In this study we develop and use a gain-of-function mouse allele of the Down syndrome cell adhesion molecule (Dscam) to complement loss-of-function models. We assay the role of Dscam in promoting cell death, spacing, and laminar targeting of neurons in the developing mouse retina. We find that ectopic or overexpression of Dscam is sufficient to drive cell death. Gain-of-function studies indicate that Dscam is not sufficient to increase spatial organization, prevent cell-to-cell pairing, or promote active avoidance in the mouse retina, despite the similarity of the Dscam loss-of-function phenotype in the mouse retina to phenotypes observed in Drosophila Dscam1 mutants. Both gain-and loss-of-function studies support a role for Dscam in targeting neurites; DSCAM is necessary for precise dendrite lamination, and is sufficient to retarget neurites of outer retinal cells after ectopic expression. We further demonstrate that DSCAM guides dendrite targeting in type 2 dopaminergic amacrine cells, by restricting the stratum in which exploring retinal dendrites stabilize, in a Dscam dosage-dependent manner. Based on these results we propose a single model to account for the numerous Dscam gain-and loss-of-function phenotypes reported in the mouse retina whereby DSCAM eliminates inappropriately placed cells and connections.

Oxygen-induced retinopathy (OIR) upregulates Müller cell vascular endothelial growth factor A (VEGFA) that causes intravitreal neovascularization similar to severe retinopathy of prematurity (ROP). Safety concerns exist with anti-VEGF treatment for ROP. We evaluated long-term knockdown of Müller cell-VEGFA with short-hairpin RNAs to VEGFA or VEGF164 via subretinal lentivirus delivery (L-VEGFAshRNA, L-VEGF164shRNA) on retinal structure and function in a rat OIR model. Lectin-stained retinal flat mounts analyzed for areas of avascular/total retina (AVA) and intravitreal neovascular/total retina (IVNV) showed initial significantly reduced IVNV by L-VEGFAshRNA and L-VEGF164shRNA compared to control, luciferase-shRNA lentivirus, without late recurrence. Spectral-domain optical coherence tomography (OCT) and immunohistochemical sections (IHC) demonstrated changes in retinal layer thicknesses in L-VEGFAshRNA or L-VEGF164shRNA  compared to control. Ganzfeld electroretinograms were increased in L-VEGFAshRNA or L-VEGF164shRNA compared to control. Erythropoietin (EPO), brain-derived neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor, neurotrophin-3 (NT-3) mRNAs were increased in L-VEGFAshRNA, but not L-VEGF164shRNA retinas. In cultured rat Müller cells, knockdown of VEGF upregulated NT-3 and EPO, whereas treatment with EPO activated neuroprotective signaling. Methods to reduce IVNV by selective knockdown of VEGFA, and particularly VEGF164, in Müller cells may have fewer deleterious effects than nonselective VEGFA inhibition to all cells in the retina.

Mature mammalian neurons have a limited ability to extend neurites and make new synaptic connections, but the mechanisms that inhibit such plasticity remain poorly understood. Here, we report that OFF-type retinal bipolar cells in mice are an exception to this rule, as they form new anatomical connections within their tiled dendritic fields well after retinal maturity. The Down syndrome cell-adhesion molecule () confines these anatomical rearrangements within the normal tiled fields, as conditional deletion of the gene permits extension of dendrite and axon arbors beyond these borders. deletion in the mature retina results in expanded dendritic fields and increased cone photoreceptor contacts, demonstrating that DSCAM actively inhibits circuit-level plasticity. Electrophysiological recordings from OFF bipolar cells showed enlarged visual receptive fields, demonstrating that expanded dendritic territories comprise functional synapses. Our results identify cell-adhesion molecule-mediated inhibition as a regulator of circuit-level neuronal plasticity in the adult retina.

Inhibition of vascular endothelial growth factor (VEGF) in retinopathy of prematurity (ROP) raises concerns for premature infants because VEGF is essential for retinovascular development as well as neuronal and glial health. This study tested the hypothesis that endothelial cell-specific knockdown of VEGF receptor 2 (VEGFR2), or downstream STAT3, would inhibit VEGF-induced retinopathy without delaying physiologic retinal vascular development. We developed an endothelial cell-specific lentiviral vector that delivered shRNAs to VEGFR2 or STAT3 and a green fluorescent protein reporter under control of the VE-cadherin promoter. The specificity and efficacy of the lentiviral vector-driven shRNAs were validated in vitro and in vivo. In the rat oxygen-induced retinopathy model highly representative of human ROP, the effects of endothelial cell knockdown of VEGFR2 or STAT3 were determined on intravitreal neovascularization (IVNV), physiologic retinal vascular development [assessed as area of peripheral avascular/total retina (AVA)], retinal structure, and retinal function. Targeted knockdown of VEGFR2 or STAT3 specifically in retinal endothelial cells by subretinal injection of lentiviral vectors into postnatal day 8 rat pup eyes efficiently inhibited IVNV, and knockdown of VEGFR2 also reduced AVA and increased retinal thickness without altering retinal function. Taken together, our results support specific knockdown of VEGFR2 in retinal endothelial cells as a novel therapeutic method to treat retinopathy.Electronic supplementary materialThe online version of this article (10.1007/s10456-018-9618-5) contains supplementary material, which is available to authorized users.

Erythropoietin (EPO) is recognized for neuroprotective and angiogenic effects and has been associated with aging and neovascular age-related macular degeneration (AMD). We hypothesized that systemic EPO facilitates the development of choroidal neovascularization (CNV). Wild type mice expressed murine EPOR (mWtEPOR) in RPE/choroids at baseline and had significantly increased serum EPO after laser treatment. To test the role of EPO signaling, we used human EPOR knock-in mice with the mWtEPOR gene replaced by either the human EPOR gene (hWtEPOR) or a mutated human EPOR gene (hMtEPOR) in a laser-induced choroidal neovascularization (LCNV) model. Loss-of-function hWtEPOR mice have reduced downstream activation, whereas gain-of-function hMtEPOR mice have increased EPOR signaling. Compared to littermate controls (mWtEPOR), hMtEPOR with increased EPOR signaling developed larger CNV lesions. At baseline, hMtEPOR mice had increased numbers of macrophages, greater expression of macrophage markers F4/80 and CD206, and following laser injury, had greater expression of cytokines CCL2, CXCL10, CCL22, IL-6, and IL-10 than mWtEPOR controls. These data support a hypothesis that injury from age- and AMD-related changes in the RPE/choroid leads to choroidal neovascularization through EPOR-mediated cytokine production.