The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation.

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

Hypoxia Inhibits Subretinal Inflammation Resolution Thrombospondin-1 Dependently.

Hypoxia is potentially one of the essential triggers in the pathogenesis of wet age-related macular degeneration (wetAMD), characterized by choroidal neovascularization (CNV) which is driven by the accumulation of subretinal mononuclear phagocytes (MP) that include monocyte-derived cells. Here we show that systemic hypoxia (10% O2) increased subretinal MP infiltration and inhibited inflammation resolution after laser-induced subretinal injury in vivo. Accordingly, hypoxic (2% O2) human monocytes (Mo) resisted elimination by RPE cells in co-culture. In Mos from hypoxic mice, Thrombospondin 1 mRNA (Thbs1) was most downregulated compared to normoxic animals and hypoxia repressed Thbs-1 expression in human monocytes in vitro. Hypoxic ambient air inhibited MP clearance during the resolution phase of laser-injury in wildtype animals, but had no effect on the exaggerated subretinal MP infiltration observed in normoxic Thbs1-/--mice. Recombinant Thrombospondin 1 protein (TSP-1) completely reversed the pathogenic effect of hypoxia in Thbs1-/--mice, and accelerated inflammation resolution and inhibited CNV in wildtype mice. Together, our results demonstrate that systemic hypoxia disturbs TSP-1-dependent subretinal immune suppression and promotes pathogenic subretinal inflammation and can be therapeutically countered by local recombinant TSP-1.

Neuroglobin effectively halts vision loss in Harlequin mice at an advanced stage of optic nerve degeneration.

Mitochondrial diseases are among the most prevalent groups of inherited neurological disorders, affecting up to 1 in 5000 adults. Despite the progress achieved on the identification of gene mutations causing mitochondrial pathologies, they cannot be cured so far. Harlequin mice, a relevant model of mitochondrial pathology due to apoptosis inducing factor depletion, suffer from progressive disappearance of retinal ganglion cells leading to optic neuropathy. In our previous work, we showed that administering adeno-associated virus encompassing the coding sequences for neuroglobin, (a neuroprotective molecule belonging to the globin family) or apoptosis-inducing factor, before neurodegeneration onset, prevented retinal ganglion cell loss and preserved visual function. One of the challenges to develop an effective treatment for optic neuropathies is to consider that by the time patients become aware of their handicap, a large amount of nerve fibers has already disappeared. Gene therapy was performed in Harlequin mice aged between 4 and 5 months with either a neuroglobin or an apoptosis-inducing factor vector to determine whether the increased abundance of either one of these proteins in retinas could preserve visual function at this advanced stage of the disease. We demonstrated that gene therapy, by preserving the connectivity of transduced retinal ganglion cells and optic nerve bioenergetics, results in the enhancement of visual cortex activity, ultimately rescuing visual impairment. This study demonstrates that: (a) An increased abundance of neuroglobin functionally overcomes apoptosis-inducing factor absence in Harlequin mouse retinas at a late stage of neuronal degeneration; (b) The beneficial effect for visual function could be mediated by neuroglobin localization to the mitochondria, thus contributing to the maintenance of the organelle homeostasis.

Mutated CCDC51 Coding for a Mitochondrial Protein, MITOK Is a Candidate Gene Defect for Autosomal Recessive Rod-Cone Dystrophy.

The purpose of this work was to identify the gene defect underlying a relatively mild rod-cone dystrophy (RCD), lacking disease-causing variants in known genes implicated in inherited retinal disorders (IRD), and provide transcriptomic and immunolocalization data to highlight the best candidate. The DNA of the female patient originating from a consanguineous family revealed no large duplication or deletion, but several large homozygous regions. In one of these, a homozygous frameshift variant, c.244_246delins17 p.(Trp82Valfs*4); predicted to lead to a nonfunctional protein, was identified in CCDC51. CCDC51 encodes the mitochondrial coiled-coil domain containing 51 protein, also called MITOK. MITOK ablation causes mitochondrial dysfunction. Here we show for the first time that CCDC51/MITOK localizes in the retina and more specifically in the inner segments of the photoreceptors, well known to contain mitochondria. Mitochondrial proteins have previously been implicated in IRD, although usually in association with syndromic disease, unlike our present case. Together, our findings add another ultra-rare mutation implicated in non-syndromic IRD, whose pathogenic mechanism in the retina needs to be further elucidated.

Insulin inhibits inflammation-induced cone death in retinal detachment.

BACKGROUND: Rhegmatogenous retinal detachment (RD) involving the macula is a major cause of visual impairment despite high surgical success rate, mainly because of cone death. RD causes the infiltration of activated immune cells, but it is not clear whether and how infiltrating inflammatory cells contribute to cone cell loss. METHODS: Vitreous samples from patients with RD and from control patients with macular hole were analyzed to characterize the inflammatory response to RD. A mouse model of RD and retinal explants culture were then used to explore the mechanisms leading to cone death. RESULTS: Analysis of vitreous samples confirms that RD induces a marked inflammatory response with increased cytokine and chemokine expression in humans, which is closely mimicked by experimental murine RD. In this model, we corroborate that myeloid cells and T-lymphocytes contribute to cone loss, as the inhibition of their accumulation by Thrombospondin 1 (TSP1) increased cone survival. Using monocyte/retinal co-cultures and TSP1 treatment in RD, we demonstrate that immune cell infiltration downregulates rod-derived cone viability factor (RdCVF), which physiologically regulates glucose uptake in cones. Insulin and the insulin sensitizers rosiglitazone and metformin prevent in part the RD-induced cone loss in vivo, despite the persistence of inflammation CONCLUSION: Our results describe a new mechanism by which inflammation induces cone death in RD, likely through cone starvation due to the downregulation of RdCVF that could be reversed by insulin. Therapeutic inhibition of inflammation and stimulation of glucose availability in cones by insulin signaling might prevent RD-associated cone death until the RD can be surgically repaired and improve visual outcome after RD. TRIAL REGISTRATION: ClinicalTrials.gov NCT03318588.

IL-1ß induces rod degeneration through the disruption of retinal glutamate homeostasis.

BACKGROUND: Age-related macular degeneration is characterized by the accumulation of subretinal macrophages and the degeneration of cones, but mainly of rods. We have previously shown that Mononuclear Phagocytes-derived IL-1ß induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1ß but the mechanism is unknown. METHODS: Retinal explants were culture in the presence of human monocytes or IL-1ß and photoreceptor cell survival was analyzed by TUNEL labeling. Glutamate concentration and transcription levels of gene involved in the homeostasis of glutamate were analyzed in cell fractions of explant cultured or not in the presence of IL-1ß. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-ß or monocytes. RESULTS: We here show that IL-1ß does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1ß leads to glutamate-induced rod photoreceptor cell death as it increases the extracellular glutamate concentrations in the retina through the inhibition of its conversion to glutamine in Müller cells, increased release from Müller cells, and diminished reuptake. The inhibition of non-NMDA receptors completely and efficiently prevented rod apoptosis in retinal explants cultured in the presence of IL-1ß or, more importantly, in vivo, in a model of subretinal inflammation. CONCLUSIONS: Our study emphasizes the importance of inflammation in the deregulation of glutamate homeostasis and provides a comprehensive mechanism of action for IL-1ß-induced rod degeneration.

Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation.

BACKGROUND: The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. METHODS: Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. RESULTS: Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and transepithelial resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and transepithelial-resistance changes were prevented by concomitant Transforming Growth Factor ß inhibition. CONCLUSIONS: Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.

Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

Angiogenesis is a cause of visual impairment and blindness in the wet form of age-related macular degeneration and in ischemic retinopathies. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization (CNV) and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF and possible adverse effects of long-term VEGF inhibition in the retina and choroid highlight a need for additional alternative therapies. Integrins αvß3 and αvß5, which regulate endothelial cell proliferation and stabilization, have been implicated in ocular angiogenesis. Lebecetin (LCT) is a 30-kDa heterodimeric C-type lectin that is isolated from Macrovipera lebetina venom and interacts with α5ß1- and αv-containing integrins. We previously showed that LCT inhibits human brain microvascular endothelial cell adhesion, migration, proliferation, and tubulogenesis. To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on CNV in the mouse CNV model and on retinal neovascularization in the oxygen-induced retinopathy model. Our data demonstrate that a single injection of LCT efficiently reduced CNV and retinal neovascularization in these models.-Montassar, F., Darche, M., Blaizot, A., Augustin, S., Conart, J.-B., Millet, A., Elayeb, M., Sahel, J.-A., Réaux-Le Goazigo, A., Sennlaub, F., Marrakchi, N., Messadi, E., Guillonneau, X. Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

Impaired vitreous composition and retinal pigment epithelium function in the FoxG1::LRP2 myopic mice.

High myopia (HM) is one of the main causes of visual impairment and blindness all over the world and an unsolved medical problem. Persons with HM are predisposed to other eye pathologies such as retinal detachment, myopic retinopathy or glaucomatous optic neuropathy, complications that may at least partly result from the extensive liquefaction of the myopic vitreous gel. To identify the involvement of the liquid vitreous in the pathogenesis of HM we here analyzed the vitreous of the recently described highly myopic low density lipoprotein receptor-related protein 2 (Lrp2)-deficient eyes. Whereas the gel-like fraction was not apparently modified, the volume of the liquid vitreous fraction (LVF) was much higher in the myopic eyes. Biochemical and proteome analysis of the LVF revealed several modifications including a marked decrease of potassium, sodium and chloride, of proteins involved in ocular tissue homeostasis and repair as well as of ADP-ribosylation factor 4 (ARF4), a protein possibly involved in LRP2 trafficking. A small number of proteins, mainly comprising known LRP2 ligands or proteins of the inflammatory response, were over expressed in the mutants. Moreover the morphology of the LRP2-deficient retinal pigment epithelium (RPE) cells was affected and the expression of ARF4 as well as of proteins involved in degradative endocytosis was strongly reduced. Our results support the idea that impairment of the RPE structure and most likely endocytic function may contribute to the vitreal modifications and pathogenesis of HM.

ANGPTL4-αvß3 interaction counteracts hypoxia-induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2.

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neuroglobin gene therapy prevents optic atrophy and preserves durably visual function in Harlequin mice.

Neuroglobin (NGB) is considered as an endogenous neuroprotective molecule against stroke, since the protein alleviates the adverse effects of hypoxic and ischemic insults. We previously demonstrated the functional link between NGB and mitochondria since it is required for respiratory chain function. Thus, here, we evaluated the relevance of this effect in the Harlequin (Hq) mouse strain, which exhibits retinal ganglion cell (RGC) loss and optic atrophy due to a respiratory chain complex I (CI) defect. A twofold decrease of NGB amounts was observed in Hq retinas. We constructed a recombinant adeno-associated virus which combines to the mouse NGB open reading frame, its 5' and 3'UTR, for guarantying mRNA stability and translation capacity. The vector was administrated intravitreally to Hq mice and NGB expression was stable for up to 7 months without negative effect on retinal architecture or function. On the contrary, RGCs and their axons were substantially preserved from degeneration; consequently, CI activity in optic nerves was protected conferring improvements in vision. Hence, we established that NGB prevents respiratory chain impairment, therefore, protecting visual function otherwise compromised by mitochondrial energetic failure.

Neuroglobin involvement in visual pathways through the optic nerve.

Neuroglobin is a member of the globin superfamily proposed to be only expressed in neurons and involved in neuronal protection from hypoxia or oxidative stress. A significant fraction of the protein localizes within the mitochondria and is directly associated with mitochondrial metabolism and integrity. The retina is the site of the highest concentration for neuroglobin and has been reported to be up to 100-fold higher than in the brain. Since neuroglobin was especially abundant in retinal ganglion cell layer, we investigated its abundance in optic nerves. Remarkably in optic nerves, neuroglobin is observed, as expected, in retinal ganglion cell axon profiles but also astrocyte processes, in physiological conditions, possess high levels of the protein. Neuroglobin mRNA and protein levels are ~10-fold higher in optic nerves than in retinas, indicating an important accumulation of neuroglobin in these support cells. Additionally, neuroglobin levels increase in Müller cells during reactive gliosis in response to eye injury. This suggests the pivotal role of neuroglobin in retinal glia involved in neuronal support and/or healing. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Retinal atrophy, inflammation, phagocytic and metabolic disruptions develop in the MerTK-cleavage-resistant mouse model.

In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTKCR) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTKCR retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTKCR and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTKCR RPE primary cultures phagocytosed less POS in vitro, while in vivo, the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina.

DRD2 activation inhibits choroidal neovascularization in patients with Parkinson's disease and age-related macular degeneration.

Neovascular age-related macular degeneration (nAMD) remains a major cause of visual impairment and puts considerable burden on patients and health care systems. l-DOPA-treated Parkinson's disease (PD) patients have been shown to be partially protected from nAMD, but the mechanism remains unknown. Using murine models that combine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced (MPTP-induced) PD and laser-induced nAMD with standard PD treatment of l-DOPA/DOPA-decarboxylase inhibitor or specific dopamine receptor inhibitors, we here demonstrate that l-DOPA treatment-induced increase of dopamine-mediated dopamine receptor D2 (DRD2) signaling inhibits choroidal neovascularization independently of MPTP-associated nigrostriatal pathway lesion. Analyzing a retrospective cohort of more than 200,000 patients with nAMD receiving anti-VEGF treatment from the French nationwide insurance database, we show that DRD2 agonist-treated PD patients have a significantly delayed age of onset of nAMD and reduced need for anti-VEGF therapies, similar to the effects of the l-DOPA treatment. While providing a mechanistic explanation for an intriguing epidemiological observation, our findings suggest that systemic DRD2 agonists might constitute an adjuvant therapy to delay and reduce the need for anti-VEGF therapy in patients with nAMD.

Neuroglobin involvement in respiratory chain function and retinal ganglion cell integrity.

Neuroglobin is a member of the globin superfamily expressed in vertebrate brain and retina. The protein is thought to be involved in neuronal protection from hypoxia or oxidative stress and could represent a key element of Alzheimer disease pathogenesis. Our aim was to determine whether neuroglobin could be directly associated with mitochondrial metabolism and integrity. We identified three different forms of neuroglobin in the retina, varying in their apparent molecular masses; all forms are abundant in mitochondrial fractions. This indicates that a significant fraction of the protein localizes within the organelle either in the matrix or in the matrix side of the inner membrane. Since neuroglobin was especially abundant in the ganglion cell layer, we transduced retinal ganglion cells with an anti-neuroglobin short hairpin RNA using in vivo electroporation. Neuroglobin knockdown leads to reduced activities of respiratory chain complexes I and III, degeneration of retinal ganglion cells, and impairment of visual function. The deleterious effect on cell survival was confirmed in primary retinal ganglion cells subjected to inhibition of neuroglobin expression. Hence, neuroglobin should be considered as a novel mitochondrial protein involved in respiratory chain function which is essential for retinal ganglion cell integrity.

Downregulation of apoptosis-inducing factor in Harlequin mice induces progressive and severe optic atrophy which is durably prevented by AAV2-AIF1 gene therapy.

The Harlequin mutant mouse, characterized by loss of function of apoptosis-inducing factor, represents a reliable genetic model that resembles pathologies caused by human mitochondrial complex I deficiency. Therefore, we extensively characterized the retinal morphology and function of Harlequin mice during the course of neuronal cell death leading to blindness, with the aim of preventing optic atrophy. Retinas and optic nerves from these mice showed an isolated respiratory chain complex I defect correlated with retinal ganglion cell loss, optic atrophy, glial and microglial cell activation. All of these changes led to irreversible vision loss. In control mice, retinas AIF1 messenger RNA was 2.3-fold more abundant than AIF2, both messenger RNAs being sorted to the mitochondrial surface. In Harlequin mouse retinas, there was a 96% decrease of both AIF1 and AIF2 messenger RNA steady-state levels. We attained substantial and long-lasting protection of retinal ganglion cell and optic nerve integrity, the preservation of complex I function in optic nerves, as well as the prevention of glial and microglial responses after intravitreal administration of an AAV2 vector containing the full-length open reading frame and the 3' untranslated region of the AIF1 gene. Therefore, we demonstrate that gene therapy for mitochondrial diseases due to mutations in nuclear DNA can be achieved, so long as the 'therapeutic gene' permits the accurate cellular localization of the corresponding messenger RNA.

Rodent Models of Retinal Degeneration: From Purified Cells in Culture to Living Animals.

Rodent models of retinal degeneration are essential for the development of therapeutic strategies. In addition to living animal models, we here also discuss models based on rodent cell cultures, such as purified retinal ganglion cells and retinal explants. These ex vivo models extend the possibilities for investigating pathological mechanisms and assessing the neuroprotective effect of pharmacological agents by eliminating questions on drug pharmacokinetics and bioavailability. The number of living rodent models has greatly increased with the possibilities to achieve transgenic modifications in animals for knocking in and out genes and mutations. The Cre-lox system has further enabled investigators to target specific genes or mutations in specific cells at specific stages. However, chemically or physically induced models can provide alternatives to such targeted gene modifications. The increased diversity of rodent models has widened our possibility to address most ocular pathologies for providing initial proof of concept of innovative therapeutic strategies.