Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo.

PURPOSE: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or "wet" Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. METHODS: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with "wet" AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8(+/-) mice expressing ß-galactosidase. Aged Mfge8(+/-) and Mfge8(-/-) mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. RESULTS: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8(-/-) mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8(-/-) mice as compared to controls. CONCLUSIONS: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8(-/-) mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells.

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.

Chromatic pupillometry in patients with retinitis pigmentosa.

OBJECTIVE:: To evaluate the chromatic pupillary response as a means of assessing outer and inner retinal function in patients with retinitis pigmentosa (RP). DESIGN:: Evaluation of diagnostic technology. PARTICIPANTS:: Thirty-two patients with RP and visual loss and 43 normal subjects. METHODS:: Patients were tested with a chromatic pupillometer using red and blue lights (1, 10, and 100 cd/m(2)), and their pupil responses were compared with those from 43 normal subjects (reported previously). Visual field and electroretinography (ERG) results were examined and compared with the pupil responses. MAIN OUTCOME MEASURES:: The percent pupil contraction of the transient response to a low-intensity (1 cd/m(2)) blue light and high-intensity (100 cd/m(2)) red light and the sustained response to a high-intensity blue light was calculated for 1 eye of each subject. RESULTS:: The pupil responses to red and blue light at all intensities were recordable in all patients except 1, whose pupil responded only to bright blue light. There was a significant difference of the pupil response between patients with RP and normal subjects in testing conditions that emphasized rod (1 cd/m(2) blue light) or cone (100 cd/m(2) red light) contribution (P<0.001). Patients with a non-recordable scotopic ERG showed significantly reduced pupil responses (P<0.001) to low-intensity blue light (1 cd/m(2)). Patients with a non-recordable or abnormal photopic ERG showed significantly reduced pupil responses (P<0.05) to high-intensity red light (100 cd/m(2)). Patients with a nonrecordable ERG had the most visual field loss and reduced pupil responses. Unexpectedly, patients with RP showed a slower re-dilation of the pupil after termination of bright blue light compared with red light, a pattern not observed in normal subjects. CONCLUSIONS:: Pupil responses to red and blue light stimuli weighted to favor cone or rod input are significantly reduced in patients with RP but are still recordable in patients having a non-recordable ERG. In addition, outer photoreceptor disease appears to unmask a post-illumination pupillary constriction to bright blue light, most likely mediated by intrinsic activation of melanopsin ganglion cells. Chromatic pupillometry provides a novel, noninvasive method for following retinal functional status, particularly in patients with severe RP and non-recordable ERG. FINANCIAL DISCLOSURE(S):: Proprietary or commercial disclosure may be found after the references.

FAM161A, associated with autosomal recessive retinitis pigmentosa,localizes at the level of the photoreceptor cilium and interacts with proteins involved in ciliopathies

Purpose: We have previously demonstrated that mutations in the FAM161A gene, encoding a protein with unknown function and no similarities with other characterized sequences, cause autosomal recessive retinitis pigmentosa (RP). The purpose of this work is to investigate the functional role of FAM161A within the retina and its relationship with other proteins involved in RP. Methods: The subcellular localization of FAM161A in the retina was assessed by immunohistochemistry of retinal sections and dissociated photoreceptors from mice, which were stained using antibodies against FAM161A and antibodies against cilium markers. The function of FAM161A was further assessed in ciliated mammalian cell lines by expression of recombinant FAM161A with various fusion tags. The binary interaction between FAM161A and a collection of ciliary and ciliopathy-associated proteins was analyzed using a yeast two-hybrid assay. The results obtained with this technique were validated using independent protein-protein interaction assays (GST-pull downs, co-transfection and co-immunoprecipitation). Results: Native FAM161A localized at the connecting cilium of photoreceptor cells, as demonstrated by immunofluorescence in both dissociated photoreceptors and retinal sections of mice. More specifically, co-staining with markers for ciliary sub-structures (RPGRIP1L, Centrin, RP1, GT335) demonstrated that FAM161A decorated the basal body and the very apical part of the connecting cilium. Upon overexpression in ciliated cultured mammalian cells, FAM161A localized to the ciliary basal body. Yeast two-hybrid analysis of the binary interaction of FAM161A and an array of ciliary proteins revealed the direct interaction of FAM161A with three proteins of which the cognate genes are mutated in retinal ciliopathies. The confirmation of these interactions using different biochemical assays is currently in progress. Conclusions: FAM161A is a ciliary basal body protein of the photoreceptor connecting cilium, rendering the associated RP as a novel retinal ciliopathy. The confined expression of FAM161A in the retina and the direct interaction of FAM161A with other retinal ciliopathy-associated proteins may explain the retinal phenotype of this specific subset of mechanistically and phenotypically connected retinal disorders.

Bax-induced apoptosis in Leber's congenital amaurosis : a dual role in rod and cone degeneration

Purpose:We previously observed that anti- and pro-apoptotic genes of the Bcl-2 family were differentially expressed during the development of LCA in the Rpe65-/- mouse model (Cottet et al. 2006). Moreover, we reported that activation and translocation of pro-apoptotic Bax to the mitochondria was associated with apoptosis of rod photoreceptors as the disease progressed (Cottet et al. 2008). In this study we challenged whether disruption of the pro-apoptotic pro-apoptotic Bax protein is sufficient to protect photoreceptor cells against apoptosis. Methods:Apoptosis of photoreceptor cells was addressed by TUNEL assay on flatmounted retinas. Counting of the rod nuclei within the ONL was performed following hematoxylin/eosin histological staining of retina sections. Expression level and localization of photoreceptor gene markers were assessed by quantitative PCR and immunohistological analyses. Results:While expression of rod photoreceptor genes was decreased in Rpe65-deficient retina, expression level remained unchanged in Rpe65-/- / Bax-/- mice. Moreover, OS dysorganization and shortening as well as decrease in ONL thickness observed in diseased retina were prevented in mice lacking functional Bax protein. TUNEL assay confirmed that Bax-dependent rod photoreceptor apoptosis was abolished in Rpe65-/- / Bax-/- mice. However, early and fast degeneration of cone cells was not prevented in Rpe65-/- / Bax-/- mice, indicating that Bax-induced apoptotic pathway was not involved in the degenerating process of cones in Rpe65-deficient retina. Conclusions:Altogether, these data show for the first time that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in LCA disease. While pro-apoptotic Bax is essential to trigger rod photoreceptor apoptosis, early degeneration of cones is not dependent on Bax-mediated apoptotic pathway in Rpe65-deficientmice.

Interleukin-1β inhibition prevents choroidal neovascularization and does not exacerbate photoreceptor degeneration.

The pro-inflammatory cytokine IL-1β has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1β in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1β expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1β was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1β significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1β on choroidal endothelial cell proliferation. Inhibition of IL-1β in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1β inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

Cell Cycle Proteins and Retinal Degeneration: Evidences of New Potential Therapeutic Targets.

During different forms of neurodegenerative diseases, including the retinal degeneration, several cell cycle proteins are expressed in the dying neurons from Drosophila to human revealing that these proteins are a hallmark of neuronal degeneration. This is true for animal models of Alzheimer's, and Parkinson's diseases, Amyotrophic Lateral Sclerosis and for Retinitis Pigmentosa as well as for acute injuries such as stroke and light damage. Longitudinal investigation and loss-of-function studies attest that cell cycle proteins participate to the process of cell death although with different impacts, depending on the disease. In the retina, inhibition of cell cycle protein action can result to massive protection. Nonetheless, the dissection of the molecular mechanisms of neuronal cell death is necessary to develop adapted therapeutic tools to efficiently protect photoreceptors as well as other neuron types.

Incidence of outer retinal tubulation in ranibizumab-treated age-related macular degeneration.

PURPOSE: To investigate the incidence of outer retinal tubulation (ORT) in ranibizumab-treated neovascular age-related macular degeneration patients. METHODS: We included 480 consecutive patients (546 eyes) with neovascular age-related macular degeneration, who were treated with variable-dosing intravitreal ranibizumab, evaluated with spectral domain optical coherence tomography, and followed-up for a minimum period of 6 months. Optical coherence tomographies were evaluated for the first appearance of ORT, precursor signs, and type of underlying lesion. Visual acuity was also recorded. RESULTS: Outer retinal tubulation was observed in 30% of eyes during a mean follow-up period of 26.7 months (SD, 13.5). Kaplan-Meier survival analysis revealed that the ORT incidence (2.5, 17.5, 28.4, and 41.6% at baseline, after 1, 2, and 4 years, respectively) continuously increased, despite visually effective anti-vascular endothelial growth factor treatment. Outer retinal tubulation was associated with a poorer functional benefit. Lower baseline visual acuity was associated with a higher risk of developing ORT. CONCLUSION: Incidence of ORT continuously increases despite visually optimal anti-vascular endothelial growth factor treatment of age-related macular degeneration. Outer retinal tubulation might be considered a prognostic factor for functional outcome and is relevant to avoid overtreatment.

Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF(-)GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF(-)GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in 'equivalent-light'' scenarios.

Development and disease of the photoreceptor cilium

Primary cilia are microtubule-rich hair-like extensions protruding from the surface of most post-mitotic cells. They act as sensory organelles that help interpret various environmental cues. Mutations in genes encoding proteins involved in ciliogenesis or protein transport to the primary cilia lead to a wide variety of diseases commonly referred to as ciliopathies,which include primary ciliary dyskinesia, situs invertus, hydrocephalus, kidney diseases, respiratory diseases, and retinal degenerations. In the retina, the photoreceptor cells have a highly specialized primary cilium called the outer segment (OS), which is essential for photosensation. Development of the photoreceptor OS shares key regulatory mechanisms with ciliogenesis in other cell types. Accumulating evidence indicates that mutations that affect OS development and/or protein transport to the OS generally lead to photoreceptor degeneration, which can be accompanied by a range of other clinical manifestations due to the dysfunction of primary cilia in different cell types. Here, we review the general mechanisms regulating ciliogenesis, and present different examples of mutations affecting OS ciliogenesis and protein transport that lead to photoreceptor degeneration. Overall, we conclude that the genetic and molecular evidence accumulated in recent years suggest a clear link between the development and function of the primary cilium and various clinical conditions. Future studies aimed at uncovering the cellular and molecular mechanisms implicated in ciliogenesis in a wide variety of animal models should greatly increase our understanding of the pathophysiology of many human diseases, including retinal degenerations.

Intraflagellar transport in the developing and mature vertebrate retina

Intraflagellar transport (IFT) is required for the assembly and maintenance of cilia. In this study we analyzed the subcellular localization of IFT proteins in retinal cells by correlative high-resolution immunofluorescence and immunoelectron microscopy. The rod photoreceptor cell was used as a model system to analyze protein distribution in cilia. To date the expression of IFT proteins has been described in the ciliary region without deciphering the precise spatial and temporal subcellular localization of IFT proteins, which was the focus of my work. rnThe establishment of the pre-embedding immunoelectron method was an important first step for the present doctoral thesis. Results of this work reveal the differential localization of IFT20, IFT52, IFT57, IFT88, IFT140 in sub-ciliary compartments and also their presence in non-ciliary compartments of retinal photoreceptor cells. Furthermore, the localization of IFT20, IFT52 and IFT57 in dendritic processes of non-ciliated neurons indicates that IFT protein complexes also operate in non-ciliated cells and may participate in intracellular vesicle trafficking in eukaryotic cells in general.rnIn addition, we have investigated the involvement of IFT proteins in the ciliogenesis of vertebrate photoreceptor cilia. Electron microscopy analyses revealed six morphologically distinct stages. The first stages are characterized by electron dense centriolar satellites and a ciliary vesicle, while the formation of a ciliary shaft and of the light sensitive outer segment disks are features of the later stages. IFT proteins were expressed during all stages of photoreceptor cell development and found to be associated with the ciliary apparatus. In addition to the centriole and basal body IFT proteins are present in the photoreceptor cytoplasm, associated with centriolar satellites, post-Golgi vesicles and with the ciliary vesicle. Therewith the data provide an evidence for the involvement of IFT proteins during ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium of photoreceptor cells. Moreover, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis indicates roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella. rn

Pathophysiology of proliferative vitreoretinopathy in retinal detachment

Because proliferative vitreoretinopathy cannot be effectively treated, its prevention is indispensable for the success of surgery for retinal detachment. The elaboration of preventive and therapeutic strategies depends upon the identification of patients who are genetically predisposed to develop the disease, as well as upon an understanding of the biological process involved and the role of local factors, such as the status of the uveovascular barrier. Detachment of the retina or vitreous activates glia to release cytokines and ATP, which not only protect the neuroretina but also promote inflammation, retinal ischemia, cell proliferation, and tissue remodeling. The vitreal microenvironment favors cellular de-differentiation and proliferation of cells with nonspecific nutritional requirements. This may render a pharmacological inhibition of their growth difficult without causing damage to the pharmacologically vulnerable neuroretina. Moreover, reattachment of the retina relies upon the local induction of a controlled wound-healing response involving macrophages and proliferating glia. Hence, the functional outcome of proliferative vitreoretinopathy will be determined by the equilibrium established between protective and destructive repair mechanisms, which will be influenced by the location and the degree of damage to the photoreceptor cells that is induced by peri-retinal gliosis.