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AimsTo determine long-term safety and efficacy outcomes of a subretinal gene therapy for CNGA3-associated achromatopsia. We present data from an open-label, nonrandomised controlled trial (NCT02610582).MethodsDetails of the study design have been previously described. Briefly, nine patients were treated in three escalating dose groups with subretinal AAV8.CNGA3 gene therapy between November 2015 and October 2016. After the first year, patients were seen on a yearly basis. Safety assessment constituted the primary endpoint. On a secondary level, multiple functional tests were carried out to determine efficacy of the therapy.ResultsNo adverse or serious adverse events deemed related to the study drug occurred after year 1. Safety of the therapy, as the primary endpoint of this trial, can, therefore, be confirmed. The functional benefits that were noted in the treated eye at year 1 were persistent throughout the following visits at years 2 and 3. While functional improvement in the treated eye reached statistical significance for some secondary endpoints, for most endpoints, this was not the case when the treated eye was compared with the untreated fellow eye.ConclusionThe results demonstrate a very good safety profile of the therapy even at the highest dose administered. The small sample size limits the statistical power of efficacy analyses. However, trial results inform on the most promising design and endpoints for future clinical trials. Such trials have to determine whether treatment of younger patients results in greater functional gains by avoiding amblyopia as a potential limiting factor.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1−/− double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1−/− double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

RationalePericytes (PCs) and vascular smooth muscle cells (vSMCs), collectively known as mural cells (MCs), are recruited through PDGFB-PDGFRB signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions.ObjectiveHere, we characterize the role of the transcription factor serum response factor (SRF) in MCs and study its function in developmental and pathological contexts.Methods and ResultsWe generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis. By postnatal day (P)6, PCs lacking SRF were morphologically abnormal and failed to properly co-migrate with angiogenic sprouts. As a consequence, PC-deficient vessels at the retinal sprouting front became dilated and leaky. By P12, also the vSMCs had lost SRF, which coincided with the formation of pathological arteriovenous (AV) shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF co-factors. We further show that MRTF-SRF signaling promotes pathological PC activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone.ConclusionsSRF is crucial for distinct functions in PCs and vSMCs. SRF directs PC migration downstream of PDGFRB signaling and mediates pathological PC activation during ischemic retinopathy. In vSMCs, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of AV shunts. These essential roles in physiological and pathological contexts provide a rational for novel therapeutic approaches through targeting SRF activity in MCs.

Little is known about the function of the auxiliary α2δ subunits of voltage-gated calcium channels in the retina. We investigated the role of α2δ-3 (Cacna2d3) using a mouse in which α2δ-3 was knocked out by LacZ insertion. Behavior experiments indicated a normal optokinetic reflex in α2δ-3 knockout animals. Strong expression of α2δ-3 could be localized to horizontal cells using the LacZ-reporter, but horizontal cell mosaic and currents carried by horizontal cell voltage-gated calcium channels were unchanged by the α2δ-3 knockout.In vivoelectroretinography revealed unaffected photoreceptor activity and signal transmission to depolarizing bipolar cells. We recorded visual responses of retinal ganglion cells with multi-electrode arrays in scotopic to photopic luminance levels and found subtle changes in α2δ-3 knockout retinas. Spontaneous activity in OFF ganglion cells was elevated in all luminance levels. Differential response strength to high- and low-contrast Gaussian white noise was compressed in ON ganglion cells during mesopic ambient luminance and in OFF ganglion cells during scotopic and mesopic ambient luminances. In a subset of ON ganglion cells, we found a sharp increase in baseline spiking after the presentation of drifting gratings in scotopic luminance. This increase happened after gratings of different spatial properties in knockout compared to wild type retinas. In a subset of ON ganglion cells of the α2δ-3 knockout, we found altered delays in rebound-like spiking to full-field contrast steps in scotopic luminance. In conclusion, α2δ-3 seems to participate in shaping visual responses mostly within brightness regimes when rods or both rods and cones are active.

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha (PDE6A). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via PDE6A gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human PDE6A cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the Pde6anmf363/nmf363-mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed in vivo by means of electroretinography. The degree of morphological rescue was investigated both in vivo using optical coherence tomography and...

Mutations in the phosphodiesterase 6A gene (PDE6A) result in retinitis pigmentosa (RP) type 43 (RP43) and are responsible for about 4% of autosomal recessive RP. There is currently no treatment for this blinding condition. The aim of this project was to use a large-animal model to test a gene supplementation viral vector designed to be translated for use in a clinical trial for the treatment of RP43. Seven Pde6a–/– puppies were given sub-retinal injections of an adeno-associated viral vector (AAV) serotype 2/8 delivering human PDE6A cDNA under control of a short rhodopsin promoter (AAV8-PDE6A). Three puppies received ∼1 × 1011 vg in one eye and four puppies ∼5 × 1011 vg/per eye, with both eyes being injected in two animals. In vivo outcome measures included vision testing and electroretinography (ERG), as well as fundus and spectral domain–optical coherence tomography imaging. Some puppies were euthanized and their eyes processed for immunohistochemistry. All puppies had improved rod-mediated vision in th...

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.

Marked attenuation of the single-flash electroretinographic (ERG) b-wave in the presence of a normal-amplitude or less-attenuated a-wave is commonly referred to as the “negative ERG.” The purpose of this study was to investigate whether the disparate origins of the negative ERG in three murine models can be discriminated using flickering stimuli. Three models were selected: (1) the Nyx nob mouse model of complete congenital stationary night blindness, (2) the oxygen-induced retinopathy (OIR) rat model of retinopathy of prematurity (ROP), and (3) the Rs1 knockout (KO) mouse model of X-linked juvenile retinoschisis. Directly after a dark-adapted, single-flash ERG luminance series, a flicker ERG frequency series (0.5–30 Hz) was performed at a fixed luminance of 0.5 log cd s/m2. This series includes frequency ranges that are dominated by activity in (A) the rod pathways (below 5 Hz), (B) the cone ON-pathway (5–15 Hz), and (C) the cone OFF-pathway (above 15 Hz). All three models produced markedly attenuated single-flash ERG b-waves. In the Nyx nob mouse, which features postsynaptic deficits in the ON-pathways, the a-wave was normal and flicker responses were attenuated in ranges A and B, but not C. The ROP rat is characterized by inner-retinal ischemia which putatively affects both ON- and OFF-bipolar cell activity; flicker responses were reduced in all ranges (A–C). Notably, the choroid supplies the photoreceptors and is thought to be relatively intact in OIR, an idea supported by the nearly normal a-wave. Finally, in the Rs1 KO mouse, which has documented abnormality of the photoreceptor-bipolar synapse affecting both ON- and OFF-pathways, the flicker responses were attenuated in all ranges (A–C). The a-wave was also attenuated, likely as a consequence to schisms in the photoreceptor layer. Consideration of both single-flash and flickering ERG responses can discriminate the functional pathology of the negative ERG in these animal models of human disease.

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

Importance Achromatopsia linked to variations in theCNGA3gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available. Objective To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encodingCNGA3(AAV8.CNGA3) in patients withCNGA3-linked achromatopsia. Design, Setting, and Participants This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants inCNGA3were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018. Intervention Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017). Main Outcomes and Measures Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment. Results Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor–mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13;P = .006, 2-sidedttest paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log;P = .003, 2-sidedttest paired samples). Conclusions and Relevance Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains. Trial Registration ClinicalTrials.gov Identifier:NCT02610582

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.

Electroretinography (ERG) is important for functional diagnostics of the retina. Types of information about retinal function obtainable by ERG differ depending on recording conditions, e.g., a combination of light stimulus and adaptation. In terms of stimulation, single-flash and flicker stimuli are frequently used because response properties have been well investigated, allowing an assessment of fundamental retinal functionality; for example, how photoreceptors and bipolar cells, including signal transmission between them, are affected under pathological conditions. Usually, ERGs are recorded with a nonzero lower cutoff frequency of amplifiers to avoid certain artifacts, and additionally, responses are averaged over time so that non-event-related signals are cancelled out. However, the improved signal quality is associated with a loss of information. Especially in steady-state flicker ERG, information about the absolute baseline of recordings is missing because the prestimulus baseline is not included on the recording trace as well as because a zero response is obtained in all cases in which the signal baseline stays constant for a sufficient amount of time. In other words, it is impossible to tell from the conventional flicker ERG whether a zero signal is obtained under conditions of maximal or no excitation of the visual system. In this chapter, we describe a direct current ERG protocol (featuring a lower cutoff frequency of zero) with repetitive single flashes mimicking conventional flicker that contains a defined onset. Using this recording protocol, it is possible to assess not only the absolute excitatory level of the retina but also the development of steady-state responses from the single flash response.

Electroretinography (ERG) is important for functional diagnostics of the retina. Types of information about retinal function obtainable by ERG differ depending on recording conditions, e.g., a combination of light stimulus and adaptation. In terms of stimulation, single-flash and flicker stimuli are frequently used because response properties have been well investigated, allowing an assessment of fundamental retinal functionality; for example, how photoreceptors and bipolar cells, including signal transmission between them, are affected under pathological conditions. Usually, ERGs are recorded with a nonzero lower cutoff frequency of amplifiers to avoid certain artifacts, and additionally, responses are averaged over time so that non-event-related signals are cancelled out. However, the improved signal quality is associated with a loss of information. Especially in steady-state flicker ERG, information about the absolute baseline of recordings is missing because the prestimulus baseline is not included on the recording trace as well as because a zero response is obtained in all cases in which the signal baseline stays constant for a sufficient amount of time. In other words, it is impossible to tell from the conventional flicker ERG whether a zero signal is obtained under conditions of maximal or no excitation of the visual system. In this chapter, we describe a direct current ERG protocol (featuring a lower cutoff frequency of zero) with repetitive single flashes mimicking conventional flicker that contains a defined onset. Using this recording protocol, it is possible to assess not only the absolute excitatory level of the retina but also the development of steady-state responses from the single flash response.

Vimentin (Vim) and glial fibrillary acidic protein (GFAP) are important components of the intermediate filament (IF) (or nanofilament) system of astroglial cells. We conducted full-field electroretinogram (ERG) recordings and found that whereas photoreceptor responses (a-wave) were normal in uninjured GFAP(-/-)Vim(-/-) mice, b-wave amplitudes were increased. Moreover, we found that Kir (inward rectifier K(+)) channel protein expression was reduced in the retinas of GFAP(-/-)Vim(-/-) mice and that Kir-mediated current amplitudes were lower in Müller glial cells isolated from these mice. Studies have shown that the IF system, in addition, is involved in the retinal response to injury and that attenuated Müller cell reactivity and reduced photoreceptor cell loss are observed in IF-deficient mice after experimental retinal detachment. We investigated whether the lack of IF proteins would affect cell survival in a retinal ischemia-reperfusion model. We found that although cell loss was induced in both genotypes, the number of surviving cells in the inner retina was lower in IF-deficient mice. Our findings thus show that the inability to produce GFAP and Vim affects normal retinal physiology and that the effect of IF deficiency on retinal cell survival differs, depending on the underlying pathologic condition.

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.

Full-field electroretinography (ERG) belongs to the gold-standard of electrophysiological test systems in ophthalmology and reflects the sum response of the entire retina to light stimulation. The assessment of the retinal function is a fundamental diagnostic technique not only in the clinical ophthalmology it is also indispensable in the ophthalmic research, in particular, in therapeutic approaches where the in vivo follow up of the benefit after treatment is absolutely necessary. Several current therapeutic approaches have demonstrated long-lasting amelioration in respective disease models and show promise for a successful translation to human patients. In this chapter we provide electroretinography protocols of experimental data which may serve as informative features for upcoming gene therapeutic approaches and clinical trials.

Treatment approaches for inherited eye diseases require local therapeutic molecule delivery by intraocular injection. One important factor that can influence the study outcome is the quality of intraocular administration. The intracompartmental structure (e.g., vitreous) of the eye allows a sustainable release of therapeutic biologicals using an intravitreal delivery. The protocol described here aims at providing the details relevant to perform a transscleral pars plana intravitreal transfer in small eyes using a genetically modified stem cell system. The fact that cells and therewith visually distinct particles are implanted, allows for the assessment of the implantation site and the distribution, and possibilities for temporal follow up studies-hence, valuable information becomes available which can be used to fine-tune the intravitreal delivery technique.

Treatment approaches for inherited eye diseases require local therapeutic molecule delivery by intraocular injection. One important factor that can influence the study outcome is the quality of intraocular administration. The intracompartmental structure (e.g., vitreous) of the eye allows a sustainable release of therapeutic biologicals using an intravitreal delivery. The protocol described here aims at providing the details relevant to perform a transscleral pars plana intravitreal transfer in small eyes using a genetically modified stem cell system. The fact that cells and therewith visually distinct particles are implanted, allows for the assessment of the implantation site and the distribution, and possibilities for temporal follow up studies-hence, valuable information becomes available which can be used to fine-tune the intravitreal delivery technique.

Full-field electroretinography (ERG) belongs to the gold-standard of electrophysiological test systems in ophthalmology and reflects the sum response of the entire retina to light stimulation. The assessment of the retinal function is a fundamental diagnostic technique not only in the clinical ophthalmology it is also indispensable in the ophthalmic research, in particular, in therapeutic approaches where the in vivo follow up of the benefit after treatment is absolutely necessary. Several current therapeutic approaches have demonstrated long-lasting amelioration in respective disease models and show promise for a successful translation to human patients. In this chapter we provide electroretinography protocols of experimental data which may serve as informative features for upcoming gene therapeutic approaches and clinical trials.

Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive in vivo retinal imaging. We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.

Purpose To investigate whether the presence of the retinal degeneration 8 (rd8) mutation in C57BL/6 mice alters the phenotype of autoimmune optic neuritis (AON). Methods C57BL/6J and C57BL/6N mice were genotyped for the rd8 mutation and fundus analyses and examination of retinal layer morphology were performed in vivo by scanning laser ophthalmoscopy and optical coherence tomography. Visual function was assessed by recording electroretinographs, and visual evoked potentials and retinae and optic nerves were assessed histopathologically. Retinal ganglion cell numbers were determined by retrograde labeling with fluorogold. Mice were then immunized with myelin oligodendrocyte glycoprotein 35-55 to induce AON before assessment of retinal ganglion cell degeneration, inflammatory infiltration of retinae and optic nerves, and demyelination. Furthermore, visual function was assessed by visual evoked potentials. Results All C57BL/6N mice were homozygous for the mutation (Crb1rd8/rd8) and had pathology typical of the rd8 mutation; however, this was not seen in the C57BL/6J (Crb1wt/wt) mice. Following induction of AON, no differences were seen between the Crb1rd8/rd8 and Crb1wt/wt mice regarding disease parameters nor regarding inner retinal degeneration either in the retina as a whole or in the inferior nasal quadrant. Conclusions The presence of the rd8 mutation in C57BL/6 mice does not affect the course of AON and should not provide a confounding factor in the interpretation of experimental results obtained in this model. However, it could be dangerous in other models of ocular pathology.

Item does not contain fulltext Mutations in the CRB1 gene lead to retinal dystrophies ranging from Leber congenital amaurosis (LCA) to early-onset retinitis pigmentosa (RP), due to developmental defects or loss of adhesion between photoreceptors and Muller glia cells, respectively. Whereas over 150 mutations have been found, no clear genotype-phenotype correlation has been established. Mouse Crb1 knockout retinas show a mild phenotype limited to the inferior quadrant, whereas Crb2 knockout retinas display a severe degeneration throughout the retina mimicking the phenotype observed in RP patients associated with CRB1 mutations. Crb1Crb2 double mutant retinas have severe developmental defects similar to the phenotype observed in LCA patients associated with CRB1 mutations. Therefore, CRB2 is a candidate modifying gene of human CRB1-related retinal dystrophy. In this study, we studied the cellular localization of CRB1 and CRB2 in human retina and tested the influence of the Crb2 gene allele on Crb1-retinal dystrophies in mice. We found that in contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Muller glia cells, and CRB2 only in Muller glia cells. Genetic ablation of one allele of Crb2 in heterozygote Crb1(+/-) retinas induced a mild retinal phenotype, but in homozygote Crb1 knockout mice lead to an early and severe phenotype limited to the entire inferior retina. Our data provide mechanistic insight for CRB1-related LCA and RP.

Achromatopsie ist eine autosomal-rezessiv vererbte Netzhauterkrankung, die durch den angeborenen kompletten Funktionsverlust der Zapfenphotorezeptoren charakterisiert ist. Etwa 80 % der Patienten weisen Mutationen in der α- oder β-Untereinheit (A3 oder B3) des cGMP-gesteuerten Kationenkanals CNG (cyclic nucleotide-gated channel) der Zapfenphotorezeptoren auf. Homolog zum humanen Krankheitsbild zeigen CNGA3-defiziente Mause einen zapfenspezifischen Funktionsverlust, der zur Degeneration der betroffenen Zapfenphotorezeptorzelle fuhrt. Am Forschungsinstitut fur Augenheilkunde in Tubingen gelang es nun erstmalig im Tiermodell fur Achromatopsie ACHM2, eine Netzhauterkrankung umfassend zu therapieren. Im vorliegenden Artikel werden der auf subretinaler Injektion von rekombinanten Adeno-assoziierten Viren (rAAV) basierende Ansatz im Detail dargelegt und die eingesetzten nicht-invasiven Diagnostikmethoden ERG, SLO und OCT zur Erfolgs- und Qualitatskontrolle beschrieben. Der nachweisbare Therapieerfolg zeigt sich in einem Funktionsgewinn des Zapfensystems und in einer weiterfuhrenden neuronalen Verarbeitung des retinalen Signals bis hin zu einem zapfendominierten Verhalten der behandelten Tiere. Die herausragenden Ergebnisse sind Ausgangspunkt fur die erste humane Translation einer Gentherapie fur Achromatopsie in Deutschland.

MPP3 and CRB1 both interact directly with PALS1/MPP5 and through this scaffold protein may form a large protein complex. To investigate the role of MPP3 in the retina we have analyzed conditional mutant Mpp3 knockout mice. Ultrastructural localization studies revealed that MPP3 is predominantly localized in apical villi of Muller glia cells. Retinas lacking MPP3 developed late onset retinal degeneration, with sporadic foci of rosette formation in the central part of the retina. Retinal degeneration in Mpp3 cKO mice was accelerated by exposure to moderate levels of white light. Electroretinography recordings in aging mice under both scotopic and photopic conditions ranged from normal to mildly subnormal, while the magnitude correlated with the strength and extent of morphological alterations. Loss of MPP3 resulted in significant loss of PALS1 at the subapical region adjacent to adherens junctions, and loss of MPP3 in Pals1 conditional knockdown retinas significantly accelerated the onset of retinal degeneration. These data suggest that MPP3 is required for maintaining proper levels of PALS1 at the subapical region, and indicate that the MPP3 gene is a candidate modulator of the Crumbs complex.

To study the relationship between retinal and tunica vasculosa lentis (TVL) disease in retinopathy of prematurity (ROP). Although the clinical hallmark of ROP is abnormal retinal blood vessels, the vessels of the anterior segment, including the TVL, are also altered.ROP was induced in Long-Evans pigmented and Sprague Dawley albino rats; room-air-reared (RAR) rats served as controls. Then, fluorescein angiographic images of the TVL and retinal vessels were serially obtained with a scanning laser ophthalmoscope near the height of retinal vascular disease, ~20 days of age, and again at 30 and 64 days of age. Additionally, electroretinograms (ERGs) were obtained prior to the first imaging session. The TVL images were analyzed for percent coverage of the posterior lens. The tortuosity of the retinal arterioles was determined using Retinal Image multiScale Analysis (Gelman et al. in Invest Ophthalmol Vis Sci 46:4734-4738, 2005).In the youngest ROP rats, the TVL was dense, while in RAR rats, it was relatively sparse. By 30 days, the TVL in RAR rats had almost fully regressed, while in ROP rats, it was still pronounced. By the final test age, the TVL had completely regressed in both ROP and RAR rats. In parallel, the tortuous retinal arterioles in ROP rats resolved with increasing age. ERG components indicating postreceptoral dysfunction, the b-wave, and oscillatory potentials were attenuated in ROP rats.These findings underscore the retinal vascular abnormalities and, for the first time, show abnormal anterior segment vasculature in the rat model of ROP. There is delayed regression of the TVL in the rat model of ROP. This demonstrates that ROP is a disease of the whole eye.

Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases.

Norrin is a retinal signaling molecule which is expressed in Müller glia and binds to Frizzled-4 to activate canonical Wnt/β-catenin signaling. Norrin is part of an essential signaling system that controls the formation of retinal capillaries during development. To evaluate neuroprotective properties of Norrin independently from its function during retinal angiogenesis, we generated transgenic mice (Rpe65-Norrin) that constitutively express Norrin in the retinal pigmented epithelium. Substantial amounts of Norrin were secreted into the outer retina, which triggered retinal Wnt/β-catenin signaling in conjunction with an increase in the expression of endothelin-2 (EDN2), endothelin receptor B (EDNRB), and glial fibrillary acidic protein (GFAP). Photoreceptors of Norrin-overexpressing mice were significantly less vulnerable to light-induced damage compared to their wild-type littermates. Following light damage, we observed less apoptotic death of photoreceptors and a better retinal function than in controls. The protective effects were abolished if either Wnt/β-catenin or EDN2 signaling was blocked by intravitreal injection of Dickkopf-1 or BQ788, respectively. Light-damaged retinae from transgenic mice contained higher amounts of brain-derived neurotrophic factor (BDNF) and pAkt than those of wild-type littermates. We conclude that constitutive overexpression of Norrin protects photoreceptors from light damage, an effect that is mediated by Wnt/β-catenin and EDN2 signaling and involves neurotrophic activities of BDNF. The findings suggest that Norrin and its associated signaling pathways have strong potentials to attenuate photoreceptor death following injury.

The absence of cyclic nucleotide-gated (CNG) channels in cone photoreceptor outer segments leads to achromatopsia, a severely disabling disease associated with the complete lack of cone photoreceptor function. In a common form, loss of the CNGA3 subunit disrupts visual transduction in cones and causes progressive degeneration. Here, we show that adeno-associated viral vector-mediated gene replacement therapy added the lacking sensual quality, cone-mediated vision, in the CNGA3 -/- mouse model of the human disease. The functional rescue of cone vision was assessed at different sites along the visual pathway. In particular, we show electrophysiologically that treated CNGA3 -/- mice became able to generate cone-mediated responses and to transfer these signals to bipolar and finally ganglion cells. In support, we found morphologically that expression of CNGA3 delayed cone cell death. Finally, we show in a behavioral test that treated mice acquired photopic vision suggesting that achromatopsia patients may as well benefit from gene replacement therapy. © 2012 Springer Science+Business Media, LLC.

To determine the potential of a commercially available optical coherence tomography (OCT) device (Spectralis™ HRA + OCT, Heidelberg Engineering) for small animal retinal imaging, we achieved to adapt this third generation OCT system to obtain and quantify high-resolution morphological sections of rodent retina in models with acquired and inherited retinal degenerations. Genetically induced (rd1, rho -/-, RPE65) and acquired retinal degeneration (light damage) was similarly clear as in histology and could be followed in a timeline fashion. We were able to detect and analyze a wide range of retinal pathology in a variety of established animal models used in vision research. As this technique allows longitudinal study designs, it will facilitate characterization of disease dynamics while reducing the numbers of study animals needed. Use of identical outcome measures and even the same diagnostic device in animal and clinical studies bears the potential to streamline translational approaches, e.g., in the assessment of putative therapeutic interventions. © 2012 Springer Science+Business Media, LLC.

Purpose Vision originates in rods and cones at the outer retina. Already at these early stages, diverse processing schemes shape and enhance image information to permit perception over a wide range of lighting conditions. In this work, we address the role of hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) in rod photoreceptors for the enhancement of rod system responsivity under conditions of light exposure. Methods To isolate HCN1 channel actions in rod system responses, we generated double mutant mice by crossbreeding Hcn1-/- mice with Cnga3-/- mice in which cones are non-functional. Retinal function in the resulting Hcn1-/- Cnga3-/- animals was followed by means of electroretinography (ERG) up to the age of four month. Retinal imaging via scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) was also performed to exclude potential morphological alterations. Results This study on Hcn1-/- Cnga3-/- mutant mice complements our previous work on HCN1 channel function in the retina. We show here in a functional rod-only setting that rod responses following bright light exposure terminate without the counteraction of HCN channels much later than normal. The resulting sustained signal elevation does saturate the retinal network due to an intensity-dependent reduction in the dynamic range. In addition, the lack of rapid adaptational feedback modulation of rod photoreceptor output via HCN1 in this double mutant limits the ability to follow repetitive (flicker) stimuli, particularly under mesopic conditions. Conclusions This work corroborates the hypothesis that, in the absence of HCN1-mediated feedback, the amplitude of rod signals remains at high levels for a prolonged period of time, leading to saturation of the retinal pathways. Our results demonstrate the importance of HCN1 channels for regular vision.

Ciliopathies, a class of rare genetic disorders, present often with retinal degeneration caused by protein transport defects between the inner segment and the outer segment of the photoreceptors. Bardet-Biedl syndrome is one such ciliopathy, genetically heterogeneous with 17 BBS genes identified to date, presenting early onset retinitis pigmentosa. By investigating BBS12-deprived retinal explants and the Bbs12(-/-) murine model, we show that the impaired intraciliary transport results in protein retention in the endoplasmic reticulum. The protein overload activates a proapoptotic unfolded protein response leading to a specific Caspase12-mediated death of the photoreceptors. Having identified a therapeutic window in the early postnatal retinal development and through optimized pharmacological modulation of the unfolded protein response, combining three specific compounds, namely valproic acid, guanabenz, and a specific Caspase12 inhibitor, achieved efficient photoreceptor protection, thereby maintaining light detection ability in vivo.

Characterization of disease models of neurodegenerative disorders requires a systematic and comprehensive phenotyping in a highly standardized manner. Therefore, automated high-resolution behavior test systems such as the homecage based LabMaster system are of particular interest. We demonstrate the power of the automated LabMaster system by discovering previously unrecognized features of a recently characterized atxn3 mutant mouse model. This model provided neurological symptoms including gait ataxia, tremor, weight loss and premature death at the age of 12 months usually detectable just 2 weeks before the mice died. Moreover, using the LabMaster system we were able to detect hypoactivity in presymptomatic mutant mice in the dark as well as light phase. Additionally, we analyzed inflammation, immunological and hematological parameters, which indicated a reduced immune defense in phenotypic mice. Here we demonstrate that a detailed characterization even of organ systems that are usually not affected in SCA3 is important for further studies of pathogenesis and required for the preclinical therapeutic studies.

Calcium mediates various neuronal functions. The complexity of neuronal Ca2+signaling is well exemplified by retinal cone photoreceptors, which, with their distinct compartmentalization, offer unique possibilities for studying the diversity of Ca2+functions in a single cell. Measuring subcellular Ca2+signals in cones under physiological conditions is not only fundamental for understanding cone function, it also bears important insights into pathophysiological processes governing retinal neurodegeneration. However, due to the proximity of light-sensitive outer segments to other cellular compartments, optical measurements of light-evoked Ca2+responses in cones are challenging. We addressed this problem by generating a transgenic mouse (HR2.1:TN-XL) in which both short- and middle-wavelength-sensitive cones selectively express the genetically encoded ratiometric Ca2+biosensor TN-XL. We show thatHR2.1:TN-XLallows recording of light-evoked Ca2+responses using two-photon imaging in individual cone photoreceptor terminals and to probe phototransduction and its diverse regulatory mechanisms with pharmacology at subcellular resolution. To further test this system, we asked whether the classical, nitric oxide (NO)-soluble guanylyl-cyclase (sGC)-cGMP pathway could modulate Ca2+in cone terminals. Surprisingly, NO reduced Ca2+resting levels in mouse cones, without evidence for direct sGC involvement. In conclusion,HR2.1:TN-XLmice offer unprecedented opportunities to elucidate light-driven Ca2+dynamics and their (dys)regulation in cone photoreceptors.

The modulatory role of large-conductance Ca2+-activated K+(BK) channels in the nervous system has been extensively studied. In the retina, it has been shown that BK channels play a pivotal role in modulating feedback from A17 amacrine cells to rod bipolar cells (RBCs). Here, we used electroretinography to examine the functional role of BK channels for rod and cone vision in the retinain vivousing a genetically engineered mouse lacking functional BK channels (Bk−/−). Under dark-adapted and light-adapted conditions, the lack of BK channels had no effect on photoreceptor activity, suggesting that these ion channels do not modulate photoreceptor responses. At the bipolar cell level, the ERG signals attributed to RBCs inBk−/−mice were not different from those in wild-type mice at low scotopic stimulus intensities. However, at high scotopic and low mesopic stimulus intensities, close to RBC saturation, a significant reduction of ERG signals reflecting RBC activity was present in theBk−/−retina. At higher mesopic stimulus intensities activating both RBCs and cone bipolar cells (CBCs), no difference in ERG signals betweenBk−/−and wild-type mice was found. In photopic stimulus paradigms, activity of ON- and OFF-CBCs inBk−/−and wild-type retinae was indistinguishable. These findings demonstrate that BK channels modulate visual responsesin vivoat the bipolar cell level at intermediate stimulus conditions.

Only in recent years have specific mutations been elucidated for feline hereditary retinal dystrophies. Molecular genetic characterization of feline diseases has so far been a slow process but with a full genome sequence for the cat recently completed and the development of a feline single nucleotide polymorphism chip, the characterization of feline monogenic disorders will be significantly simplified. This review summarizes current knowledge with regard to specific hereditary retinal dystrophies in cats and gives an overview of how cats can be used as models in translational research.

Purpose Hypoxic preconditioning activates hypoxia-inducible transcription factors (HIFs) in the retina and protects photoreceptors from light-induced retinal degeneration. The authors tested whether photoreceptor-specific activation of HIFs in normoxia is sufficient for protection. Methods Rod-specific Vhl knockdown mice were generated using the Cre-lox system with the rod opsin promoter controlling expression of CRE recombinase to stabilize HIF transcription factors in normoxic rods. Cell death was induced by light exposure and quantified by ELISA. Rhodopsin was quantified by spectrophotometry. Gene expression was analyzed by real-time PCR, and levels of proteins were determined by Western blotting. Morphology was investigated by light microscopy and retinal function tested by ERG. Results The rod-specific Vhl knockdown stabilized HIF-α proteins and induced expression of HIF target genes in retinas of 10-week-old mice under normoxic conditions. Retinal morphology and function were normal. At 36 hours after exposure to excessive light, Vhl knockdowns showed significantly less photoreceptor cell death than did wild-type controls. Ten days after light exposure, however, photoreceptor degeneration in Vhl knockdowns was similar to that of control animals. Vhl knockdowns expressed Fgf2 at higher basal levels before light exposure. After light exposure, however, expression of Fgf2 was not significantly different from that of wild-type controls. Conclusions Artificial activation of HIF transcription factors in normoxic photoreceptors results in an increased basal expression of Fgf2 that may contribute to a transient protection of rods against light damage. Full photoreceptor protection may require a hypoxia-like response in additional retinal cell types and/or the differential regulation of additional mechanisms.

Loss of vision and blindness in human patients is often caused by the degeneration of neuronal cells in the retina. In mouse models, photoreceptors can be protected from death by hypoxic preconditioning. Preconditioning in low oxygen stabilizes and activates hypoxia inducible transcription factors (HIFs), which play a major role in the hypoxic response of tissues including the retina. We show that a tissue-specific knockdown of von Hippel-Lindau protein (VHL) activated HIF transcription factors in normoxic conditions in the retina. Sustained activation of HIF1 and HIF2 was accompanied by persisting embryonic vasculatures in the posterior eye and the iris. Embryonic vessels persisted into adulthood and led to a severely abnormal mature vessel system with vessels penetrating the photoreceptor layer in adult mice. The sustained hypoxia-like response also activated the leukemia inhibitory factor (LIF)-controlled endogenous molecular cell survival pathway. However, this was not sufficient to protect the retina against massive cell death in all retinal layers of adult mice. Caspases 1, 3 and 8 were upregulated during the degeneration as were several VHL target genes connected to the extracellular matrix. Misregulation of these genes may influence retinal structure and may therefore facilitate growth of vessels into the photoreceptor layer. Thus, an early and sustained activation of a hypoxia-like response in retinal cells leads to abnormal vasculature and severe retinal degeneration in the adult mouse retina.

Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

Usher syndrome denotes a clinically and genetically heterogeneous combination of retinitis pigmentosa and sensorineural deafness. The division into subtypes I, II, and III is based on the degree of hearing loss: Type I is characterized by deafness from birth together with ataxia and retarded motor development, type II by a stationary deafness of a moderate degree, and type III by a progressive deafness with adult onset. In Germany, Usher syndrome currently bears particular relevance because in January 2009 a new compulsory screening of auditory function in newborn infants was introduced. Consequently, it can be expected that a higher number of patients with Usher syndrome will be identified in early childhood and referred to ophthalmologists. The focus of this work is to introduce the typical clinical picture of Usher syndrome, summarize diagnostic options, and give an overview of therapeutic strategies.

Purpose To investigate whether sheets of fetal retinal allografts can integrate into the dystrophic Abyssinian cat retina with progressive rod cone degeneration. Methods Fetal retinal sheets (cat gestational day 42), incubated with BDNF microspheres, were transplanted to the subretinal space of four cats at an early disease stage. Cats were studied by fundus examinations, bilateral full-field flash ERGs, and indocyanine green and fluorescein angiograms up to 4 months following surgery. E42 donor and transplanted eyes were analyzed by histology and immunohistochemistry for retinal markers. Results Funduscopy and angiography showed good integration of the transplants in two of four cats, including extension of host blood vessels into the transplant and some scarring in the host. In these two, transplants were found in the subretinal space with laminated areas, with photoreceptor outer segments in normal contacts with the host retinal pigment epithelium. In some areas, transplants appeared to be well-integrated within the host neural retina. Neither of these two cats showed functional improvement in ERGs. In the other two cats, only remnants of donor tissue were left. Transplants stained for all investigated cellular markers. No PKC immunoreactivity was detected in the fetal donor retina at E42, but developed in the 4-month-old grafts. Conclusions Fetal sheet transplants can integrate well within a degenerating cat retina and develop good lamination of photoreceptors. Functional improvement was not demonstrated by ERG in cats with well-laminated grafts. Transplants need to be further evaluated in cat host retinas with a more advanced retinal degeneration using longer follow-up times.