Retinal Photoreceptor Degeneration Research Articles

Cd9 Protects Photoreceptors from Injury and Potentiates Edn2 Expression.

PurposeCd9 is a tetraspanin membrane protein that plays various roles in tissue development and disease pathogenesis, especially in cancer, but the expression patterns and function of Cd9 in retinal development and disease are not well understood. We asked its roles during retinal photoreceptor degeneration by using CD9-knockout mice.MethodsCd9 knockout mice and rd1 mice were used to examine roles of Cd9 for progression of photoreceptor degeneration. Reverse transcription-polymerase chain reaction and immunohistochemistry were mainly used as analytical methods.ResultsCd9 transcripts were only weakly expressed in retina at embryonic day 14, but its expression level subsequently increased and peaked at around postnatal day 12. In 6-week-old female mice derived retina, mRNA expression decreased slightly but was maintained at a significant level. Published RNA-sequencing data and immunohistochemistry indicated that Cd9 was expressed abundantly in Müller glia and weakly in other retinal neurons. Notably, when photoreceptors were damaged, Cd9 expression was increased in rod photoreceptors and decreased in Müller glia. Cd9 knockout mice retinas developed normally; however, once the retina suffered damage, degeneration of photoreceptors was more severe in Cd9 knockout retinas than control retinas. Induction of Edn2, which is known to protect against photoreceptor damage, was severely hampered. In addition, induction of Socs3, which is downstream of gp130 (Il6st), was weaker in Cd9 knockout retinas.ConclusionsTaken together, these findings indicate that, although Cd9 was dispensable for normal gross morphological development, it protected rod photoreceptors and enhanced Edn2 expression, possibly through modulation of gp130 signaling.

Fundus autofluorescence, spectral-domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model.

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.

CD36 Deficiency Inhibits Retinal Inflammation and Retinal Degeneration in Cx3cr1 Knockout Mice.

Background: CD36, a member of the class B scavenger receptor family, participates in Toll-like receptor signaling on mononuclear phagocytes (MP) and can promote sterile pathogenic inflammation. We here analyzed the effect of CD36 deficiency on retinal inflammation and photoreceptor degeneration, the hallmarks of age-related macular degeneration (AMD), that characterize Cx3cr1−/−mice.Methods: We analyzed subretinal MP accumulation, and cone- and rod-degeneration in light-challenged and aged, CD36 competent or deficient, hyper-inflammatory Cx3cr1−/− mice, using histology and immune-stained retinal flatmounts. Monocytes (Mo) were subretinally adoptively transferred to evaluate their elimination rate from the subretinal space and Interleukin 6 (IL-6) secretion from cultured Mo-derived cells (MdCs) of the different mouse strains were analyzed.Results: CD36 deficient Cx3cr1−/− mice were protected against age- and light-induced subretinal inflammation and associated cone and rod degeneration. CD36 deficiency in Cx3cr1−/− MPs inhibited their prolonged survival in the immune-suppressive subretinal space and reduced the exaggerated IL-6 secretion observed in Cx3cr1−/− MPs that we previously showed leads to increased subretinal MP survival.Conclusion: Cd36 deficiency significantly protected hyperinflammatory Cx3cr1−/− mice against subretinal MP accumulation and associated photoreceptor degeneration. The observed CD36-dependent induction of pro-inflammatory IL-6 might be at least partially responsible for the prolonged MP survival in the immune-suppressive environment and its pathological consequences on photoreceptor homeostasis.

Retinal degeneration development in Cnga1 deficient mouse

AbstractRetinitis pigmentosa (RP; OMIM 268,000) is a group of retina degenerative diseases with chronic, progressive degeneration of retinal pigmented epithelium and photoreceptors, with about 2.5 million people affected worldwide. Being highly genetic heterogeneous, RP exhibits various clinical phenotypes. Cyclic nucleotide gated (CNG) channel is involved in the visual photo‐transduction upon hyperpolarizing the rod photoreceptor. CNG family comprises six homologous members in mammals, A‐type subunits (CNGA1–4) and B‐type subunits (CNGB1 and CNGB3). Here we report the first Cnga1 mouse model identified from the library if previous ENU‐mutagenesis screens, which shows retinal degeneration in mice starting with 4 week of age. Additionally, using the ACmaster we observed changes in the axial length of the eye, the anterior chamber and lens thickness, all being reduced.

Activation of Sigma 1 Receptor Extends Survival of Cones and Improves Visual Acuity in a Murine Model of Retinitis Pigmentosa.

PurposeRetinitis pigmentosa (RP), a retinal photoreceptor degeneration, typically affects rod function and subsequently cones. Activation of sigma 1 receptor (Sig1R) has been shown to preserve cone function through 6 weeks in the rd10 mouse model of RP, when mice were treated systemically with the Sig1R ligand (+)-pentazocine (PTZ). This study determined the extent to which cone function is preserved in rd10 mice when Sig1R is activated.MethodsRd10 mice were administered (+)-PTZ (alternate days beginning at postnatal day [P]14) over a period of 180 days. Mouse visual function and structure were measured in vivo using optokinetic tracking response, scotopic and photopic electroretinography plus photopic assessment using “natural” noise stimuli, and optical coherence tomography (OCT). Immunofluorescent methods were used to detect cones in retinal cryosections.ResultsVisual acuity was maintained in rd10(+)-PTZ-treated mice through P56, whereas rd10 nontreated mice showed marked decline by P28. Cone responses were detected in (+)-PTZ-treated mice through P60, which were more robust when tested with natural noise stimuli; cone responses were minimal in nontreated rd10 mice. OCT revealed significantly thicker retinas in (+)-PTZ-treated rd10 mice through P60 compared to nontreated mice. Cones were detected by immunofluorescence in (+)-PTZ-treated rd10 retinas through P120.ConclusionsThe extent to which cone rescue could be sustained in (+)-PTZ-treated rd10 mice was evaluated comprehensively, showing that activation of Sig1R is associated with prolonged visual acuity, extended detection of cone function, and detection of cones in retinal histologic sections. The data reflect promising long-term neuroprotection when Sig1R is activated.

Systemic taurine treatment provides neuroprotection against retinal photoreceptor degeneration and visual function impairments.

ObjectiveRetinitis pigmentosa causes progressive photoreceptor degeneration in the subjects while no clinical therapy exists. The present study sought to evaluate the potential protective effects of taurine on a pharmacologically induced RP animal model.MethodsPhotoreceptor degeneration in mice was induced by an intraperitoneal injection of N-methyl-N-nitrosourea (MNU). The MNU-administrated mouse received taurine treatment and then they were examined by electroretinography, spectral-domain optical coherence tomography, optokinetic test, and histological and immunohistochemistry assay.ResultsProminent taurine deficiency was found in the retinas of MNU-administered mice. Intravenous taurine treatment increased significantly the retinal taurine level. Morphological studies showed that taurine could alleviate the retinal disorganizations in the MNU-induced mice. Taurine also ameliorated the visual impairments in the MNU-induced mice as evidenced by functional examinations. Immunostaining experiments demonstrated that both the M-cone and S-cone populations in the degenerative retinas are rescued by taurine. In particular, the M-cone photoreceptors in superior-temporal quadrant and the S-cone photoreceptors in inferior-nasal quadrant were preferentially rescued. Mechanism study showed that the photoreceptor apoptosis and oxidative stress in the degenerative retina were effectively alleviated by taurine treatment.ConclusionTaurine is protective against the MNU-induced photoreceptor degeneration. Systemic taurine administration may act as a promising therapeutic potion for retinopathies with chronic cycle.

Photoreceptors in a mouse model of Leigh syndrome are capable of normal light-evoked signaling

Mitochondrial dysfunction is an important cause of heritable vision loss. Mutations affecting mitochondrial bioenergetics may lead to isolated vision loss or life-threatening systemic disease, depending on a mutation's severity. Primary optic nerve atrophy resulting from death of retinal ganglion cells is the most prominent ocular manifestation of mitochondrial disease. However, dysfunction of other retinal cell types has also been described, sometimes leading to a loss of photoreceptors and retinal pigment epithelium that manifests clinically as pigmentary retinopathy. A popular mouse model of mitochondrial disease that lacks NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), a subunit of mitochondrial complex I, phenocopies many traits of the human disease Leigh syndrome, including the development of optic atrophy. It has also been reported that ndufs4-/- mice display diminished light responses at the level of photoreceptors or bipolar cells. By conducting electroretinography (ERG) recordings in live ndufs4-/- mice, we now demonstrate that this defect occurs at the level of retinal photoreceptors. We found that this deficit does not arise from retinal developmental anomalies, photoreceptor degeneration, or impaired regeneration of visual pigment. Strikingly, the impairment of ndufs4-/- photoreceptor function was not observed in ex vivo ERG recordings from isolated retinas, indicating that photoreceptors with complex I deficiency are intrinsically capable of normal signaling. The difference in electrophysiological phenotypes in vivo and ex vivo suggests that the energy deprivation associated with severe mitochondrial impairment in the outer retina renders ndufs4-/- photoreceptors unable to maintain the homeostatic conditions required to operate at their normal capacity.

Computational modeling of retinal hypoxia and photoreceptor degeneration in patients with age-related macular degeneration.

Although drusen have long been acknowledged as a primary hallmark of dry age-related macular degeneration (AMD) their role in the disease remains unclear. We hypothesize that drusen accumulation increases the barrier to metabolite transport ultimately resulting in photoreceptor cell death. To investigate this hypothesis, a computational model was developed to evaluate steady-state oxygen distribution in the retina. Optical coherence tomography images from fifteen AMD patients and six control subjects were segmented and translated into 3D in silico representations of retinal morphology. A finite element model was then used to determine the steady-state oxygen distribution throughout the retina for both generic and patient-specific retinal morphology. Oxygen levels were compared to the change in retinal thickness at a later time point to observe possible correlations. The generic finite element model of oxygen concentration in the retina agreed closely with both experimental measurements from literature and clinical observations, including the minimal pathological drusen size identified by AREDS (64 μm). Modeling oxygen distribution in the outer retina of AMD patients showed a substantially stronger correlation between hypoxia and future retinal thinning (Pearson correlation coefficient, r = 0.2162) than between drusen height and retinal thinning (r = 0.0303) indicating the potential value of this physiology-based approach. This study presents proof-of-concept for the potential utility of finite element modeling in evaluating retinal health and also suggests a potential link between transport and AMD pathogenesis. This strategy may prove useful as a prognostic tool for predicting the clinical risk of AMD progression.

Microglia are not required for prion-induced retinal photoreceptor degeneration

Degeneration of photoreceptors in the retina is a major cause of blindness in humans. Often retinal degeneration is due to inheritance of mutations in genes important in photoreceptor (PR) function, but can also be induced by other events including retinal trauma, microvascular disease, virus infection or prion infection. The onset of apoptosis and degeneration of PR neurons correlates with invasion of the PR cellular areas by microglia or monocytes, suggesting a causal role for these cells in pathogenesis of PR degenerative disease. To study the role of microglia in prion-induced retinal disease, we fed prion-infected mice a CSF-1 receptor blocking drug, PLX5622, to eliminate microglia in vivo, and the effects on retinal degeneration were analyzed over time. In mice not receiving drug, the main inflammatory cells invading the degenerating PR areas were microglia, not monocytes. Administration of PLX5622 was highly effective at ablating microglia in retina. However, lack of microglia during prion infection did not prevent degeneration of PR cells. Therefore, microglia were not required for the PR damage process during prion infection. Indeed, mice lacking microglia had slightly faster onset of PR damage. Similar results were seen in C57BL/10 mice and transgenic mice expressing GFP or RFP on microglia and monocytes, respectively. These results were supported by experiments using prion-infected Cx3cr1 knockout mice without PLX5622 treatment, where microglial expansion in retina was delayed, but PR degeneration was not. Contrary to predictions, microglia were not a causative factor in retinal damage by prion infection. Instead, newly generated PrPSc accumulated around the inner segment region of the PR cells and appeared to correlate with initiation of the pathogenic process in the absence of microglia.

Adeno-Associated Viral Gene Therapy for Inherited Retinal Disease.

Inherited retinal diseases (IRDs) are a group of rare, heterogenous eye disorders caused by gene mutations that result in degeneration of the retina. There are currently limited treatment options for IRDs; however, retinal gene therapy holds great promise for the treatment of different forms of inherited blindness. One such IRD for which gene therapy has shown positive initial results is choroideremia, a rare, X-linked degenerative disorder of the retina and choroid. Mutation of the CHM gene leads to an absence of functional Rab escort protein 1 (REP1), which causes retinal pigment epithelium cell death and photoreceptor degeneration. The condition presents in childhood as night blindness, followed by progressive constriction of visual fields, generally leading to vision loss in early adulthood and total blindness thereafter. A recently developed adeno-associated virus-2 (AAV2) vector construct encoding REP1 (AAV2-REP1) has been shown to deliver a functional version of the CHM gene into the retinal pigment epithelium and photoreceptor cells. Phase 1 and 2 studies of AAV2-REP1 in patients with choroideremia have produced encouraging results, suggesting that it is possible not only to slow or stop the decline in vision following treatment with AAV2-REP1, but also to improve visual acuity in some patients.

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

Dysfunction in mitochondrial oxidative phosphorylation (OXPHOS) underlies a wide spectrum of human ailments known as mitochondrial diseases. Deficiencies in complex I of the electron transport chain (ETC) contribute to 30–40% of all cases of mitochondrial diseases, and leads to eye disease including optic nerve atrophy and retinal degeneration. The mechanisms responsible for organ damage in mitochondrial defects may include energy deficit, oxidative stress, and an increase in the NADH/NAD+ redox ratio due to decreased NAD+ regeneration. Currently, there is no effective treatment to alleviate human disease induced by complex I defect.Photoreceptor cells have the highest energy demand and dependence on OXPHOS for survival, and the lowest reserve capacity indicating that they are sensitive to OXPHOS defects. We investigated the effect of mitochondrial OXPHOS deficiency on retinal photoreceptors in a model of mitochondrial complex I defect (apoptosis inducing factor, AIF-deficient mice, Harlequin mice), and tested the protective effect of a mitochondrial redox compound (methylene blue, MB) on mitochondrial and photoreceptor integrity. MB prevented the reduction in the retinal thickness and protein markers for photoreceptor outer segments, Muller and ganglion cells, and altered mitochondrial integrity and function induced by AIF deficiency. In rotenone-induced complex I deficient 661 W cells (an immortalized mouse photoreceptor cell line) MB decreased the NADH/NAD+ ratio and oxidative stress without correcting the energy deficit, and improved cell survival. MB deactivated the mitochondrial stress response pathways, the unfolding protein response and mitophagy. In conclusion, preserving mitochondrial structure and function alleviates retinal photoreceptor degeneration in mitochondrial complex I defect.

The Retinol-Binding Protein Receptor 2 (Rbpr2) Is Required for Photoreceptor Survival and Visual Function in the Zebrafish.

Vitamin A/retinol (ROL) and its metabolites (retinoids) play critical roles in eye development and photoreception. Short-term dietary vitamin A deficiency (VAD) manifests clinically as night blindness, while prolonged VAD is known to cause retinal pigment epithelium (RPE) and photoreceptor degeneration. Therefore, sustained uptake of dietary vitamin A, for ocular retinoid production, is essential for photoreceptor health and visual function. The mechanisms influencing the uptake, storage, and supply of dietary vitamin A, for ocular retinoid production, however, are not fully understood. We investigated, in zebrafish, the physiological role of the retinol-binding protein receptor 2 (Rbpr2), for the uptake of dietary ROL, which is necessary for vision. NIH3T3 cells expressing zebrafish Rbpr2 showed plasma membrane localization patterns and were capable of ROL uptake from its bound form. Using whole-mount in situ hybridization, Rbpr2 was found to be expressed exclusively in the liver, intestine, and pancreas, of staged zebrafish larvae. At 5.5days post fertilization, TALEN-generated rbpr2 mutants (rbpr2 -/- ) had smaller eyes and shorter OS lengths and showed loss of PNA (cones) and rhodopsin (rods) by immunofluorescence staining. Finally, tests for visual function using optokinetic response (OKR) showed no consistent OKR in rbpr2 -/- larval zebrafish. Our analysis, therefore, suggests that Rbpr2 is capable of ROL uptake and loss of this membrane receptor in zebrafish results in photoreceptor defects that adversely affect visual function.

Inflammation-Induced Photoreceptor Cell Death.

Neuroinflammation is an important aspect of many diseases of the eye, and experimental animal models have been widely used to determine its impact on retinal homeostasis and neuron survival. Physical separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE) results in activation and infiltration of macrophages. Numerous studies have shown the critical role of macrophages in retinal disease processes. In retinal detachment, accumulation of macrophages in the subretinal space is associated with changes in cytokine and chemokine profile which lead to photoreceptor cell death. Targeted disruption of macrophage chemotaxis significantly reduces retinal detachment-induced photoreceptor degeneration. Apoptosis is the predominant mechanism of cell death; however regulated necrosis is also a contributor of photoreceptor loss. Therefore, effective neuroprotective approaches could integrate combined inhibition of both apoptotic and regulated necrosis pathways.

Conditional Müller Cell Ablation Leads to Retinal Iron Accumulation.

PurposeRetinal iron accumulation is observed in a wide range of retinal degenerative diseases, including AMD. Previous work suggests that Müller glial cells may be important mediators of retinal iron transport, distribution, and regulation. A transgenic model of Müller cell loss recently demonstrated that primary Müller cell ablation leads to blood–retinal barrier leakage and photoreceptor degeneration, and it recapitulates clinical features observed in macular telangiectasia type 2 (MacTel2), a rare human disease that features Müller cell loss. We used this mouse model to determine the effect of Müller cell loss on retinal iron homeostasis.MethodsChanges in total retinal iron levels after Müller cell ablation were measured using inductively coupled plasma mass spectrometry. Corresponding changes in the expression of iron flux and iron storage proteins were determined using quantitative PCR, Western analysis, and immunohistochemistry.ResultsMüller cell loss led to blood–retinal barrier breakdown and increased iron levels throughout the neurosensory retina. There were corresponding changes in mRNA and/or protein levels of ferritin, transferrin receptor, ferroportin, Zip8, and Zip14. There were also increased iron levels within the RPE of retinal sections from a patient with MacTel2 and both RPE and neurosensory retina of a patient with diabetic retinopathy, which, like MacTel2, causes retinal vascular leakage.ConclusionThis study shows that Müller cells and the blood–retinal barrier play pivotal roles in the regulation of retinal iron homeostasis. The retinal iron accumulation resulting from blood–retinal barrier dysfunction may contribute to retinal degeneration in this model and in diseases such as MacTel2 and diabetic retinopathy.

Neural Responses to Multielectrode Stimulation of Healthy and Degenerate Retina.

Simultaneous stimulation of multiple retinal electrodes in normally sighted animals shows promise in improving the resolution of retinal prostheses. However, the effects of simultaneous stimulation on degenerate retinae remain unknown. Therefore, we investigated the characteristics of cortical responses to multielectrode stimulation of the degenerate retina. Four adult cats were bilaterally implanted with retinal electrode arrays in the suprachoroidal space after unilateral adenosine triphosphate (ATP)-induced retinal photoreceptor degeneration. Functional and structural changes were characterized by using electroretinogram a-wave amplitude and optical coherence tomography. Multiunit activity was recorded from both hemispheres of the visual cortex. Responses to single- and multielectrode stimulation of the ATP-injected and fellow control eyes were characterized and compared. The retinae of ATP-injected eyes displayed structural and functional changes consistent with mid- to late-stage photoreceptor degeneration and remodeling. Responses to multielectrode stimulation of the ATP-injected eyes exhibited shortened latencies, lower saturated spike counts, and higher thresholds, compared to stimulation of the fellow control eyes. Electrical receptive field sizes were significantly larger in the ATP-injected eye than in the control eye, and positively correlated with the extent of degeneration. Significant differences exist between cortical responses to stimulation of healthy and degenerate retinae. Our results highlight the importance of using a retinal degeneration model when evaluating the efficacy of novel stimulation paradigms.

Retinal electrostimulation in rats: Activation thresholds from superior colliculus and visual cortex recordings.

Retinal neuromodulation is an emerging therapeutic approach to restore functional vision to those suffering retinal photoreceptor degeneration. The retina encodes visual information and transmits it to the brain. Replicating this retinal code through electrical stimulation is essential to improving the performance of visual prostheses. In doing so, the first step relies on precise neural recordings from visual centers that allow studying the response of these neurons to electrical stimulation of the retina. This paper demonstrates the feasibility of a rat model to conduct highly reliable electrophysiological studies in the field of retinal neuromodulation. A disc electrode, implanted in the retrobulbar space was used to stimulate the retina of Long-Evans rats. Buzsaki multi-electro arrays were inserted in the superior colliculus (SC) to record electrical activity propagated from the retinal ganglion cells (RGCs). Activation thresholds calculated from local field potentials (visual cortex) and from neural spikes (SC) were contrasted. Both values were comparable to those in humans and in other animal models, and were slightly higher when estimated from SC recordings. However, differences were not statistically significant.

Polysialinsäure zur Immunmodulation im Tiermodell der feuchten altersabhängigen Makuladegeneration (AMD)

Background Chronic activation of the innate immune system is a hallmark of retinal degenerative diseases, including age-related macular degeneration (AMD). Overt microglia and macrophage reactivity, as well as dysregulation of the alternative complement system, trigger and sustain retinal degeneration. It is now accepted that there exists a vicious cycle of mononuclear phagocyte reactivity, abnormal intrinsic complement activation, damage of Bruch's membrane, dysfunction of the retinal pigment epithelium, photoreceptor degeneration and choroidal neovascularization (CNV). Targeting the innate immune system may, therefore, be a complementary approach to anti-angiogenic therapy. This article presents data from polysialic acid treatment experiments in the laser-induced mouse model of wet AMD. Material and Methods The laser-CNV mouse model was used to simulate events of neovascular AMD. Polysialic acid was applied by intravitreal injection and microglia activity was assessed with Iba1 staining of retinal and RPE/choroidal flat mounts. Neovascular leakage was determined by fluorescein angiography. Results Intravitreal injection of polysialic acid reduced retinal and RPE/choroidal lesion-associated microglia activity in the laser-induced mouse model of AMD. Polysialic acid treatment also diminished vascular leakage at laser spots in this model. Conclusion Local retinal immunomodulation with polysialic acid presents a novel concept for treatment of inflammatory conditions related to neovascular AMD.

A large deletion in RPGR causes XLPRA in Weimaraner dogs.

BackgroundProgressive retinal atrophy (PRA) belongs to a group of inherited retinal disorders associated with gradual vision impairment due to degeneration of retinal photoreceptors in various dog breeds. PRA is highly heterogeneous, with autosomal dominant, recessive or X-linked modes of inheritance. In this study we used exome sequencing to investigate the molecular genetic basis of a new type of PRA, which occurred spontaneously in a litter of German short-hair Weimaraner dogs.ResultsWhole exome sequencing in two PRA-affected Weimaraner dogs identified a large deletion comprising the first four exons of the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene known to be involved in human retinitis pigmentosa and canine PRA. Screening of 16 individuals in the corresponding pedigree of short-hair Weimaraners by qPCR, verified the deletion in hemizygous or heterozygous state in one male and six female dogs, respectively. The mutation was absent in 88 additional unrelated Weimaraners. The deletion was not detectable in the parents of one older female which transmitted the mutation to her offspring, indicating that the RPGR deletion represents a de novo mutation concerning only recent generations of the Weimaraner breed in Germany.ConclusionOur results demonstrate the value of an existing DNA biobank combined with exome sequencing to identify the underlying genetic cause of a spontaneously occurring inherited disease. Identification of the genetic cause has allowed the development of a diagnostic test, which should help to eradicate the PRA causing mutation from the respective canine line. Thus, planning of future pairings is facilitated and manifestation of this type of PRA can be prevented.Electronic supplementary materialThe online version of this article (doi:10.1186/s40575-016-0037-x) contains supplementary material, which is available to authorized users.

553. Comparision of AAV Serotypes for Gene Delivery to iPSC Derived RPE

Inherited retinal degenerations, such as retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), are characterized by progressive impairment of visual function associated with degeneration of retinal pigment epithelium (RPE) and photoreceptors. These monogenic diseases are genetically heterogeneous due to mutations in genes expressed affecting those cells. Gene therapy holds great potential for the treatment of inherited retinal diseases for which there currently is none. Efficient retinal gene transfer has been achieved with several recombinant viral vectors, including those derived from adenovirus, retrovirus, herpesvirus, and adeno-associated virus (AAV). Among these, AAV vectors appear particularly amenable to retinal gene transfer. However the success of any preclinical study depends on the availability of relevant animal models. In situations where suitable animal models are unavailable recent studies have generated proof-of concept data using personalized cell models: induced pluripotent stem cells (iPSC) derived from affected individuals and un-affected controls. With the increasing number of retinal gene therapy paradigms and recombinant vectors, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. To date, most in vitro assays using recombinant vector transduction have targeted iPSCs. To elicit additional features relevant to the disease, we differentiated iPSCs to RPE and evaluated the transduction efficiencies of a panel of AAV serotypes. Characterization of iPSC-derived RPE by qRT-PCR, immunohistochemistry, western blot analysis and flow cytometry showed the expression of typical RPE markers, phagocytic ability and gene-expression patterns similar to those of native RPE. Comparison of transduction efficiencies of different AAVs in iPSC-derived RPE was carried out using an enhanced green-fluorescent protein (eGFP) reporter gene driven by the cytomegalovirus immediate-early (CMV) promoter. At 24 to 48 hours post transduction, cells expressing GFP were identified by Typhoon scanner, and flow cytometry. Relative GFP expression was evaluated by using the software, Image-J. Of the tested AAV serotypes, AAV2 transduced iPSC-derived RPE cells most efficiently, followed by AAV7m8, AAV1 and AAV6. Differentiation into retinal neuronal cells types may require use of alternative AAV serotypes in order to obtain transduction efficiencies relevant to determination of therapeutic efficacy.