No abstract

Vascular endothelial growth factor (VEGF-A) is a major pathogenic factor and a therapeutic target for age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. Despite intensive effort in the field, the cellular mechanisms of VEGF action remain virtually uninvestigated. This situation makes it difficult to design cellular target-based therapeutics for these diseases. In light of the recent finding that VEGF is a potential neurotrophic factor, revealing the cellular mechanisms of VEGF action becomes necessary to preserve its beneficial effect and inhibit its pathological function in long-term anti-VEGF therapeutics for ocular vascular diseases. We therefore generated conditional VEGF knockout mice with an inducible Cre/lox system and determined the significance of Müller cell-derived VEGF in retinal development and maintenance and ischaemia-induced neovascularizartion and vascular leakage. Retinal development in the conditional VEGF knockout mice was analysed by examining retinal and choroidal vasculatures and retinal morphology and function. Ischaemia-induced retinal neovascularization and vascular leakage in the conditional VEGF knockout mice were analysed with fluorescein angiography, quantification of proliferative neovascular cells, immunohistochemistry, and immunoblotting using an oxygen-induced retinopathy model. Our results demonstrated that disruption of Müller cell-derived VEGF resulted in no apparent defects in retinal and choroidal vasculatures and retinal morphology and function, significant inhibition of the ischaemia-induced retinal neovascularization and vascular leakage, and attenuation of the ischaemia-induced breakdown of the blood-retina barrier. These results suggest that the retinal Müller cell-derived VEGF is a major contributor to ischaemia-induced retinal vascular leakage and pre-retinal and intra-retinal neovascularization. The observation that a significant, but not complete, reduction of VEGF in the retina does not cause detectable retinal degeneration suggests that appropriate doses of anti-VEGF agents may be important to the safe treatment of retinal vascular diseases.

Background: Autophagy is a conserved process of lysosome-mediated intracellular degradation. Results: Dysregulation of autophagy is associated with retinal cell death by all-trans-retinal and by light exposure. Conclusion: Autophagy protects the retina from light-induced retinal degeneration. Significance: Dynamic autophagy regulation may influence retinal cell survival under stress and disease conditions.

Intraflagellar transport (IFT) 20 is required for the formation of photoreceptor outer segments, and deletion of this gene in mature cells leads to opsin accumulation in the cell body. IFT20 interacts with opsin photopigments both in the context of the IFT particle and independent of the particle, suggesting that opsin receptors are cargo for the IFT system.

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rodspecific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to lightinduced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection. Insulin receptor (IR)2 signaling provides a trophic signal for transformed retinal neurons in culture (1), but the role of the IR in vivo is unknown. IR activation has been shown to rescue retinal neurons from apoptosis through a phosphoinositide 3-kinase (PI3K) cascade (1). We previously reported that light induces tyrosine phosphorylation of the retinal IR and that this activation leads to the binding of PI3K to rod outer segment (ROS) membranes (2). More recently, we demonstrated that IR activation is mediated through the G-protein-coupled receptor rhodopsin (3). IR signaling is also involved in 17␤-estradiolmediated neuroprotection in the retina (4). Recent evidence suggests a down-regulation of IR kinase activity in diabetic retinopathy that is associated with the deregulation of downstream signaling molecules (5). Deletion of several downstream effector molecules of the IR signaling pathway, such as IRS-2 (6), Akt2 (7), and Bcl-xl (8), in the retina resulted in a photoreceptor degeneration phenotype. These studies clearly indicate the importance of the IR signaling pathway in the retina.The IR is highly conserved, and the high degree of IR signaling homology between Caenorhabditis elegans, Drosophila, and humans suggests functional conservation in mammalian retina. The IR regulates neuronal survival in C. elegans (9). In Drosophila, the IR serves an important function to guide retinal photoreceptor axons from the retina to the brain during development (10), and the IR influences the size and number of photoreceptors (11). The lack of IR activation leads to neurod...

Transgenic mice were generated that express Cre recombinase in the RPE in an inducible fashion. These mice will be useful for studies of the RPE-specific role of genes that are expressed in the RPE as well as other cells, particularly for avoiding embryonic lethality and dissecting the function of genes that play dual roles in development and adulthood.

Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking KIF3A, membrane proteins involved in phototransduction did not traffic to the outer segments resulting in complete absence of a photopic electroretinogram and progressive cone degeneration. Rod photoreceptors lacking KIF3A degenerated rapidly between 2 and 4 weeks postnatally, but the phototransduction components including rhodopsin trafficked to the outer segments during the course of degeneration. Furthermore, KIF3A deletion did not affect synaptic anterograde trafficking. The results indicate that trafficking of membrane proteins to the outer segment is dependent on kinesin-II in cone, but not rod photoreceptors, even though rods and cones share similar structures, and closely related phototransduction polypeptides.

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life.DOI: http://dx.doi.org/10.7554/eLife.00324.001