation to its role in axonal and dendritic degeneration.This research was funded by ELKARTEK KK-2019/00086, Research groups of the UPV/EHU (GIU 2018/50)and MINECO-Retos (PID2019-111139RB-I00) to E.V. Programa de perfeccionamiento de personal InvestigadorDoctor, Gobierno Vasco to X.P

Characteristics of Whale Muller Glia in Primary and Immortalized Cultures

[EN] Muller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Muller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Muller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Muller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Muller glia to neurons. Here, the Muller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Muller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Muller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Muller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Muller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular markers of Muller cells, with an increase in dedifferentiation markers like alpha-SMA and beta-III tubulin, while they preserve their GS expression depending on the culture passage. Here we also discuss the possible influence of the animal's age and size on these cells, and on their senescence.This study was supported by ELKARTEK (KK-2019/00086), MINECO-Retos (PID2019-111139RB-I00), Grupos UPV/EHU (GIU 2018/150), and Proyectos de Investigación Básica y/o Aplicada (PIBA_2020_1_0026) to EV, Basque Government postdoctoral grant (POS_2020_2_0031) to XP, UPV/EHU- Bordeaux predoctoral grant (PIFBUR20/10) to SB, and UPV/EHU postdoctoral grant (ESPDOC20/058) to NR

Dexamethasone Protects Retinal Ganglion Cells But Not Muller Glia Against Hyperglycemia In Vitro

Diabetic retinopathy (DR) is a common complication of diabetes, for which hyperglycemia is a major etiological factor. It is known that retinal glia (Muller cells) and retinal ganglion cells (RGCs) are affected by diabetes, and there is evidence that DR is associated with neural degeneration. Dexamethasone is a glucocorticoid used to treat many inflammatory and autoimmune conditions, including several eye diseases like DR. Thus, our goal was to study the effect of dexamethasone on the survival of RGCs and Muller glial cells isolated from rat retinas and maintained in vitro under hyperglycemic conditions. The behavior of primary RGC cell cultures, and of mixed RGC and Muller cell co-cultures, was studied in hyperglycemic conditions (30 mM glucose), both in the presence and absence of Dexamethasone (1 mu M). RGC and Muller cell survival was evaluated, and the conditioned media of these cultures was collected to quantify the inflammatory cytokines secreted by these cells using a multiplex assay. The role of IL-1 beta, IL-6 and TNF alpha in RGC death was also evaluated by adding these cytokines to the co-cultures. RGC survival decreased significantly when these cells were grown in high glucose conditions, reaching 54% survival when they were grown alone and only 33% when co-cultured with Muller glia. The analysis of the cytokines in the conditioned media revealed an increase in IL-1 beta, IL-6 and TNF alpha under hyperglycemic conditions, which reverted to the basal concentration in co-cultures maintained in the presence of dexamethasone. Finally, when these cytokines were added to co-cultures they appeared to have a direct effect on RGC survival. Hence, these cytokines could be implicated in the death of RGCs when glucose concentrations increase and dexamethasone might protect RGCs from the cell death induced in these conditions.This work was funded by the support of Retos-MINECO Fondos Feder (RTC-2016-48231) and Grupos Consolidados del Gobierno Vasco (IT437-10) to E.V

RNA Localization and Local Translation in Glia in Neurological and Neurodegenerative Diseases: Lessons from Neurons

Cell polarity is crucial for almost every cell in our body to establish distinct structural and functional domains. Polarized cells have an asymmetrical morphology and therefore their proteins need to be asymmetrically distributed to support their function. Subcellular protein distribution is typically achieved by localization peptides within the protein sequence. However, protein delivery to distinct cellular compartments can rely, not only on the transport of the protein itself but also on the transport of the mRNA that is then translated at target sites. This phenomenon is known as local protein synthesis. Local protein synthesis relies on the transport of mRNAs to subcellular domains and their translation to proteins at target sites by the also localized translation machinery. Neurons and glia specially depend upon the accurate subcellular distribution of their proteome to fulfil their polarized functions. In this sense, local protein synthesis has revealed itself as a crucial mechanism that regulates proper protein homeostasis in subcellular compartments. Thus, deregulation of mRNA transport and/or of localized translation can lead to neurological and neurodegenerative diseases. Local translation has been more extensively studied in neurons than in glia. In this review article, we will summarize the state-of-the art research on local protein synthesis in neuronal function and dysfunction, and we will discuss the possibility that local translation in glia and deregulation thereof contributes to neurological and neurodegenerative diseases.This paper was partially funded by grants awarded to J.B. (MICINN grants SAF2016-76347-R, RYC-2016-19837 and PID2019-110721RB-I00; The Alzheimer’s Association grant AARG-19-618303) and E.A. (MICINN grant PID2019-108465RB-I00; Basque Government grant PIBA-2020-1-0012). M.B.-U. is a UPV/EHU fellow; A.G.-B. is a FPU (FPU17/04891) fellow; M.G. and A.d.l.C. are GV fellows

The Effect of Plasma Rich in Growth Factors on Microglial Migration, Macroglial Gliosis and Proliferation, and Neuronal Survival

Plasma rich in growth factors (PRGF) is a subtype of platelet-rich plasma that has being employed in the clinic due to its capacity to accelerate tissue regeneration. Autologous PRGF has been used in ophthalmology to repair a range of retinal pathologies with some efficiency. In the present study, we have explored the role of PRGF and its effect on microglial motility, as well as its possible pro-inflammatory effects. Organotypic cultures from adult pig retinas were used to test the effect of the PRGF obtained from human as well as pig blood. Microglial migration, as well as gliosis, proliferation and the survival of retinal ganglion cells (RGCs) were analyzed by immunohistochemistry. The cytokines present in these PRGFs were analyzed by multiplex ELISA. In addition, we set out to determine if blocking some of the inflammatory components of PRGF alter its effect on microglial migration. In organotypic cultures, PRGF induces microglial migration to the outer nuclear layers as a sign of inflammation. This phenomenon could be due to the presence of several cytokines in PRGF that were quantified here, such as the major pro-inflammatory cytokines IL-1beta, IL-6 and TNFalpha. Heterologous PRGF (human) and longer periods of cultured (3days) induced more microglia migration than autologous porcine PRGF. Moreover, the migratory effect of microglia was partially mitigated by: 1) heat inactivation of the PRGF; 2) the presence of dexamethasone; or 3) anti-cytokine factors. Furthermore, PRGF seems not to affect gliosis, proliferation or RGC survival in organotypic cultures of adult porcine retinas. PRGF can trigger an inflammatory response as witnessed by the activation of microglial migration in the retina. This can be prevented by using autologous PRGF or if this is not possible due to autoimmune diseases, by mitigating its inflammatory effect. In addition, PRGF does not increase either the proliferation rate of microglial cells or the survival of neurons. We cannot discard the possible positive effect of microglial cells on retinal function. Further studies should be performed to warrant the use of PRGF on the nervous systemWe acknowledge the support of MINECO-Retos Fondos Fender (RTC-2016-48231), Gobierno Vasco (PUE_2018_1_0004), ELKARTEK (KK-2019/00086), MINECO-Retos (PID2019-111139RB-I00) and PIBA (2020-1-0026) to E

Plasma Rich in Growth Factors (PRGF) Increases the Number of Retinal Muller Glia in Culture but Not the Survival of Retinal Neurons

Plasma rich in growth factors (PRGF) is a subtype of platelet-rich plasma (PRP) that stimulates tissue regeneration and may promote neuronal survival. It has been employed in ophthalmology to achieve tissue repair in some retinal pathologies, although how PRGF acts in the retina is still poorly understood. As a part of the central nervous system, the retina has limited capacity for repair capacity following damage, and retinal insult can provoke the death of retinal ganglion cells (RGCs), potentially producing irreversible blindness. RGCs are in close contact with glial cells, such as Muller cells, that help maintain homeostasis in the retina. In this study, the aim was to determine whether PRGF can protect RGCs and whether it increases the number of Muller cells. Therefore, PRGF were tested on primary cell cultures of porcine RGCs and Muller cells, as well as on co-cultures of these two cell types. Moreover, the inflammatory component of PRGF was analyzed and the cytokines in the different PRGFs were quantified. In addition, we set out to determine if blocking the inflammatory components of PRGF alters its effect on the cells in culture. The presence of PRGF compromises RGC survival in pure cultures and in co-culture with Muller cells, but this effect was reversed by heat-inactivation of the PRGF. The detrimental effect of PRGF on RGCs could be in part due to the presence of cytokines and specifically, to the presence of pro-inflammatory cytokines that compromise their survival. However, other factors are likely to be present in the PRGF that have a deleterious effect on the RGCs since the exposure to antibodies against these cytokines were insufficient to protect RGCs. Moreover, PRGF promotes Muller cell survival. In conclusion, PRGF hinders the survival of RGCs in the presence or absence of Muller cells, yet it promotes Muller cell survival that could be the reason of retina healing observed in the in vivo treatments, with some cytokines possibly implicated. Although PRGF could stimulate tissue regeneration, further studies should be performed to evaluate the effect of PRGF on neurons and the implication of its potential inflammatory role in such processesWe acknowledge the support of MINECO-Retos Fondos Fender (RTC-2016-48231), Gobierno Vasco (PUE_2018_1_0004), ELKARTEK (KK-2019/00086), PIBA 2020-1-0026 and MINECO-Retos (PID2019-111139RB-I00) to E

Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays

Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using anti-MMP-9 antibody microarrays (AbMAs). After validation with eight healthy control tear samples characterized by ELISA, 20 samples were tested from individuals diagnosed with ocular inflammation due to: cataracts, glaucoma, meibomian gland dysfunction, allergy, or dry eye. Concentration values of tear MMP-9 were obtained for each sample, and 12 patients surpassed the pathological threshold (30 ng/mL). A significant elevation of MMP-9 concentration in the tears of glaucoma patients compared with healthy controls was observed. In order to evaluate the diagnostic ability, an ROC curve analysis was performed using our data, determining the optimal threshold for the test at 33.6 ng/mL of tear MMP-9. In addition, a confusion matrix was applied, estimating sensitivity at 60%, specificity at 88%, and accuracy at 68%. In conclusion, we demonstrated that the AbMAs system allows the quantification of MMP-9 in pathologies that involve inflammation of the ocular surface.This research was funded by Basque Government, BIKAINTEK, grant number 48-AF-W2-2019-00006; by the University of the Basque Country, PIFIND19/02, grant number 201900016247; by ELKARTEK, grant number (KK-2019/00086), by MINECO-Retos, grant number (PID2019-111139RBI00) to E.V.; and by FISS, grant number FISS-21-RD21/0002/0041, to I.R.-A. and A.A

A Pilot Study of a Panel of Ocular Inflammation Biomarkers in Patients with Primary Sjögren’s Syndrome

Ocular diseases have a strong impact on individuals, the effects of which extend from milder visual impairment to blindness. Due to this and to their prevalence, these conditions constitute important health, social and economic challenges. Thus, improvements in their early detection and diagnosis will help dampen the impact of these conditions, both on patients and on healthcare systems alike. In this sense, identifying tear biomarkers could establish better non-invasive approaches to diagnose these diseases and to monitor responses to therapy. With this in mind, we developed a solid phase capture assay, based on antibody microarrays, to quantify S100A6, MMP-9 and CST4 in human tear samples, and we used these arrays to study tear samples from healthy controls and patients with Sjögren’s Syndrome, at times concomitant with rheumatoid arthritis. Our results point out that the detection of S100A6 in tear samples seems to be positively correlated to rheumatoid arthritis, consistent with the systemic nature of this autoinflammatory pathology. Thus, we provide evidence that antibody microarrays may potentially help diagnose certain pathologies, possibly paving the way for significant improvements in the future care of these patients.This research was funded by the Basque Government (BIKAINTEK, grant number 48-AF-W2-2019-00006), by the University of the Basque Country (PIFIND19/02, grant number 201900016247), and by ELKARTEK (KK-2019/00086) and MINECO-Retos (PID2019-111139RB-I00) grants to E.V., as well as by FISS-21-RD21/0002/0041 to A.A