Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity and neurogenesis. In zebrafish and chick, transition from the quiescence to reactivity is essential for retinal regeneration, while in mice a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I (NFI) transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice following injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110 -3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the ␤-subunit of the insulin receptor (IR␤) in ROS membranes, which leads to the association of PI3K enzyme activity with IR␤. Retinas from light-or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IR␤, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P 2 as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IR␤ and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IR␤ immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR␤ in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P 3 . We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stressinduced apoptosis.Many cell proliferation and cell survival pathways are initiated upon activation of tyrosine kinase receptors, which transduce their signals by recruiting a variety of cytoplasmic signaling proteins (1, 2). Many of the signaling proteins contain phosphotyrosine binding domains, Src homology 2 (SH2) 1 domains, and Src homology 3 (SH3) domains, which are involved in mediating protein-protein interactions (4, 5).The phosphotyrosine-dependent interaction between different phosphotyrosine binding and SH2 domain-containing proteins with activated receptors initiates cellular changes that take place in response to a wide range of extracellular signals (2).One of the SH2 domain-containing proteins, phosphoinositide 3-kinase (PI3K), consists of an ϳ85-kDa regulatory subunit (p85) and a ϳ110-kDa catalytic subunit (p110), the latter being responsible for the phosphorylation of phosphoinositides at the D3 position and of serine residues in proteins (7, 8). The p85 subunit contains an SH3 domain capable of binding to proline-rich sequences, a p110 binding domain, and two SH2 domains. PI3K was initially found to be associated with middle-T/pp60c-Src (9), pp60v-Src, and platelet-derived growth factor receptors in both normal and transformed NIH3T3 fibroblast cells (10, 11). PI3K activity increases in response to platelet-derived growth factor binding to its receptor, in large part because the p85-p110 complex is translocated from the cytosol to the plasma membrane, by the direct binding of the p85 SH2 domain to tyro...

No abstract

Members of the interleukin‐6 cytokine family, including leukemia inhibitory factor (LIF), signal through gp130. The neuroprotective role of gp130 activation has been widely demonstrated in both CNS and PNS, but the mechanism by which this is accomplished is not well established. We investigated temporal and cell‐specific activation of signaling pathways induced by LIF in the mature mouse retina. Intravitreal injection of LIF preserved photoreceptor function and prevented photoreceptor cell death from light‐induced oxidative damage in a dose‐dependent manner (2 days post‐injection). A therapeutic dose of LIF induced rapid and sustained activation of signal transducer and activator of transcription (STAT) 3. Activated STAT3 was localized to all the retinal neurons and glial cells, including photoreceptors. Activation of extracellular signal‐regulated kinase 1 and 2 was robust but transient in Müller glial cells, and undetectable at the time of light exposure. Akt was not activated by LIF. We also show that at the time of neuroprotection, STAT3 but not extracellular signal‐regulated kinase 1 and 2 or the Akt pathways was active in LIF‐treated retinas, and activated STAT3 was clearly localized in transcriptionally active areas of photoreceptor nuclei. Our data suggest that photoreceptor protection in response to LIF can be directly mediated by activation of STAT3 in photoreceptors.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the western world. Recent evidence suggests that RPE and photoreceptors have an interconnected metabolism and that mitochondrial damage in RPE is a trigger for degeneration in both RPE and photoreceptors in AMD. To test this hypothesis, this study was designed to induce mitochondrial damage in RPE in mice to determine whether this is sufficient to cause RPE and photoreceptor damage characteristic of AMD. In this study, we conditionally deleted the gene encoding the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD encoded by Sod2 ) in the retinal pigment epithelium (RPE) of albino BALB/cJ mice. VMD2-Cre;Sod2 flox/flox BALB/cJ mice were housed in either 12-h dark, 12-h 200 lux white lighting (normal light), or 12-h dark, 12-h <10 lux red lighting (dim light). Electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT) were performed to assess retinal function and morphology. Immunofluorescence was used to examine protein expression; quantitative RT-PCR was used to measure gene expression. Sod2 knockout (KO) mice had reduced RPE function with age and increased oxidative stress compared to wild type (WT) controls as expected by the cell-specific deletion of Sod2. This was associated with alterations in RPE morphology and the structure and function of RPE mitochondria. In addition, data show a compensatory increase in RPE glycolytic metabolism. The metabolic shift in RPE correlated with severe disruption of photoreceptor mitochondria including a reduction in TOMM20 expression, mitochondrial fragmentation, and reduced COXIII/β-actin levels. These findings demonstrate that mitochondrial oxidative stress can lead to RPE dysfunction and metabolic reprogramming of RPE. Secondary to these changes, photoreceptors also undergo metabolic stress with increased mitochondrial damage. These data are consistent with the hypothesis of a linked metabolism between RPE and photoreceptors and suggest a mechanism of retinal degeneration in dry AMD.

Retinal degenerative diseases are generally characterized by a permanent loss of light-sensitive retinal neurons known as photoreceptors, or their support cells, the retinal pigmented epithelium (RPE). Metabolic dysfunction has been implicated as a common mechanism of degeneration. In this study, we used the drug metformin in a gain-of-function approach to activate adenosine monophosphate-activated protein kinase (AMPK). We found that treatment protected photoreceptors and the RPE from acute injury and delayed inherited retinal degeneration. Protection was associated with decreased oxidative stress, decreased DNA damage, and increased mitochondrial energy production. To determine whether protection was a local or a systemic effect of metformin, we used AMPK retinal knockout mice and found that local expression of AMPK catalytic subunit α2 was required for metformin-induced protection. Our data demonstrate that increasing the activity of AMPK in retinal neurons or glia can delay or prevent degeneration of photoreceptors and the RPE from multiple types of cell-death triggers.

PurposeAMD is the leading cause of irreversible blindness in older individuals in the Western world, and there are currently no therapies to halt disease progression. Studies suggest that the commonly prescribed antidiabetic drug, metformin, is associated with decreased risk of several ocular diseases, but no work has investigated the effect of metformin use on development of AMD. Thus, we aim to investigate whether metformin use is associated with decreased risk of developing AMD.MethodsIn this retrospective case-control study, we used medical records from patients older than 55 who have visited a University of Florida health clinic. Three controls were matched for every AMD case, defined by International Classification of Diseases, Ninth Revision code, based on the Charlson Comorbidity Index to ensure comparable baseline overall health status. Univariate and conditional multivariable logistic regressions were used to determine the association between a variety of covariates, including metformin use, and AMD diagnosis.ResultsMetformin use was associated with decreased odds of developing AMD, independently of the other covariates investigated, with an odds ratio of 0.58 and a 95% confidence interval of 0.43 to 0.79. Other medications assessed were not associated with decreased odds of developing AMD.ConclusionsPatients who had taken metformin had decreased odds of developing AMD, suggesting that metformin may have a therapeutic role in AMD development or progression in those who are at risk. Further work should include clinical trials to investigate prospectively whether metformin has a protective effect in those at risk for developing AMD.

Aims/hypothesis We sought to determine the impact of longstanding type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells. Methods Wild-type, gp130−/− and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp+ chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood. Results BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b+/CD45hi/CCR2+/Ly6Chi inflammatory monocytes. Diabetic gp130−/− mice were protected from development of diabetes-induced changes in their HSCs. Conclusions/interpretation The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.