Primary Retinal Cell Cultures as a Model to Study Retina Biology.

Since its inception, primary retinal cultures have been an in vitro tool for modeling the in vivo environment of the retina for biological studies on development and disease. They offer simple and controlled experimental approaches when compared to in vivo models. In this review we highlight the strengths and weaknesses of primary retinal culture models, and the features of dispersed retinal cell cultures.

Retina stem cells, hopes and obstacles.

Retinal degeneration is a major contributor to visual dysfunction worldwide. Although it comprises several eye diseases, loss of retinal pigment epithelial (RPE) and photoreceptor cells are the major contributors to their pathogenesis. Early therapies included diverse treatments, such as provision of anti-vascular endothelial growth factor and many survival and trophic factors that, in some cases, slow down the progression of the degeneration, but do not effectively prevent it. The finding of stem cells (SC) in the eye has led to the proposal of cell replacement strategies for retina degeneration. Therapies using different types of SC, such as retinal progenitor cells (RPCs), embryonic SC, pluripotent SCs (PSCs), induced PSCs (iPSCs), and mesenchymal stromal cells, capable of self-renewal and of differentiating into multiple cell types, have gained ample support. Numerous preclinical studies have assessed transplantation of SC in animal models, with encouraging results. The aim of this work is to revise the different preclinical and clinical approaches, analyzing the SC type used, their efficacy, safety, cell attachment and integration, absence of tumor formation and immunorejection, in order to establish which were the most relevant and successful. In addition, we examine the questions and concerns still open in the field. The data demonstrate the existence of two main approaches, aimed at replacing either RPE cells or photoreceptors. Emerging evidence suggests that RPCs and iPSC are the best candidates, presenting no ethical concerns and a low risk of immunorejection. Clinical trials have already supported the safety and efficacy of SC treatments. Serious concerns are pending, such as the risk of tumor formation, lack of attachment or integration of transplanted cells into host retinas, immunorejection, cell death, and also ethical. However, the amazing progress in the field in the last few years makes it possible to envisage safe and effective treatments to restore vision loss in a near future.

Retinoid X receptor activation promotes photoreceptor survival and modulates the inflammatory response in a mouse model of retinitis pigmentosa.

Photoreceptor cell (PHR) death is a hallmark of most retinal neurodegenerative diseases, in which inflammation plays a critical role. Activation of retinoid X receptors (RXR) modulates and integrates multiple cell functions, and has beneficial effects in animal models of chronic inflammatory diseases. Nonetheless, the mechanisms involved and their role in retina neuroprotection are poorly understood. In this work we assessed whether RXR activation prevents inflammation and/or PHR death in retinitis pigmentosa, an inherited retina neurodegeneration, using as an ex vivo model, retinas from the rd1 mice, a murine model of this disease. We demonstrated that rd1 retinas had lower levels of RXR alpha isoform than their wt counterparts at early developmental times, whereas its distribution pattern remained similar. In mixed neuro-glial cultures obtained from either rd1 or wt retinas, both PHR and Müller glial cells (MGC) expressed RXRalpha, and RXR activation by its synthetic pan-agonist PA024 selectively increased mRNA levels of RXRgamma isoform. PA024 decreased PHR death in rd1 mixed cultures; it reduced the amount of non-viable neurons, delayed the onset of PHR apoptosis, and decreased Bax mRNA levels. PA024 also reduced MGC reactivity in vitro before and at the onset of degeneration, decreasing GFAP expression, increasing glutamine synthetase mRNA levels, and promoting the transcription of the anti-inflammatory cytokine, Il-10. These results suggest that RXR activation rescues rd1 PHR and decreases MGC reactivity, promoting an anti-inflammatory environment in the rd1 retina, thus supporting the potential of RXR agonists as pharmacological tools for treating retina degenerative diseases.

Pigment epithelium-derived factor (PEDF) and derived peptides promote survival and differentiation of photoreceptors and induce neurite-outgrowth in amacrine neurons.

Pigment epithelium-derived factor (PEDF) is a cytoprotective protein for the retina. We hypothesize that this protein acts on neuronal survival and differentiation of photoreceptor cells in culture. The purpose of the present study was to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry through TUNEL assay, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. Our findings support the neurotrophic PEDF peptides as neuronal guardians for the retina, highlighting their potential as promoters of retinal differentiation, and inhibitors of retinal cell death and its blinding consequences. Cover Image for this issue: https://doi.org/10.1111/jnc.15089.

Damaging effects of BMAA on retina neurons and Müller glial cells.

B-N-methylamino-L-alanine (BMAA), a cyanotoxin produced by most cyanobacteria, has been proposed to cause long term damages leading to neurodegenerative diseases, including Amyotrophic Lateral Sclerosis/Parkinsonism Dementia complex (ALS/PDC) and retinal pathologies. Previous work has shown diverse mechanisms leading to BMAA-induced degeneration; however, the underlying mechanisms of toxicity affecting retina cells are not fully elucidated. We here show that BMAA treatment of rat retina neurons in vitro induced nuclear fragmentation and cell death in both photoreceptors (PHRs) and amacrine neurons, provoking mitochondrial membrane depolarization. Pretreatment with the N-Methyl-D-aspartate (NMDA) receptor antagonist MK-801 prevented BMAA-induced death of amacrine neurons, but not that of PHRs, implying activation of NMDA receptors participated only in amacrine cell death. Noteworthy, BMAA stimulated a selective axonal outgrowth in amacrine neurons, simultaneously promoting growth cone destabilization. BMAA partially decreased the viability of Müller glial cells (MGC), the main glial cell type in the retina, induced marked alterations in their actin cytoskeleton and impaired their capacity to protect retinal neurons. BMAA also induced cell death and promoted axonal outgrowth in differentiated rat pheochromocytoma (PC12) cells, implying these effects were not limited to amacrine neurons. These results suggest that BMAA is toxic for retina neurons and MGC and point to the involvement of NMDA receptors in amacrine cell death, providing new insight into the mechanisms involved in BMAA neurotoxic effects in the retina.

Ceramide-1-phosphate promotes the migration of retina Müller glial cells.

Müller glial cells, the major glial cell type in the retina, are activated by most retina injuries, leading to an increased proliferation and migration that contributes to visual dysfunction. The molecular cues involved in these processes are still ill defined. We demonstrated that sphingosine-1-phosphate (S1P), a bioactive sphingolipid, promotes glial migration. We now investigated whether ceramide-1-phosphate (C1P), also a bioactive sphingolipid, was involved in Müller glial cell migration. We evaluated cell migration in primary Müller glial cultures, prepared from newborn rat retinas, by the scratch wound assay. Addition of either 10 µM C8-ceramide-1-phosphate (C8-C1P) or 5 µM C16-C1P (a long chain, natural C1P) stimulated glial migration. Inhibiting PI3K almost completely blocked C8-C1P-elicited migration whereas inhibition of ERK1-2/MAPK pathway diminished it and p38MAPK inhibition did not affect it. Pre-treatment with a cytoplasmic phospholipase A2 (cPLA2) inhibitor markedly reduced C8-C1P-induced migration. Inhibiting ceramide kinase (CerK), the enzyme catalyzing C1P synthesis, partially decreased glial migration. Combined addition of S1P and C8-C1P promoted glial migration to the same extent as when they were added separately, suggesting they converge on their downstream signaling to stimulate Müller glia migration. These results suggest that C1P addition stimulated migration of glial Müller cells, promoting the activation of cPLA2, and the PI3K and ERK/MAPK pathways. They also suggest that CerK-dependent C1P synthesis was one of the factors contributing to glial migration, thus uncovering a novel role for C1P in controlling glial motility.

A Defective Crosstalk Between Neurons and Müller Glial Cells in the rd1 Retina Impairs the Regenerative Potential of Glial Stem Cells.

Müller glial cells (MGC) are stem cells in the retina. Although their regenerative capacity is very low in mammals, the use of MGC as stem cells to regenerate photoreceptors (PHRs) during retina degenerations, such as in retinitis pigmentosa, is being intensely studied. Changes affecting PHRs in diseased retinas have been thoroughly investigated; however, whether MGC are also affected is still unclear. We here investigated whether MGC in retinal degeneration 1 (rd1) mouse, an animal model of retinitis pigmentosa, have impaired stem cell properties or structure. rd1 MGC showed an altered morphology, both in culture and in the whole retina. Using mixed neuron-glial cultures obtained from newborn mice retinas, we determined that proliferation was significantly lower in rd1 than in wild type (wt) MGC. Levels of stem cell markers, such as Nestin and Sox2, were also markedly reduced in rd1 MGC compared to wt MGC in neuron-glial cultures and in retina cryosections, even before the onset of PHR degeneration. We then investigated whether neuron-glial crosstalk was involved in these changes. Noteworthy, Nestin expression was restored in rd1 MGC in co-culture with wt neurons. Conversely, Nestin expression decreased in wt MGC in co-culture with rd1 neurons, as occurred in rd1 MGC in rd1 neuron-glial mixed cultures. These results imply that MGC proliferation and stem cell markers are reduced in rd1 retinas and might be restored by their interaction with "healthy" PHRs, suggesting that alterations in rd1 PHRs lead to a disruption in neuron-glial crosstalk affecting the regenerative potential of MGC.

Ceramide Induces the Death of Retina Photoreceptors Through Activation of Parthanatos.

Ceramide (Cer) has a key role inducing cell death and has been proposed as a messenger in photoreceptor cell death in the retina. Here, we explored the pathways induced by C2-acetylsphingosine (C2-Cer), a cell-permeable Cer, to elicit photoreceptor death. Treating pure retina neuronal cultures with 10 µM C2-Cer for 6 h selectively induced photoreceptor death, decreasing mitochondrial membrane potential and increasing the formation of reactive oxygen species (ROS). In contrast, amacrine neurons preserved their viability. Noteworthy, the amount of TUNEL-labeled cells and photoreceptors expressing cleaved caspase-3 remained constant and pretreatment with a pan-caspase inhibitor did not prevent C2-Cer-induced death. C2-Cer provoked polyADP ribosyl polymerase-1 (PARP-1) overactivation. Inhibiting PARP-1 decreased C2-Cer-induced photoreceptor death; C2-Cer increased polyADP ribose polymer (PAR) levels and induced the translocation of apoptosis inducing factor (AIF) from mitochondria to photoreceptor nuclei, which was prevented by PARP-1 inhibition. Pretreatment with a calpain and cathepsin inhibitor and with a calpain inhibitor reduced photoreceptor death, whereas selective cathepsin inhibitors granted no protection. Combined pretreatment with a PARP-1 and a calpain inhibitor evidenced the same protection as each inhibitor by itself. Neither autophagy nor necroptosis was involved in C2-Cer-elicited death; no increase in LDH release was observed upon C2-Cer treatment and pretreatment with inhibitors of necroptosis and autophagy did not rescue photoreceptors. These results suggest that C2-Cer induced photoreceptor death by a novel, caspase-independent mechanism, involving activation of PARP-1, decline of mitochondrial membrane potential, calpain activation, and AIF translocation, all of which are biochemical features of parthanatos.

Microsaccadic behavior when developing a complex dynamical activity.

Microsaccade are sensitive to changes of perceptual inputs as well as modulations of cognitive states. There are just a few works analyzing microsaccade while subjects are processing complex information and fewer when doing predictions about upcoming events. To evaluate whether contextual predictability would change microsaccadic behavior, we evaluated microsaccade of twenty one persons when reading 40 regular sentences and 40 proverbs. Analysis of microsaccade during reading proverbs and regular sentences revealed that microsaccade rate on words before maxjump, during maxjump and words after maxjump varied depending on the kind of sentence and on the word predictability. Maxjump was defined as the word with the largest difference between the cloze predictability of two consecutive words. Low and high predictable words demanded less or more microsaccade on words previous, during and on maxjump depending of the semantic context and of the readers' predictions of upcoming words.In summary, the present study shows that microsaccade' rate evidenced significant differences when reading proverbs and regular sentences. Hence, evaluation of microsaccade during reading sentences with different contextual predictability might provide information about specific effect of cue attention on complex task.

Patients with Mild Alzheimer's Disease Fail When Using Their Working Memory: Evidence from the Eye Tracking Technique.

Patients with Alzheimer's disease (AD) develop progressive language, visuoperceptual, attentional, and oculomotor changes that can have an impact on their reading comprehension. However, few studies have examined reading behavior in AD, and none have examined the contribution of predictive cueing in reading performance. For this purpose we analyzed the eye movement behavior of 35 healthy readers (Controls) and 35 patients with probable AD during reading of regular and high-predictable sentences. The cloze predictability of words N - 1, and N + 1 exerted an influence on the reader's gaze duration. The predictabilities of preceding words in high-predictable sentences served as task-appropriate cues that were used by Control readers. In contrast, these effects were not present in AD patients. In Controls, changes in predictability significantly affected fixation duration along the sentence; noteworthy, these changes did not affect fixation durations in AD patients. Hence, only in healthy readers did predictability of upcoming words influence fixation durations via memory retrieval. Our results suggest that Controls used stored information of familiar texts for enhancing their reading performance and imply that contextual-word predictability, whose processing is proposed to require memory retrieval, only affected reading behavior in healthy subjects. In AD patients, this loss reveals impairments in brain areas such as those corresponding to working memory and memory retrieval. These findings might be relevant for expanding the options for the early detection and monitoring in the early stages of AD. Furthermore, evaluation of eye movements during reading could provide a new tool for measuring drug impact on patients' behavior.

Protective effects of retinoid x receptors on retina pigment epithelium cells.

Age-related macular degeneration (AMD) is among the main pathologies leading to blindness in adults and has currently no cure or effective treatment. Selective apoptosis of retina pigment epithelial (RPE) cells results in the progressive loss of photoreceptor neurons, with the consequent gradual vision loss. Oxidative stress plays an important role in this process. We have previously determined that activation of RXRs protects rat photoreceptor neurons from oxidative stress-induced apoptosis. In this study we investigated whether RXR ligands prevented apoptosis in an RPE cell line, D407 cells, exposed to hydrogen peroxide (H2O2). H2O2 induced apoptosis of D407 cells, promoting p65NFκB nuclear translocation, increasing Bax mRNA expression, activating caspase-3 and altering cell morphology. We show, for the first time, that HX630, a RXR pan-agonist, protected D407 cells from H2O2-induced apoptosis, preventing p65NFκB nuclear translocation, increasing Bclxl and PPARγ mRNA levels and simultaneously decreasing Bax mRNA levels and caspase-3 activation. Pretreatment with a RXR antagonist blocked HX630 protection. LG100754, which binds RXRs but only activates heterodimers and is an antagonist of RXR homodimers, also had a protective effect. In addition, only agonists known to bind to RXR/PPARγ were protective. As a whole, our results suggest that RXR activation protects RPE cells from oxidative stress-induced apoptosis and this protection might involve signaling through a heterodimeric receptor, such as RXR/PPARγ. These data also imply that RXR agonists might provide potential pharmacological tools for treating retina degenerative diseases.

Synthesis of docosahexaenoic acid from eicosapentaenoic acid in retina neurons protects photoreceptors from oxidative stress.

Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2 O2 ). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in retina photoreceptors, and its precursor, eicosapentaenoic acid (EPA) have multiple beneficial effects. Here, we show that retina neurons in vitro express the desaturase FADS2 and can synthesize DHA from EPA. Moreover, addition of EPA to these cultures protects photoreceptors from oxidative stress and promotes their differentiation through its metabolization to DHA.

Sphingosine-1-Phosphate Is a Crucial Signal for Migration of Retina Müller Glial Cells.

PURPOSE: Migration of Müller glial cells is enhanced in proliferative retinopathies, but the mechanisms involved are ill defined. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid synthesized by sphingosine kinase (SphK), which promotes proliferation, migration, and inflammation, acting as an intracellular mediator and activating a family of membrane receptors (S1PRs). We investigated whether S1P regulated glial migration. METHODS: Müller glial cell cultures from rat retinas were supplemented with 5 µM S1P, and migration was evaluated by scratch-wound assays. Cultures were treated with SphK inhibitor 2 (SphKI 2), a SphK1 inhibitor, or with W146 and BML-241, S1P1 and S1P3 antagonists, respectively, to investigate whether Müller glial cells synthesized S1P and S1P-activated S1PRs to stimulate migration. The effects of LY294002, U0126, and SB203580, which are phosphatidylinositol-3 kinase (PI3K), extracellular signal regulated kinase/mitogen-activated protein kinase (ERK/MAPK), and p38 MAPK inhibitors, respectively, on glial migration were determined. RESULTS: Sphingosine-1-phosphate addition prompted the formation of lamellipodia and enhanced glial migration. SphKI 2 almost completely prevented glial migration in controls; BML-241 inhibited this migration both in controls and in S1P-supplemented cultures, whereas W146 had no significant effect. Pretreatment with LY294002 and U0126 abrogated glial migration; SB203580 decreased it partially, although not significantly. CONCLUSIONS: Our results suggest that Müller glial cells synthesize S1P, which signals through S1P3 and the PI3K and ERK/MAPK pathways to induce glial migration. As a whole, our data point to a central role for S1P in controlling glial cell motility. Because deregulation of this process is involved in several retinal pathologies, S1P signaling emerges as a potential tool for treating these diseases.

Light, lipids and photoreceptor survival: live or let die?

Due to its constant exposure to light and its high oxygen consumption the retina is highly sensitive to oxidative damage, which is a common factor in inducing the death of photoreceptors after light damage or in inherited retinal degenerations. The high content of docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, has been suggested to contribute to this sensitivity. DHA is crucial for developing and preserving normal visual function. However, further roles of DHA in the retina are still controversial. Current data support that it can tilt the scale either towards degeneration or survival of retinal cells. DHA peroxidation products can be deleterious to the retina and might lead to retinal degeneration. However, DHA has also been shown to act as, or to be the source of, a survival molecule that protects photoreceptors and retinal pigment epithelium cells from oxidative damage. We have established that DHA protects photoreceptors from oxidative stress-induced apoptosis and promotes their differentiation in vitro. DHA activates the retinoid X receptor (RXR) and the ERK/MAPK pathway, thus regulating the expression of anti and pro-apoptotic proteins. It also orchestrates a diversity of signaling pathways, modulating enzymatic pathways that control the sphingolipid metabolism and activate antioxidant defense mechanisms to promote photoreceptor survival and development. A deeper comprehension of DHA signaling pathways and context-dependent behavior is required to understand its dual functions in retinal physiology.

Lack of contextual-word predictability during reading in patients with mild Alzheimer disease.

In the present work we analyzed the effect of contextual word predictability on the eye movement behavior of patients with mild Alzheimer disease (AD) compared to age-matched controls, by using the eyetracking technique and lineal mixed models. Twenty AD patients and 40 age-matched controls participated in the study. We first evaluated gaze duration during reading low and highly predictable sentences. AD patients showed an increase in gaze duration, compared to controls, both in sentences of low or high predictability. In controls, highly predictable sentences led to shorter gaze durations; by contrary, AD patients showed similar gaze durations in both types of sentences. Similarly, gaze duration in controls was affected by the cloze predictability of word N and N+1, whereas it was the same in AD patients. In contrast, the effects of word frequency and word length were similar in controls and AD patients. Our results imply that contextual-word predictability, whose processing is proposed to require memory retrieval, facilitated reading behavior in healthy subjects, but this facilitation was lost in early AD patients. This loss might reveal impairments in brain areas such as those corresponding to working memory, memory retrieval, and semantic memory functions that are already present at early stages of AD. In contrast, word frequency and length processing might require less complex mechanisms, which were still retained by AD patients. To the best of our knowledge, this is the first study measuring how patients with early AD process well-defined words embedded in sentences of high and low predictability. Evaluation of the resulting changes in eye movement behavior might provide a useful tool for a more precise early diagnosis of AD.

Eye movement alterations during reading in patients with early Alzheimer disease.

PURPOSE: Eye movements follow a reproducible pattern during normal reading. Each eye movement ends up in a fixation point, which allows the brain to process the incoming information and to program the following saccade. Alzheimer disease (AD) produces eye movement abnormalities and disturbances in reading. In this work, we investigated whether eye movement alterations during reading might be already present at very early stages of the disease. METHODS: Twenty female and male adult patients with the diagnosis of probable AD and 20 age-matched individuals with no evidence of cognitive decline participated in the study. Participants were seated in front of a 20-inch LCD monitor and single sentences were presented on it. Eye movements were recorded with an eye tracker, with a sampling rate of 1000 Hz and an eye position resolution of 20 arc seconds. RESULTS: Analysis of eye movements during reading revealed that patients with early AD decreased the amount of words with only one fixation, increased their total number of first- and second-pass fixations, the amount of saccade regressions and the number of words skipped, compared with healthy individuals (controls). They also reduced the size of outgoing saccades, simultaneously increasing fixation duration. CONCLUSIONS: The present study shows that patients with mild AD evidenced marked alterations in eye movement behavior during reading, even at early stages of the disease. Hence, evaluation of eye movement behavior during reading might provide a useful tool for a more precise early diagnosis of AD and for dynamical monitoring of the pathology.

Retinoid X receptor activation is essential for docosahexaenoic acid protection of retina photoreceptors.

We have established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes survival of rat retina photoreceptors during early development in vitro and upon oxidative stress by activating the ERK/MAPK signaling pathway. Here we have investigated whether DHA turns on this pathway through activation of retinoid X receptors (RXRs) or by inducing tyrosine kinase (Trk) receptor activation. We also evaluated whether DHA release from phospholipids was required for its protective effect. Addition of RXR antagonists (HX531, PA452) to rat retinal neuronal cultures inhibited DHA protection during early development in vitro and upon oxidative stress induced with Paraquat or H2O2. In contrast, the Trk inhibitor K252a did not affect DHA prevention of photoreceptor apoptosis. These results imply that activation of RXRs was required for DHA protection whereas Trk receptors were not involved in this protection. Pretreatment with 4-bromoenol lactone, a phospholipase A2 inhibitor, blocked DHA prevention of oxidative stress-induced apoptosis of photoreceptors. It is noteworthy that RXR agonists (HX630, PA024) also rescued photoreceptors from H2O2-induced apoptosis. These results provide the first evidence that activation of RXRs prevents photoreceptor apoptosis and suggest that DHA is first released from phospholipids and then activates RXRs to promote the survival of photoreceptors.

Retinoic acid promotes apoptosis and differentiation in photoreceptors by activating the P38 MAP kinase pathway.

PURPOSE: Retinoic acid (RA) has a critical role during development of the retina. We investigated RA effects on photoreceptor apoptosis and differentiation, and the intracellular pathways involved. METHODS: Rat retinal neuronal cultures were supplemented with RA with or without docosahexaenoic acid (DHA), a photoreceptor survival factor, and photoreceptor apoptosis and differentiation were evaluated at different times of development. To investigate the intracellular pathways activated by RA, the levels of phosphorylated (P) ERK and P-p38 in cultures with or without RA, and the effect of pretreatment with SB203580, a p38 specific inhibitor, on apoptosis and differentiation were evaluated. RESULTS: RA addition at day 0, when cells still were proliferating, selectively increased apoptosis in photoreceptors, whereas addition at day 2 no longer caused cell death. RA stimulated opsin and peripherin expression, and neurite outgrowth regardless of the time of development. Addition of RA at day 0, but not at day 2, rapidly increased P-p38 levels, but did not affect P-ERK levels. p38 inhibition completely prevented RA-induced apoptosis, and partially decreased differentiation. DHA prevented apoptosis and additively increased differentiation, without affecting RA activation of p38. CONCLUSIONS: Our results show that RA activation of the p38 intracellular pathway was essential for its early induction of apoptosis and partially responsible for promoting differentiation. DHA prevention of this apoptosis suggests that RA effects during early development must be counterbalanced by survival factors to prevent photoreceptor death, in an interplay that might help to establish the final number of photoreceptors.

Leukemia inhibitory factor coordinates the down-regulation of the visual cycle in the retina and retinal-pigmented epithelium.

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.

Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors.

Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.